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Sample records for cd40l dna combined

  1. Multimeric soluble CD40 ligand (sCD40L) efficiently enhances HIV specific cellular immune responses during DNA prime and boost with attenuated poxvirus vectors MVA and NYVAC expressing HIV antigens.

    PubMed

    Gómez, Carmen E; Nájera, José L; Sánchez, Raquel; Jiménez, Victoria; Esteban, Mariano

    2009-05-21

    The attenuated poxvirus vectors MVA and NYVAC are now in clinical trials against HIV/AIDS. Due to the vectors restricted replication capacity in human cells, approaches to enhance their immunogenicity are highly desirable. Here, we have analyzed the ability of a soluble form of hexameric CD40L (sCD40L) to stimulate specific immune responses to HIV antigens when inoculated in mice during priming with DNA and in the booster with MVA or NYVAC, expressing the vectors HIV-1 Env, Gag, Pol and Nef antigens from clade B. Our findings revealed that sCD40L in DNA/poxvirus combination enhanced the magnitude about 2-fold (DNA-B/MVA-B) and 4-fold (DNA-B/NYVAC-B), as well as the breath of the HIV antigen specific cellular immune responses. sCD40L was necessary in both prime and boost inoculations triggering a potent polarization of the Th response towards a Th1 type. In DNA-B/NYVAC-B regime the addition of sCD40L significantly enhanced the humoral immune response against HIV gp160, but not in DNA-B/MVA-B combination. These findings provided evidence for the immunostimulatory benefit of sCD40L when DNA and the poxvirus vectors MVA and NYVAC are used as immunogens.

  2. Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway

    PubMed Central

    Wang, Xiao-Lin; Zhou, Yuan-Li; Sun, Wei; Li, Li

    2016-01-01

    CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques. PMID:27120457

  3. Modulation of Single-Cell IgG Secretion Frequency and Rates in Human Memory B Cells by CpG DNA, CD40L, IL-21 and Cell Division∥

    PubMed Central

    Henn, Alicia D.; Rebhahn, Jonathan; Brown, Miguel A.; Murphy, Alison J.; Coca, Mircea N.; Hyrien, Ollivier; Pellegrin, Tina; Mosmann, Tim; Zand, Martin S.

    2009-01-01

    During the recall response by CD27+ IgG class switched human memory B cells, total IgG secreted is a function of (1) the number of IgG secreting cells (IgG-SC) and (2) the secretion rate of each cell. Here we report the quantitative ELISPOT method (qELISPOT) for simultaneous estimation of single cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgMneg memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. B cell receptor cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27hi B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27hi B cell blasts. In contrast, the addition of IL-21 to CD40L +IL-4 activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG+ck-activated B memory B cell populations, is suggestive of an “On/Off switch” regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG secreting cells and the distribution of their IgG secretion rates. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this

  4. Stratification of ST-elevation myocardial infarction patients based on soluble CD40L longitudinal changes.

    PubMed

    Napoleão, Patrícia; Cabral, Luís B P; Selas, Mafalda; Freixo, Cláudia; Monteiro, Maria do Céu; Criado, Maria Begoña; Costa, Marina C; Enguita, Francisco J; Viegas-Crespo, Ana Maria; Saldanha, Carlota; Carmo, Miguel Mota; Ferreira, Rui Cruz; Pinheiro, Teresa

    2016-10-01

    Involvement of soluble CD40 ligand (sCD40L) in thrombosis and inflammation on the context of coronary artery disease is currently being revised. In that perspective, we had studied the association of sCD40L with markers of platelet activation and markers of endothelial and vascular function. On that cohort, a stratification of patients with acute myocardial infarction (AMI) 1 month after percutaneous coronary intervention (PCI) was observed based on concentrations of sCD40L. The study intended to identify the groups of AMI patients with different profiles of sCD40L concentrations and verify how medication, clinical evolution, biochemical data, and markers of regulation of endothelial function at genetic (endothelial nitric oxide synthase polymorphisms) and post-transcriptional levels (circulating microRNAs) affect sCD40L serum levels. Lower quartiles of sCD40L (<2.3 ng/mL) were associated with higher concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP), high frequency of G894T polymorphism, and altered expression of a set of microRNAs assumed to be involved in the regulation of endothelial and cardiac function and myocardium hypertrophy, relative to patients in sCD40L upper quartiles. A characteristic sCD40L variation pattern in STEMI patients was identified. Low levels of sCD40L 1 month after PCI distinguish STEMI patients with worse prognosis, a compromised cardiac healing, and a persistent endothelial dysfunction, as given by the association between sCD40L, NT-proBNP, G894T polymorphism, and specific profile of miRNA expression. These results suggest sCD40L could have a prognostic value in STEMI patients. PMID:27172386

  5. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  6. Interruption of classic CD40L-CD40 signalling but not of the novel CD40L-Mac-1 interaction limits arterial neointima formation in mice.

    PubMed

    Willecke, F; Tiwari, S; Rupprecht, B; Wolf, D; Hergeth, S; Hoppe, N; Dufner, B; Schulte, L; Anto Michel, N; Bukosza, N; Marchini, T; Jäckel, M; Stachon, P; Hilgendorf, I; Zeschky, K; Schleicher, R; Langer, H F; von Zur Muhlen, C; Bode, C; Peter, K; Zirlik, A

    2014-08-01

    The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 µm² vs 35469 ± 11870 µm²). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis.

  7. Chorioamnionitis is associated with increased CD40L expression on cord blood platelets.

    PubMed

    Sitaru, Ana-Gabriela; Speer, Christian P; Holzhauer, Susanne; Obergfell, Achim; Walter, Ulrich; Grossmann, Ralf

    2005-12-01

    Chorioamnionitis (CA) is a severe infection responsible not only for premature birth but also for many severe neonatal diseases. The aim of the present study was to investigate the expression of CD40L and P-selectin on platelets and the plasma levels of their soluble forms in the umbilical cord blood in infants with documented chorioamnionitis. Umbilical cord blood samples were obtained from 10 healthy term newborns, 10 non-infected preterm infants, 10 preterm infants with premature rupture of membranes and 9 preterm infants with clinical and histological CA. The expression of CD40L and P-selectin on platelets was analyzed by flow cytometry. Soluble P-selectin (sCD62P), soluble CD40L (sCD40L) and interleukine-6 (IL-6) were measured in plasma by ELISA assays. Neonates with CA had significantly higher percentages of platelets expressing CD40L in basal conditions (5.3 +/- 2.9% vs. 1.6 +/- 0.7% and in non-infected preterm infants p < 0.05), while the percentages of P-selectin positive platelets were similar among all groups. In contrast, the level of sP-selectin was higher in infants with CA (222 +/- 128 ng/ml vs. 104 +/- 71 ng/ml in non-infected preterm infants, p < 0.05) but no differences were found in the levels of sCD40L. As expected, the levels of IL-6, a pro-inflammatory cytokine were significantly higher in samples obtained from preterm neonates whose mothers had also elevated inflammatory parameters. Our observations suggest that platelets are involved in the complex inflammatory pathogenesis of CA. Neither P-selectin expression on cord blood platelets nor plasma sP-selectin or sCD40L were suitable platelet markers in CA, whereas CD40L was significantly elevated in histologically proven CA.

  8. T cell-expressed CD40L potentiates the bone anabolic activity of intermittent PTH treatment.

    PubMed

    Robinson, Jerid W; Li, Jau-Yi; Walker, Lindsey D; Tyagi, Abdul Malik; Reott, Michael A; Yu, Mingcan; Adams, Jonathan; Weitzmann, M Neale; Pacifici, Roberto

    2015-04-01

    T cells are known to potentiate the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. One of the involved mechanisms is increased T cell secretion of Wnt10b, a potent osteogenic Wnt ligand that activates Wnt signaling in stromal cells (SCs). However, additional mechanisms might play a role, including direct interactions between surface receptors expressed by T cells and SCs. Here we show that iPTH failed to promote SC proliferation and differentiation into osteoblasts (OBs) and activate Wnt signaling in SCs of mice with a global or T cell-specific deletion of the T cell costimulatory molecule CD40 ligand (CD40L). Attesting to the relevance of T cell-expressed CD40L, iPTH induced a blunted increase in bone formation and failed to increase trabecular bone volume in CD40L(-/-) mice and mice with a T cell-specific deletion of CD40L. CD40L null mice exhibited a blunted increase in T cell production of Wnt10b and abrogated CD40 signaling in SCs in response to iPTH treatment. Therefore, expression of the T cell surface receptor CD40L enables iPTH to exert its bone anabolic activity by activating CD40 signaling in SCs and maximally stimulating T cell production of Wnt10b.

  9. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

  10. Soluble CD40L in children and adolescents with type 1 diabetes: relation to microvascular complications and glycemic control.

    PubMed

    El-Asrar, Mohamed A; Adly, Amira Am; Ismail, Eman A

    2012-12-01

    CD40-soluble CD40 ligand (sCD40L) interactions might constitute an important mediator for vascular inflammation that initiates diabetic microangiopathy. Little is known about the relation between sCD40L and glycemic control. Therefore, this study aimed to evaluate sCD40L levels in patients with type 1 diabetes and its relation to microvascular complications and metabolic control. Sixty patients with type 1 diabetes were compared with 30 healthy control subjects. Detailed medical history, thorough clinical examination, and laboratory assessment of high-sensitivity C-reactive protein, glycemic control, and the presence of microvascular complications were performed. Measurement of serum sCD40L levels was done using enzyme-linked immunosorbent assay. Patients were divided into two groups according to the presence of microvascular complications. Serum sCD40L levels were significantly elevated in patients with type 1 diabetes in both groups compared with healthy controls (p < 0.001). Patients with microvascular complications had higher serum sCD40L concentrations than non-complicated cases (median, 13 000 vs. 450 pg/mL; p < 0.001). Serum sCD40L cutoff value of 530 pg/mL was able to differentiate complicated from non-complicated cases (p < 0.001). Patients with microalbuminuria or peripheral neuropathy showed higher levels of sCD40L when compared with patients without these complications (p < 0.05). Serum sCD40L levels were positively correlated with hemoglobin A1c and urinary albumin excretion (p < 0.001). We suggest that serum sCD40L levels are elevated in type 1 diabetes, particularly in patients with microvascular complications and a significant correlation with glycemic control exists. Therefore, measurement of serum sCD40L levels in poorly controlled patients would help to identify those at high risk of developing microvascular complications.

  11. Immune response is an important aspect of the antitumor effect produced by a CD40L-encoding oncolytic adenovirus.

    PubMed

    Diaconu, Iulia; Cerullo, Vincenzo; Hirvinen, Mari L M; Escutenaire, Sophie; Ugolini, Matteo; Pesonen, Saila K; Bramante, Simona; Parviainen, Suvi; Kanerva, Anna; Loskog, Angelica S I; Eliopoulos, Aristides G; Pesonen, Sari; Hemminki, Akseli

    2012-05-01

    Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (T(H)1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)-mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the T(H)1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8(+) T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of T(H)1 cytokines.

  12. Preliminary evidence for a matrix metalloproteinase-2 (MMP-2)-dependent shedding of soluble CD40 ligand (sCD40L) from activated platelets.

    PubMed

    Reinboldt, Stephan; Wenzel, Folker; Rauch, Bernhard H; Hohlfeld, Thomas; Grandoch, Maria; Fischer, Jens W; Weber, Artur-Aron

    2009-09-01

    Platelets are the major source of soluble CD40 ligand (sCD40L) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets to release sCD40L. However, the enzyme involved in sCD40L shedding has not been identified yet. Using a panel of pharmacological inhibitors of serine, cysteine, aspartate, or metalloproteinases, preliminary evidence is presented for the hypothesis that matrix metalloproteinase-2 (MMP-2) might be the protease, primarily responsible for CD40L cleavage from platelet surface. PMID:19811225

  13. Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype.

    PubMed

    Chimote, Ameet A; Hajdu, Peter; Kottyan, Leah C; Harley, John B; Yun, Yeoheung; Conforti, Laura

    2016-05-01

    Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

  14. The Serum Levels of the Soluble Factors sCD40L and CXCL1 Are Not Indicative of Endometriosis

    PubMed Central

    Pateisky, Petra; Pils, Dietmar; Kuessel, Lorenz; Szabo, Ladislaus; Walch, Katharina; Obwegeser, Reinhard; Wenzl, René; Yotova, Iveta

    2016-01-01

    Endometriosis is a benign but troublesome gynecological condition, characterized by endometrial-like tissue outside the uterine cavity. Lately, the discovery and validation of noninvasive diagnostic biomarkers for endometriosis is one of the main priorities in the field. As the disease elicits a chronic inflammatory reaction, we focused our interest on two factors well known to be involved in inflammation and neoplastic processes, namely, soluble CD40 Ligand and CXCL1, and asked whether differences in the serum levels of sCD40L and CXCL1 in endometriosis patients versus controls can serve as noninvasive disease markers. A total of n = 60 women were included in the study, 31 endometriosis patients and 29 controls, and the serum levels of sCD40L and CXCL1 were measured by enzyme-linked immunosorbent assay. Overall, there were no statistically significant differences in the levels of expression of both sCD40L and CXCL1 between patients and controls. This study adds useful clinical data showing that the serum levels of the soluble factors sCD40L and CXCL1 are not associated with endometriosis and are not suitable as biomarkers for disease diagnosis. However, we found a trend toward lower levels of sCD40L in the deep infiltrating endometriosis subgroup making it a potentially interesting target worth further investigation. PMID:27190986

  15. Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

    PubMed

    Davidson, Donna C; Hirschman, Michael P; Sun, Anita; Singh, Meera V; Kasischke, Karl; Maggirwar, Sanjay B

    2012-01-01

    Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

  16. CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

    PubMed Central

    Zaccard, Colleen R.; Watkins, Simon C.; Kalinski, Pawel; Fecek, Ronald J.; Yates, Aarika L.; Salter, Russell D.; Ayyavoo, Velpandi; Rinaldo, Charles R.; Mailliard, Robbie B.

    2014-01-01

    The ability of dendritic cells (DC) to mediate CD4+ T cell help for cellular immunity is guided by instructive signals received during DC maturation, and the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. Here we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type-1 immunity (DC1) are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or recombinant CD40L. This immunologic process of DC ‘reticulation’ facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by DC1, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread. PMID:25548234

  17. PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development.

    PubMed

    Awe, Olufolakemi; Hufford, Matthew M; Wu, Hao; Pham, Duy; Chang, Hua-Chen; Jabeen, Rukhsana; Dent, Alexander L; Kaplan, Mark H

    2015-10-15

    PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4(+) T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1(lck-/-)) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1(lck-/-) mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1(lck-/-) mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1(lck-/-) mice, anti-CD40L treatment of immunized Sfpi1(lck-/-) mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity.

  18. Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40L provides universal protection against diverse subtypes.

    PubMed

    Fan, X; Hashem, A M; Chen, Z; Li, C; Doyle, T; Zhang, Y; Yi, Y; Farnsworth, A; Xu, K; Li, Z; He, R; Li, X; Wang, J

    2015-01-01

    The influenza viral hemagglutinin (HA) is comprised of two subunits. Current influenza vaccine predominantly induces neutralizing antibodies (Abs) against the HA1 subunit, which is constantly evolving in unpredictable fashion. The other subunit, HA2, however, is highly conserved but largely shielded by the HA head domain. Thus, enhancing immune response against HA2 could potentially elicit broadly inhibitory Abs. We generated a recombinant adenovirus (rAd) encoding secreted fusion protein, consisting of codon-optimized HA2 subunit of influenza A/California/7/2009(H1N1) virus fused to a trimerized form of murine CD40L, and determined its ability of inducing protective immunity upon intranasal administration. We found that mice immunized with this recombinant viral vaccine were completely protected against lethal challenge with divergent influenza A virus subtypes including H1N1, H3N2, and H9N2. Codon-optimization of HA2 as well as the use of CD40L as a targeting ligand/molecular adjuvant were indispensable to enhance HA2-specific mucosal IgA and serum IgG levels. Moreover, induction of HA2-specific T-cell responses was dependent on CD40L, as rAd secreting HA2 subunit without CD40L failed to induce any significant levels of T-cell cytokines. Finally, sera obtained from immunized mice were capable of inhibiting 13 subtypes of influenza A viruses in vitro. These results provide proof of concept for a prototype HA2-based universal influenza vaccine. PMID:25052763

  19. Plasma Prostaglandin E2 Levels Correlated with the Prevention of Intravenous Immunoglobulin Resistance and Coronary Artery Lesions Formation via CD40L in Kawasaki Disease

    PubMed Central

    Kuo, Ho-Chang; Wang, Chih-Lu; Yang, Kuender D.; Lo, Mao-Hung; Hsieh, Kai-Sheng; Li, Sung-Chou

    2016-01-01

    Background A form of systemic vasculitis, Kawasaki disease (KD) occurs most frequently in children under the age of five years old. Previous studies have found that Prostaglandin E2 (PGE2) correlates with KD, although the related mechanisms are still unknown. CD40L may also be a marker of vasculitis in KD, so this study focuses on PGE2 and CD40L expression in KD. Materials and Methods This study consisted of a total of 144 KD patients, whose intravenous immunoglobulin (IVIG)/coronary arterial lesion (CAL) formation resistance was evaluated. PGE2 levels were evaluated in vitro to study the effect of CD40L on CD4+ T lymphocytes. Results PGE2 levels significantly increased after IVIG treatment (p<0.05), especially in patients who responded to initial IVIG treatment (p = 0.004) and for patients without CAL formation (p = 0.016). Furthermore, an in vitro study revealed that IVIG acted as a trigger for PGE2 expression in the acute-stage mononuclear cells of KD patients. According to our findings, both IVIG and PGE2 can impede surface CD40L expressions on CD4+ T lymphocytes (p<0.05). Conclusions The results of this study are among the first to find that plasma PGE2 is correlated with the prevention of IVIG resistance and CAL formation through CD40L in KD. PMID:27525421

  20. Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and CD40L signaling.

    PubMed

    Epron, G; Ame-Thomas, P; Le Priol, J; Pangault, C; Dulong, J; Lamy, T; Fest, T; Tarte, K

    2012-01-01

    Interleukin-15 (IL-15) has been extensively studied for its role in the survival and proliferation of NK and T cells through a unique mechanism of trans-presentation by producer cells. Conversely, whereas activated B cells have been described as IL-15-responding cells, the cellular and molecular context sustaining this effect remains unexplored. In this study, we found that, whereas human B cells could not respond to soluble IL-15, monocytes and lymphoid tissue-derived macrophages but not stromal cells efficiently trans-present IL-15 to normal B cells and cooperate with T-cell-derived CD40L to promote IL-15-dependent B-cell proliferation. Furthermore, CD40L signaling triggers a Src-independent upregulation of STAT5 expression and favors a Src-dependent phosphorylation of STAT5 in response to IL-15. In follicular lymphoma (FL), immunohistochemical studies reported a strong relationship between malignant B cells, infiltrating macrophages and T cells. We show here an overexpression of IL-15 in purified tumor-associated macrophages, and STAT5A in purified tumor B cells. Moreover, FL B cells respond to IL-15 trans-presented by monocytes/macrophages, in particular, in the presence of CD40L-mediated signaling. This cooperation between IL-15 and CD40L reinforces the importance of tumor microenvironment and unravels a mechanism of FL growth that should be considered if using IL-15 as a drug in this disease.

  1. Dendritic cells induce Tc1 cell differentiation via the CD40/CD40L pathway in mice after exposure to cigarette smoke.

    PubMed

    Kuang, Liang-Jian; Deng, Ting-Ting; Wang, Qin; Qiu, Shi-Lin; Liang, Yi; He, Zhi-Yi; Zhang, Jian-Quan; Bai, Jing; Li, Mei-Hua; Deng, Jing-Min; Liu, Guang-Nan; Liu, Ji-Feng; Zhong, Xiao-Ning

    2016-09-01

    Dendritic cells and CD8(+) T cells participate in the pathology of chronic obstructive pulmonary disease, including emphysema, but little is known of the involvement of the CD40/CD40L pathway. We investigated the role of the CD40/CD40L pathway in Tc1 cell differentiation induced by dendritic cells in a mouse model of emphysema, and in vitro. C57BL/6J wild-type and CD40(-/-) mice were exposed to cigarette smoke (CS) or not (control), for 24 wk. In vitro experiments involved wild-type and CD40(-/-) dendritic cells treated with CS extract (CSE) or not. Compared with the control groups, the CS mice (both wild type and CD40(-/-)) had a greater percentage of lung dendritic cells and higher levels of major histocompatability complex (MHC) class I molecules and costimulatory molecules CD40 and CD80. Relative to the CS CD40(-/-) mice, the CS wild type showed greater signs of lung damage and Tc1 cell differentiation. In vitro, the CSE-treated wild-type cells evidenced more cytokine release (IL-12/p70) and Tc1 cell differentiation than did the CSE-treated CD40(-/-) cells. Exposure to cigarette smoke increases the percentage of lung dendritic cells and promotes Tc1 cell differentiation via the CD40/CD40L pathway. Blocking the CD40/CD40L pathway may suppress development of emphysema in mice exposed to cigarette smoke. PMID:27448664

  2. Valproic Acid Inhibits the Release of Soluble CD40L Induced by Non-Nucleoside Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus Infected Individuals

    PubMed Central

    Davidson, Donna C.; Schifitto, Giovanni; Maggirwar, Sanjay B.

    2013-01-01

    Despite the use of highly active antiretroviral therapies (HAART), a majority of Human Immunodeficiency Virus Type 1 (HIV) infected individuals continually develop HIV – Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistent with this notion, we have previously shown that levels of the inflammatory mediator soluble CD40 ligand (sCD40L) are elevated in the plasma and cerebrospinal fluid (CSF) of HIV infected, cognitively impaired individuals, and that excess sCD40L can contribute to blood brain barrier (BBB) permeability in vivo, thereby signifying the importance of this inflammatory mediator in the pathogenesis of HAND. Here we demonstrate that the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) induces the release of circulating sCD40L in both HIV infected individuals and in an in vitro suspension of washed human platelets, which are the main source of circulating sCD40L. Additionally, EFV was found to activate glycogen synthase kinase 3 beta (GSK3β) in platelets, and we now show that valproic acid (VPA), a known GSK3β inhibitor, was able to attenuate the release of sCD40L in HIV infected individuals receiving EFV, and in isolated human platelets. Collectively these results have important implications in determining the pro-inflammatory role that some antiretroviral regimens may have. The use of antiretrovirals remains the best strategy to prevent HIV-associated illnesses, including HAND, however these drugs have clear limitations to this end, and thus, these results underscore the need to develop adjunctive therapies for HAND that can also minimize the undesired negative effects of the antiretrovirals. PMID:23555843

  3. A novel CD40LG deletion causes the hyper-IgM syndrome with normal CD40L expression in a 6-month-old child.

    PubMed

    López-Herrera, Gabriela; Maravillas-Montero, José Luis; Vargas-Hernández, Alexander; Berrón-Ruíz, Laura; Ramírez-Sánchez, Emmanuel; Yamazaki-Nakashimada, Marco Antonio; Espinosa-Rosales, Francisco Javier; Santos-Argumedo, Leopoldo

    2015-05-01

    The X-linked hyper-IgM syndrome (XHIGM) is the most common form of HIGM. Patients are clinically diagnosed on the basis of recurrent sinopulmonary infections, accompanied with low levels of IgG and IgA, normal to elevated levels of IgM, and the presence of peripheral B cells. Here, we have reported a novel deletion of four nucleotides in CD40LG exon 3, c.375_378delCAAA, which led to a frameshift mutation with a premature stop codon, p.Asn101*126. The deletion resulted in a truncated protein, in which majority of the extracellular domain was lost. However, detection of surface CD40L was still possible as the intracellular, transmembrane, and part of the extracellular domains were not affected. This indicated that this mutation did not affect protein stability and that immunodetection of CD40L expression is not enough for the diagnosis of XHIGM. Our study strongly suggests that genetic diagnosis for XHIGM should always be performed when clinical data support this diagnosis and CD40L protein is present.

  4. Agreement of skin test with IL-4 production and CD40L expression by T cells upon immunotherapy of subjects with systemic reactions to Hymenoptera stings.

    PubMed

    Urra, José M; Cabrera, Carmen M; Alfaya, Teresa; Feo-Brito, Francisco

    2016-02-01

    Venom immunotherapy is the only curative intervention for subjects with Hymenoptera venom allergy who suffering systemic reactions upon bee or wasp stings. Venom immunotherapy can restore normal immunity against venom allergens, as well as providing to allergic subjects a lifetime tolerance against venoms. Nevertheless, it is necessary using safety assays to monitoring the development of tolerance in the VIT protocols to avoid fatal anaphylactic reactions. The purpose of this study was to assess the modifications in several markers of tolerance induction in subjects with Hymenoptera venom allergy undergoing immunotherapy. The studies were performed at baseline time and after six month of VIT. Intradermal skin tests, basophil activation tests, specific IgE levels; and the T-cell markers (IL-4 and IFN-γ producing cells; and expression of the surface activation markers CD40L and CTLA-4) were assayed. At six month of immunotherapy all parameters studied had significant alterations. All decreased, except the IFN-γ producing cells. In addition, modifications in intradermal skin test showed a significant correlation with both, CD40L expression on CD4 T lymphocytes (p=0.043) and IL-4 producing T lymphocytes (p=0.012). Neither basophil activation test nor serum levels of sIgE demonstrated any correlation with the immunological parameters studied nor among them. These results suggest that both IL-4 production and CD40L expression could be two good indicators of the beneficial effects of venom immunotherapy which translate into skin tests.

  5. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    PubMed

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  6. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    PubMed

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies. PMID:27622331

  7. Anthocyanins and their physiologically relevant metabolites alter the expression of IL‐6 and VCAM‐1 in CD40L and oxidized LDL challenged vascular endothelial cells

    PubMed Central

    Amin, Hiren P.; Czank, Charles; Raheem, Saki; Zhang, Qingzhi; Botting, Nigel P.; Cassidy, Aedín

    2015-01-01

    Scope In vitro and in vivo studies suggest that dietary anthocyanins modulate cardiovascular disease risk; however, given anthocyanins extensive metabolism, it is likely that their degradation products and conjugated metabolites are responsible for this reported bioactivity. Methods and results Human vascular endothelial cells were stimulated with either oxidized LDL (oxLDL) or cluster of differentiation 40 ligand (CD40L) and cotreated with cyanidin‐3‐glucoside and 11 of its recently identified metabolites, at 0.1, 1, and 10 μM concentrations. Protein and gene expression of IL‐6 and VCAM‐1 was quantified by ELISA and RT‐qPCR. In oxLDL‐stimulated cells the parent anthocyanin had no effect on IL‐6 production, whereas numerous anthocyanin metabolites significantly reduced IL‐6 protein levels; phase II conjugates of protocatechuic acid produced the greatest effects (>75% reduction, p ≤ 0.05). In CD40L‐stimulated cells the anthocyanin and its phase II metabolites reduced IL‐6 protein production, where protocatechuic acid‐4‐sulfate induced the greatest reduction (>96% reduction, p ≤ 0.03). Similarly, the anthocyanin and its metabolites reduced VCAM‐1 protein production, with ferulic acid producing the greatest effect (>65% reduction, p ≤ 0.04). Conclusion These novel data provide evidence to suggest that anthocyanin metabolites are bioactive at physiologically relevant concentrations and have the potential to modulate cardiovascular disease progression by altering the expression of inflammatory mediators. PMID:25787755

  8. CD40L induces matrix-metalloproteinase-9 but not tissue inhibitor of metalloproteinases-1 in cervical carcinoma cells: imbalance between NF-kappaB and STAT3 activation.

    PubMed

    Smola-Hess, S; Schnitzler, R; Hadaschik, D; Smola, H; Mauch, C; Krieg, T; Pfister, H

    2001-07-15

    Matrix-metalloproteinases (MMPs) are essentially required for tumor cell invasion and metastasis. Production of precursor enzymes is regulated on transcriptional level, while activation of the pro-enzymes is tightly controlled by posttranscriptional mechanisms. The enzyme activity can be blocked by specific tissue inhibitors of MMPs (TIMPs). In cervical carcinomas strong up-regulation of the type IV collagenase MMP-9 had been demonstrated. We show that activation of CD40, a receptor highly expressed on cervical carcinomas, induces MMP-9 in cervical carcinoma cells, whereas TIMP-1 production inhibiting MMP-9 activity was not affected. This gene induction pattern corresponded to the differential activation of the transcription factor nuclear factor kappaB (NF-kappaB) regulating MMP-9, but not signal transducer and activator of transcription 3 (STAT3), which is involved in TIMP-1 gene regulation. Transient expression of the CD40-inducible NF-kappaB subunit p65 was sufficient for MMP-9 induction. Agents that suppressed CD40-mediated NF-kappaB activation also reduced MMP-9 induction, further supporting an important role of NF-kappaB in CD40-mediated MMP-9 induction. Our data suggest that CD40 expression in carcinoma cells might convert a CD40L-dependent immunological defense signal into a tumor-promoting signal. Selective CD40-mediated signaling through NF-kappaB but not STAT3 correlates to a shift of the balance between MMP-9 and TIMP-1 toward the protease.

  9. CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming

    PubMed Central

    Martín-Fontecha, Alfonso; Baumjohann, Dirk; Guarda, Greta; Reboldi, Andrea; Hons, Miroslav; Lanzavecchia, Antonio; Sallusto, Federica

    2008-01-01

    There is growing evidence that the maturation state of dendritic cells (DCs) is a critical parameter determining the balance between tolerance and immunity. We report that mouse CD4+ effector memory T (TEM) cells, but not naive or central memory T cells, constitutively expressed CD40L at levels sufficient to induce DC maturation in vitro and in vivo in the absence of antigenic stimulation. CD4+ TEM cells were excluded from resting lymph nodes but migrated in a CD62P-dependent fashion into reactive lymph nodes that were induced to express CD62P, in a transient or sustained fashion, on high endothelial venules. Trafficking of CD4+ TEM cells into chronic reactive lymph nodes maintained resident DCs in a mature state and promoted naive T cell responses and experimental autoimmune encephalomyelitis (EAE) to antigens administered in the absence of adjuvants. Antibodies to CD62P, which blocked CD4+ TEM cell migration into reactive lymph nodes, inhibited DC maturation, T cell priming, and induction of EAE. These results show that TEM cells can behave as endogenous adjuvants and suggest a mechanistic link between lymphocyte traffic in lymph nodes and induction of autoimmunity. PMID:18838544

  10. Gene expression and TB pathogenesis in rhesus macaques: TR4, CD40, CD40L, FAS (CD95), and TNF are host genetic markers in peripheral blood mononuclear cells that are associated with severity of TB lesions.

    PubMed

    Roodgar, Morteza; Ross, Cody T; Tarara, Ross; Lowenstine, Linda; Dandekar, Satya; Smith, David Glenn

    2015-12-01

    Tuberculosis (TB) pathologic lesions in rhesus macaques resemble those in humans. The expression levels of several host TB candidate genes in the peripheral blood mononuclear cells (PBMCs) of six rhesus macaques experimentally infected with Mycobacterium tuberculosis were quantified pre-infection and at several dates post-infection. Quantitative measures of TB histopathology in the lungs including: granuloma count, granuloma size, volume of granulomatous and non-granulomatous lesions, and direct bacterial load, were used as the outcomes of a multi-level Bayesian regression model in which expression levels of host genes at various dates were used as predictors. The results indicate that the expression levels of TR4, CD40, CD40L, FAS (CD95) and TNF in PBMC were associated with quantitative measures of the severity of TB histopathologic lesions in the lungs of the study animals. Moreover, no reliable association between the expression levels of IFNE in PBMCs and the severity of TB lesions in the lungs of the study animals was found. In conclusion, PBMC expression profiles derived from the above-listed host genes might be appropriate biomarkers for probabilistic diagnosis and/or prognosis of TB severity in rhesus macaques.

  11. Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows.

    PubMed

    Pujol, Julien; Bouillenne, Fabrice; Farnir, Frédéric; Dufrasne, Isabelle; Mainil, Jacques; Galleni, Moreno; Lekeux, Pierre; Bureau, Fabrice; Fiévez, Laurence

    2015-11-15

    Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4(+) T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4(+) T cell responses but also CD8(+) cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8(+) T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4(+) and CD8(+) T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens.

  12. Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.

    PubMed

    Kim, Jung-Sik; Byun, Nari; Chung, Hyunwoo; Kim, Hyun-Je; Kim, Jong-Min; Chun, Taehoon; Lee, Won-Woo; Park, Chung-Gyu

    2016-04-22

    Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

  13. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    PubMed

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  14. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

    PubMed Central

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  15. CD4+ T cell–independent DNA vaccination against opportunistic infections

    PubMed Central

    Zheng, Mingquan; Ramsay, Alistair J.; Robichaux, Myles B.; Norris, Karen A.; Kliment, Corrine; Crowe, Christopher; Rapaka, Rekha R.; Steele, Chad; McAllister, Florencia; Shellito, Judd E.; Marrero, Luis; Schwarzenberger, Paul; Zhong, Qiu; Kolls, Jay K.

    2005-01-01

    Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell–independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens. PMID:16308571

  16. Complexes between nuclear factor-κB p65 and signal transducer and activator of transcription 3 are key actors in inducing activation-induced cytidine deaminase expression and immunoglobulin A production in CD40L plus interleukin-10-treated human blood B cells.

    PubMed

    Lafarge, S; Hamzeh-Cognasse, H; Richard, Y; Pozzetto, B; Cogné, M; Cognasse, F; Garraud, O

    2011-11-01

    The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces.

  17. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    ERIC Educational Resources Information Center

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  18. Combining Microarray and Genomic Data to Predict DNA Binding Motifs

    SciTech Connect

    Mao, Linyong; Mackenzie, Ronald C.; Roh, J. H.; Eraso, Jesus M.; Kaplan, Samuel; Resat, Haluk

    2005-10-01

    The ability to detect regulatory elements within genome sequences is important in understanding how gene expression is controlled in biological systems. In this work, we combine microarray data analysis with genome sequence analysis to predict DNA sequences in the photosynthetic bacterium Rhodobacter sphaeroides that bind the regulators PrrA, PpsR and FnrL. These predictions were made by using hierarchical clustering to detect genes that share similar expression patterns. The DNA sequences upstream of these genes were then searched for possible transcription factor recognition motifs that may be involved in their co-regulation. The approach used promises to be widely applicable for the prediction of cis-acting DNA binding elements. Using this method we were independently able to detect and extend the previously described consensus sequences that have been suggested to bind FnrL and PpsR. In addition we have predicted sequences that may be recognized by the global regulator PrrA. Our results support the earlier suggestions that the DNA binding sequence of PrrA may have a variable sized gap between its conserved block elements. Using the predicted DNA binding sequences, we have performed a whole genome scale analysis to determine the relative importance of the interplay between these three regulators PpsR, FnrL and PrrA. Results of this analysis showed that, compared to the regulation by PpsR and FnrL, a much larger number of genes are candidates to be regulated by PrrA. Our study demonstrates by example that integration of multiple data types can be a powerful approach for inferring transcriptional regulatory patterns in microbial systems, and it allowed us to detect the photosynthesis related regulatory patterns in R. sphaeroides.

  19. Identifying DNA Binding Motifs by Combining Data from Different Sources

    SciTech Connect

    Mao, Linyong; Resat, Haluk; Nagib Callaos; Katsuhisa Horimoto; Jake Chen; Amy Sze Chan

    2004-07-19

    A transcription factor regulates the expression of its target genes by binding to their operator regions. It functions by affecting the interactions between RNA polymerases and the gene's promoter. Many transcription factors bind to their targets by recognizing a specific DNA sequence pattern, which is referred to as a consensus sequence or a motif. Since it would remove the possible biases, combining biological data from different sources can be expected to improve the quality of the information extracted from the biological data. We analyzed the microarray gene expression data and the organism's genome sequence jointly to determine the transcription factor recognition sequences with more accuracy. Utilizing such a data integration approach, we have investigated the regulation of the photosynthesis genes of the purple non-sulphur photosynthetic bacterium Rhodobacter sphaeroides. The photosynthesis genes in this organism are tightly regulated as a function of environmental growth conditions by three major regulatory systems, PrrB/PrrA, AppA/PpsR and FnrL. In this study, we have detected a previously undefined PrrA consensus sequence, improved the previously known DNA-binding motif of PpsR, and confirmed the consensus sequence of the global regulator FnrL.

  20. Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains.

    PubMed

    Lee, Ja Yil; Wang, Feng; Fazio, Teresa; Wind, Shalom; Greene, Eric C

    2012-10-01

    We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ∼20pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.

  1. Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience.

    PubMed

    Ciniselli, Chiara Maura; Pizzamiglio, Sara; Malentacchi, Francesca; Gelmini, Stefania; Pazzagli, Mario; Hartmann, Christina C; Ibrahim-Gawel, Hady; Verderio, Paolo

    2015-06-15

    In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.

  2. Effect of Danhong Injection Combined with Naoxintong Tablets on Prognosis and Inflammatory Factor Expression in Acute Coronary Syndrome Patients Undergoing Percutaneous Coronary Intervention

    PubMed Central

    Lv*, Yun; Pan, Yaping; Gao*, Yan; Lu, Jingqian; Li, Yi; Bai, Jie; Zhai, Jing

    2015-01-01

    Background Danhong is a Chinese medical component that has been broadly used to treat various cerebrovascular diseases. This work aimed to investigate the effect of Danhong injection combined with Naoxintong tablets on the short-term prognosis and expression of inflammatory factor-soluble CD40 ligand (sCD40L) in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). Methods A total of 100 ACS patients with PCI were randomly divided equally into treatment and control groups. The control group was treated with conventional secondary prevention of coronary heart disease. Based on secondary prevention, Danhong injection combined with Naoxintong tablets was administered in the treatment group. The incidences of major adverse cardiovascular events and cardiac functions, including ejection fraction (EF) and six-minute walk test distance, during hospital discharge and at the third postoperative month were observed. The serum sCD40 levels at different times were also noted. Results There were 2 patients in the treatment group and 7 in the control group that were lost during follow-up, so the collected data were from only 48 patients in the treatment and 43 in the control group. During hospital discharge and at the third postoperative month, no significant difference in death, myocardial infarction, stroke, angina pectoris and readmission were observed between the two groups (p > 0.05). Upon hospital discharge, EF, six-minute walk test distance and serum sCD40L level in the two groups were not significantly different (p > 0.05). At the third postoperative month, EF and six-minute walk test distance in treatment group were significantly higher than those in the control group (p < 0.05), and the serum sCD40L level in the treatment group was significantly lower than that in the control group (p < 0.01). In addition, serum sCD40L levels in the two groups at the third postoperative month were significantly lower than those during hospital

  3. Enhancement of anti-proliferative activities of Metformin, when combined with Celecoxib, without increasing DNA damage.

    PubMed

    Ullah, Asad; Ashraf, Muhammad; Javeed, Aqeel; Anjum, Aftab Ahmad; Attiq, Ali; Ali, Sarwat

    2016-07-01

    Pathophysiological changes in diabetes like hyperglycemia, oxidative stress, insulin resistance and compensatory hyperinsulinemia predispose cells to malignant transformation and damage DNA repair mechanism. This study was designed to explore the potential synergistic toxic effects of anti-diabetic drug (Metformin), and an analgesic drug (Celecoxib) at cellular level. MTT assay run on Vero cell line revealed that the combinations of Metformin and Celecoxib augment the anti-proliferative effects, whereas Single cell gel electrophoresis spotlighted that Metformin produce non-significant DNA damage with the threshold concentration of 400μg/ml in peripheral blood mononuclear cells (lymphocytes and monocytes), while Celecoxib produced significant (P<0.05) DNA damage (class III comets) above the concentration of 75μg/ml, however the DNA damage or DNA tail protrusions by combinations of both drugs were less than what was observed with Celecoxib alone. Metformin or Celecoxib did not appear mutagenic against any mutant strains (TA 100 and TA 98) but their combination exhibited slight mutagenicity at much higher concentration. The results obtained at concentrations higher than the therapeutic level of drugs and reflect that Metformin in combination with Celecoxib synergistically inhibits the cell proliferation in a concentration dependent pattern. Since, this increase in cytotoxicity did not confer an increase in DNA damage; this combination could be adopted to inhibit the growth of malignant cell without producing any genotoxic or mutagenic effects at cellular level. PMID:27327526

  4. Graphene quantums dots combined with endonuclease cleavage and bidentate chelation for highly sensitive electrochemiluminescent DNA biosensing.

    PubMed

    Lou, Jing; Liu, Shanshan; Tu, Wenwen; Dai, Zhihui

    2015-01-20

    A novel strategy for highly sensitive electrochemiluminescence (ECL) detection of DNA was proposed based on site-specific cleavage of BamHI endonuclease combined with the excellent ECL activity of graphene quantum dots (GQDs) and bidentate chelation of the dithiocarbamate DNA (DTC-DNA) probe assembly. The difference between photoluminescence and ECL spectral peaks suggested that a negligible defect existed on the GQDs surface for generation of an ECL signal. The formed DTC-DNA was directly attached to the gold surface by bidentate anchoring (S-Au-S bonds), which conferred a strong affinity between the ligands and the gold surface, increasing the robustness of DNA immobilization on the gold surface. BamHI endonuclease site-specifically recognized and cleaved the duplex symmetrical sequence, which made the double-stranded DNA fragments and GQDs break off from the electrode surface, inducing a decrease of the ECL signal. Using hepatitis C virus-1b genotype complementary DNA (HCV-1b cDNA) as a model, a novel signal-off ECL DNA biosensor was developed based on variation of the ECL intensity before and after digestion of the DNA hybrid. Electrochemical impedance spectroscopy confirmed the successful fabrication of the ECL DNA biosensor. This ECL biosensor for HCV-1b cDNA determination exhibited a linear range from 5 fM to 100 pM with a detection limit of 0.45 fM at a signal-to-noise ratio of 3 and showed satisfactory selectivity and good stability, which validated the feasibility of the designed strategy. The proposed strategy may be conveniently combined with other specific biological recognition events for expansion of the biosensing application, especially in clinical diagnoses. PMID:25523862

  5. Triggered polycatenated DNA scaffolds for DNA sensors and aptasensors by a combination of rolling circle amplification and DNAzyme amplification.

    PubMed

    Bi, Sai; Li, Li; Zhang, Shusheng

    2010-11-15

    The concept of triggered polycatenated DNA scaffolds has been elegantly introduced into ultrasensitive biosensing applications by a combination of rolling circle amplification (RCA) and DNAzyme amplification. As compared to traditional methods in which one target could only initiate the formation of one circular template for RCA reaction, in the present study two species of linear single-stranded DNA (ssDNA) monomers are self-assembled into mechanically interlocked polycatenated nanostructures on capture probe-tagged magnetic nanoparticles (MNPs) only upon the introduction of one base mutant DNA sequence as initiator for single-nucleotide polymorphisms (SNPs) analysis. The resultant topologically polycatenated DNA ladder is further available for RCA process by using the serially ligated circular DNA as template for the synthesis of hemin/G-quadruplex HRP-mimicking DNAzyme chains, which act as biocatalytic labels for the luminol-H(2)O(2) chemiluminescence (CL) system. Notably, the problem of high background induced by excess hemin itself is circumvented by immobilizing the biotinylated RCA products on streptavidin-modified MNPs via biotin-streptavidin interaction. Similarly, a universal strategy is contrived by substitutedly employing aptamer as initiator for the construction of polycatenated DNA scaffolds to accomplish ultrasensitive detection of proteins based on structure-switching of aptamer upon target binding, which is demonstrated by using thrombin as a model analyte in this study. Overall, with two successive amplification steps and one magnetic separation procedure, this flexible biosensing system exhibits not only high sensitivity and specificity with the detection limits of SNPs and thrombin as low as 71 aM and 6.6 pM, respectively, but also excellent performance in real human serum assay with no PCR preamplification for SNPs assay. Given the unique and attractive characteristics, this study illustrates the potential of DNA nanotechnology in bioanalytical

  6. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    PubMed

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  7. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    SciTech Connect

    Barends, Thomas R. M.; Brosi, Richard W. W.; Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten; Seidel, Ralf; Shoeman, Robert L.; Zimmermann, Sabine; Bittl, Robert; Schlichting, Ilme; Reinstein, Jochen

    2013-08-01

    The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

  8. Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

    PubMed

    Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

    2015-08-01

    DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity.

  9. Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage.

    PubMed

    Yamada, Hisatsugu; Tanabe, Kazuhito; Nishimoto, Sei-ichi

    2008-01-01

    An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.

  10. STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)

    NASA Astrophysics Data System (ADS)

    Heller, Iddo; Sitters, Gerrit; Broekmans, Onno D.; Farge, Géraldine; Menges, Carolin; Wende, Wolfgang; Hell, Stefan W.; Peterman, Erwin J.; Wuite, Gijs J.

    2014-09-01

    Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.

  11. Combined activation of methyl paraben by light irradiation and esterase metabolism toward oxidative DNA damage.

    PubMed

    Okamoto, Yoshinori; Hayashi, Tomohiro; Matsunami, Shinpei; Ueda, Koji; Kojima, Nakao

    2008-08-01

    Methyl paraben (MP) is often used as a preservative in foods, drugs, and cosmetics because of its high reliability in safety based on the rapid excretion and nonaccumulation following administration. Light irradiation sometimes produces unexpected activity from chemicals such as MP; furthermore, there is ample opportunity for MP to be exposed to sunlight. Here, we investigated whether MP shows DNA damage after sunlight irradiation. Two major photoproducts, p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH), were detected after sunlight irradiation to an aqueous MP solution. Both photoproducts were inactive in the in vitro DNA damage assay that measures oxidized guanine formed in calf thymus DNA in the presence of divalent copper ion, a known mediator of oxidative DNA damage. Simulated MP metabolism using dermal tissues after light irradiation produced these two photoproducts, which reacted with a microsomal fraction (S9) of the skin. A metabolite from MP-3OH, not PHBA, caused distinct DNA damage in the in vitro assay. This active metabolite was identified as protocatechuic acid, a hydrolyzed MP-3OH product. In addition, NADH, a cellular reductant, enhanced DNA damage by approximately five times. These results suggest that reactive oxygen species generated by the redox cycle via metal ion and catechol autoxidation are participating in oxidative DNA damage. This study reveals that MP might cause skin damage involving carcinogenesis through the combined activation of sunlight irradiation and skin esterases.

  12. [Combined hopping-superexchange mechanism of charge transfer in DNA; a model with nearest interactions].

    PubMed

    Lakhno, V D; Sultanov, V B

    2007-01-01

    In the framework of the earlier developed combined hopping-superexchange mechanism of charge transfer in DNA, a model with all nearest interactions between nucleobases is proposed. It is shown that the transfer rates for various types of nucleotide sequences calculated within this model are in a good agreement with experimental data.

  13. Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

    PubMed

    Yin, Xiaotao; Wang, Wei; Zhu, Xiaoming; Wang, Yu; Wu, Shuai; Wang, Zicheng; Wang, Lin; Du, Zhiyan; Gao, Jiangping; Yu, Jiyun

    2015-09-18

    To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and β-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic. PMID:26253468

  14. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    PubMed Central

    Grossmann, Claudius; Tenbusch, Matthias; Nchinda, Godwin; Temchura, Vladimir; Nabi, Ghulam; Stone, Geoffrey W; Kornbluth, Richard S; Überla, Klaus

    2009-01-01

    Background Targeting of protein antigens to dendritic cells (DC) via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR) and CD40 ligands (CD40L) as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C. PMID:19650904

  15. The Updated Phylogenies of the Phasianidae Based on Combined Data of Nuclear and Mitochondrial DNA

    PubMed Central

    Shen, Yong-Yi; Dai, Kun; Cao, Xue; Murphy, Robert W.; Shen, Xue-Juan; Zhang, Ya-Ping

    2014-01-01

    The phylogenetic relationships of species in the Phasianidae, Order Galliformes, are the object of intensive study. However, convergent morphological evolution and rapid species radiation result in much ambiguity in the group. Further, matrilineal (mtDNA) genealogies conflict with trees based on nuclear DNA retrotransposable elements. Herein, we analyze 39 nearly complete mitochondrial genomes (three new) and up to seven nuclear DNA segments. We combine these multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids. The nuclear DNA tree is largely congruent with the tree derived from mt genomes. However, branching orders of mt/nuclear trees largely conflict with those based on retrotransposons. For example, Gallus/Bambusicola/Francolinus forms the sister-group of Coturnix/Alectoris in the nuclear/mtDNA trees, yet the tree based on retrotransposable elements roots the former at the base of the tree and not with the latter. Further, while peafowls cluster with Gallus/Coturnix in the mt tree, they root at the base of the phasianids following Gallus in the tree based on retrotransposable elements. The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in our study reveal the complex topology of the Phasianidae. PMID:24748132

  16. A DNA-based system for selecting and displaying the combined result of two input variables

    PubMed Central

    Liu, Huajie; Wang, Jianbang; Song, Shiping; Fan, Chunhai; Gothelf, Kurt V.

    2015-01-01

    Oligonucleotide-based technologies for biosensing or bio-regulation produce huge amounts of rich high-dimensional information. There is a consequent need for flexible means to combine diverse pieces of such information to form useful derivative outputs, and to display those immediately. Here we demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather than through logic operations. The multiplicative example demonstrated here illustrates a much more general capability—to generate a unique output for any distinct pair of DNA inputs. The system thereby functions as a lookup table and could be a key component in future, more powerful data-processing systems for diagnostics and sensing. PMID:26646059

  17. Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

    PubMed Central

    Liu, Guo-Xia; Fang, Gui-Qing; Xu, Wei

    2014-01-01

    Combinations of chemotherapeutic drugs with nucleic acid has shown great promise in cancer therapy. In the present study, paclitaxel (PTX) and DNA were co-loaded in the hyaluronic acid (HA) and folate (FA)-modified liposomes (HA/FA/PPD), to obtain the dual targeting biomimetic nanovector. The prepared HA/FA/PPD exhibited nanosized structure and narrow size distributions (247.4 ± 4.2 nm) with appropriate negative charge of −25.40 ± 2.7 mV. HA/FA/PD (PTX free HA/FA/PPD) showed almost no toxicity on murine malignant melanoma cell line (B16) and human hepatocellular carcinoma cell line (HepG2) (higher than 80% cell viability), demonstrating the safety of the blank nanovector. In comparison with the FA-modified PTX/DNA co-loaded liposomes (FA/PPD), HA/FA/PPD showed significant superiority in protecting the nanoparticles from aggregation in the presence of plasma and degradation by DNase I. Moreover, HA/FA/PPD could also significantly improve the transfection efficiency and cellular internalization rates on B16 cells comparing to that of FA/PPD (p < 0.05) and PPD (p < 0.01), demonstrating the great advantages of dual targeting properties. Furthermore, fluorescence microscope and flow cytometry results showed that PTX and DNA could be effectively co-delivered into the same tumor cell via HA/FA/PPD, contributing to PTX/DNA combination cancer treatment. In conclusion, the obtained HA/FA/PPD in the study could effectively target tumor cells, enhance transfection efficiency and subsequently achieve the co-delivery of PTX and DNA, displaying great potential for optimal combination therapy. PMID:25177862

  18. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban; Medintz, Igor L.

    2016-04-01

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  19. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  20. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  1. A platform for combined DNA and protein microarrays based on total internal reflection fluorescence.

    PubMed

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax.

  2. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  3. Molecular recognition of DNA-protein complexes: a straightforward method combining scanning force and fluorescence microscopy.

    PubMed

    Sanchez, Humberto; Kanaar, Roland; Wyman, Claire

    2010-06-01

    Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein-DNA complexes.

  4. Augmentation of French grunt diet description using combined visual and DNA-based analyses

    USGS Publications Warehouse

    Hargrove, John S.; Parkyn, Daryl C.; Murie, Debra J.; Demopoulos, Amanda W.J.; Austin, James D.

    2012-01-01

    Trophic linkages within a coral-reef ecosystem may be difficult to discern in fish species that reside on, but do not forage on, coral reefs. Furthermore, dietary analysis of fish can be difficult in situations where prey is thoroughly macerated, resulting in many visually unrecognisable food items. The present study examined whether the inclusion of a DNA-based method could improve the identification of prey consumed by French grunt, Haemulon flavolineatum, a reef fish that possesses pharyngeal teeth and forages on soft-bodied prey items. Visual analysis indicated that crustaceans were most abundant numerically (38.9%), followed by sipunculans (31.0%) and polychaete worms (5.2%), with a substantial number of unidentified prey (12.7%). For the subset of prey with both visual and molecular data, there was a marked reduction in the number of unidentified sipunculans (visual – 31.1%, combined &ndash 4.4%), unidentified crustaceans (visual &ndash 15.6%, combined &ndash 6.7%), and unidentified taxa (visual &ndash 11.1%, combined &ndash 0.0%). Utilising results from both methodologies resulted in an increased number of prey placed at the family level (visual &ndash 6, combined &ndash 33) and species level (visual &ndash 0, combined &ndash 4). Although more costly than visual analysis alone, our study demonstrated the feasibility of DNA-based identification of visually unidentifiable prey in the stomach contents of fish.

  5. DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents

    SciTech Connect

    Setlow, R. B.; Ahmed, F. E.

    1980-01-01

    Normal human and XP2 fibroblasts were treated with UV plus UV-mimetic chemicals. The UV dose used was sufficient to saturate the UV excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a UV specific endonuclease. Since the repair of damage from UV and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of UV and chemical damage and that after a combined treatment the total amount of repair would be the same as from UV or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170.

  6. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer.

    PubMed

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-08-28

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  7. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer

    PubMed Central

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-01-01

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer.

  8. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer

    PubMed Central

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-01-01

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  9. Protection of pigs against Taenia solium cysticercosis using recombinant antigen or in combination with DNA vaccine.

    PubMed

    Guo, Ying-Jun; Sun, Shu-Han; Zhang, Yi; Chen, Zhu-Huan; Wang, Kai-Yu; Huang, Li; Zhang, Shu; Zhang, Hong-Ying; Wang, Qing-Min; Wu, Dan; Zhu, Wei-Jia

    2004-09-28

    In the present study, we investigated the duration of protection afforded to pigs immunized in two different prime-boost regimens: one is homologus priming and boosting with a protein vaccine, and the other is priming with a DNA vaccine and boosting with the protein vaccine. Groups of pigs that received the same vaccination regimen were then challenged with Taenia solium eggs at 6, 12 or 20 weeks post-immunization (wpi), respectively. The results showed that all vaccinated pigs challenged at 6 or 12 wpi showed significant (P < 0.05) reduction in the development of cysts. When challenged at 20 wpi, pigs primed with the DNA vaccine (pcDNA3-cC1) followed by two boosters of the protein vaccine (GST-cC1) showed significant (P < 0.05) protection against the challenge of T. solium eggs, whereas pigs receiving three injections of the protein vaccine showed no significant protection compared to non-vaccinated controls (P > 0.05). Antibody isotype assays showed that DNA prime-protein boost regimen induced a predominantly IgG2 response, compared to an IgG1 biased response for the protein prime-protein boost regimen. In addition, peripheral blood mononuclear cells (PBMC) obtained from the DNA prime-protein boost group proliferated strongly in response to GST-cC1 protein, and this responsiveness persisted until 20 wpi. Taken together, our data suggest that the use of a prime-boost strategy combining DNA and protein vaccines may be better than protein alone for the longevity of protection against the challenge of T. solium eggs.

  10. Novel DNA Topoisomerase IIα Inhibitors from Combined Ligand- and Structure-Based Virtual Screening

    PubMed Central

    Drwal, Malgorzata N.; Marinello, Jessica; Manzo, Stefano G.; Wakelin, Laurence P. G.; Capranico, Giovanni; Griffith, Renate

    2014-01-01

    DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, “poisons”, binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors. PMID:25489853

  11. Combining allele frequency uncertainty and population substructure corrections in forensic DNA calculations.

    PubMed

    Cowell, Robert

    2016-07-01

    In forensic DNA calculations of relatedness of individuals and in DNA mixture analyses, at least two sources of uncertainty are present concerning the allele frequencies used for evaluating genotype probabilities when evaluating likelihoods. They are: (i) imprecision in the estimates of the allele frequencies in the population by using an inevitably finite database of DNA profiles to estimate them; and (ii) the existence of population substructure. Green and Mortera [6] showed that these effects may be taken into account individually using a common Dirichlet model within a Bayesian network formulation, but that when taken in combination this is not the case; however they suggested an approximation that could be used. Here we develop a slightly different approximation that is shown to be exact in the case of a single individual. We demonstrate the numerical closeness of the approximation using a published database of allele counts, and illustrate the effect of incorporating the approximation into calculations of a recently published statistical model of DNA mixtures. PMID:27231804

  12. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    PubMed

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471. PMID:27275414

  13. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    PubMed

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.

  14. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  15. Combining natural and man-made DNA tracers to advance understanding of hydrologic flow pathway evolution

    NASA Astrophysics Data System (ADS)

    Dahlke, H. E.; Walter, M. T.; Lyon, S. W.; Rosqvist, G. N.

    2014-12-01

    Identifying and characterizing the sources, pathways and residence times of water and associated constituents is critical to developing improved understanding of watershed-stream connections and hydrological/ecological/biogeochemical models. To date the most robust information is obtained from integrated studies that combine natural tracers (e.g. isotopes, geochemical tracers) with controlled chemical tracer (e.g., bromide, dyes) or colloidal tracer (e.g., carboxilated microspheres, tagged clay particles, microorganisms) applications. In the presented study we explore how understanding of sources and flow pathways of water derived from natural tracer studies can be improved and expanded in space and time by simultaneously introducing man-made, synthetic DNA-based microtracers. The microtracer used were composed of polylactic acid (PLA) microspheres into which short strands of synthetic DNA and paramagnetic iron oxide nanoparticles are incorporated. Tracer experiments using both natural tracers and the DNA-based microtracers were conducted in the sub-arctic, glacierized Tarfala (21.7 km2) catchment in northern Sweden. Isotopic hydrograph separations revealed that even though storm runoff was dominated by pre-event water the event water (i.e. rainfall) contributions to streamflow increased throughout the summer season as glacial snow cover decreased. This suggests that glaciers are a major source of the rainwater fraction in streamflow. Simultaneous injections of ten unique DNA-based microtracers confirmed this hypothesis and revealed that the transit time of water traveling from the glacier surface to the stream decreased fourfold over the summer season leading to instantaneous rainwater contributions during storm events. These results highlight that integrating simultaneous tracer injections (injecting tracers at multiple places at one time) with traditional tracer methods (sampling multiple times at one place) rather than using either approach in isolation can

  16. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

    PubMed Central

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. PMID:27369379

  17. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection.

    PubMed

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H; Brown, Tom

    2016-09-30

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. PMID:27369379

  18. DNA bases assembled on the Au(110)/electrolyte interface: a combined experimental and theoretical study.

    PubMed

    Salvatore, Princia; Nazmutdinov, Renat R; Ulstrup, Jens; Zhang, Jingdong

    2015-02-19

    Among the low-index single-crystal gold surfaces, the Au(110) surface is the most active toward molecular adsorption and the one with fewest electrochemical adsorption data reported. Cyclic voltammetry (CV), electrochemically controlled scanning tunneling microscopy (EC-STM), and density functional theory (DFT) calculations have been employed in the present study to address the adsorption of the four nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T), on the Au(110)-electrode surface. Au(110) undergoes reconstruction to the (1 × 3) surface in electrochemical environment, accompanied by a pair of strong voltammetry peaks in the double-layer region in acid solutions. Adsorption of the DNA bases gives featureless voltammograms with lower double-layer capacitance, suggesting that all the bases are chemisorbed on the Au(110) surface. Further investigation of the surface structures of the adlayers of the four DNA bases by EC-STM disclosed lifting of the Au(110) reconstruction, specific molecular packing in dense monolayers, and pH dependence of the A and G adsorption. DFT computations based on a cluster model for the Au(110) surface were performed to investigate the adsorption energy and geometry of the DNA bases in different adsorbate orientations. The optimized geometry is further used to compute models for STM images which are compared with the recorded STM images. This has provided insight into the physical nature of the adsorption. The specific orientations of A, C, G, and T on Au(110) and the nature of the physical adsorbate/surface interaction based on the combination of the experimental and theoretical studies are proposed, and differences from nucleobase adsorption on Au(111)- and Au(100)-electrode surfaces are discussed.

  19. DNA bases assembled on the Au(110)/electrolyte interface: a combined experimental and theoretical study.

    PubMed

    Salvatore, Princia; Nazmutdinov, Renat R; Ulstrup, Jens; Zhang, Jingdong

    2015-02-19

    Among the low-index single-crystal gold surfaces, the Au(110) surface is the most active toward molecular adsorption and the one with fewest electrochemical adsorption data reported. Cyclic voltammetry (CV), electrochemically controlled scanning tunneling microscopy (EC-STM), and density functional theory (DFT) calculations have been employed in the present study to address the adsorption of the four nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T), on the Au(110)-electrode surface. Au(110) undergoes reconstruction to the (1 × 3) surface in electrochemical environment, accompanied by a pair of strong voltammetry peaks in the double-layer region in acid solutions. Adsorption of the DNA bases gives featureless voltammograms with lower double-layer capacitance, suggesting that all the bases are chemisorbed on the Au(110) surface. Further investigation of the surface structures of the adlayers of the four DNA bases by EC-STM disclosed lifting of the Au(110) reconstruction, specific molecular packing in dense monolayers, and pH dependence of the A and G adsorption. DFT computations based on a cluster model for the Au(110) surface were performed to investigate the adsorption energy and geometry of the DNA bases in different adsorbate orientations. The optimized geometry is further used to compute models for STM images which are compared with the recorded STM images. This has provided insight into the physical nature of the adsorption. The specific orientations of A, C, G, and T on Au(110) and the nature of the physical adsorbate/surface interaction based on the combination of the experimental and theoretical studies are proposed, and differences from nucleobase adsorption on Au(111)- and Au(100)-electrode surfaces are discussed. PMID:25611676

  20. Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.

    PubMed

    Mazier, S; Villette, S; Goffinont, S; Renouard, S; Maurizot, J C; Genest, D; Spotheim-Maurizot, M

    2008-11-01

    The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site. PMID:18959464

  1. Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein-DNA complexes.

    PubMed

    Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

    2009-01-01

    Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA.

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

    PubMed

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

    2011-06-01

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

  3. Combined analysis of DNA methylation and cell cycle in cancer cells

    PubMed Central

    Desjobert, Cécile; El Maï, Mounir; Gérard-Hirne, Tom; Guianvarc'h, Dominique; Carrier, Arnaud; Pottier, Cyrielle; Arimondo, Paola B; Riond, Joëlle

    2015-01-01

    DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase. PMID:25531272

  4. Chromosomal and DNA ploidy characterization of salivary gland neoplasms by combined FISH and flow cytometry.

    PubMed

    El-Naggar, A K; Dinh, M; Tucker, S L; Gillenwater, A; Luna, M A; Batsakis, J G

    1997-08-01

    Concurrent DNA ploidy by flow cytometry and interphase FISH analysis of chromosomes 6 through 12, 17, 18, X, and Y were prospectively performed on 22 salivary gland neoplasms (four benign and 18 malignant) to investigate the diagnostic and biological implications of their alterations in these neoplasms. Our results show that benign neoplasms lack DNA aneuploidy and numerical chromosomal abnormalities. Low-grade malignant neoplasms, except for two lesions, manifested small chromosomal gains and losses and were generally DNA diploid or near-diploid aneuploid, whereas all high-grade tumors showed marked polysomy and were DNA aneuploid. Marked intratumoral and intertumoral chromosomal heterogeneity also were noted in and between individual tumors. Although polysomy was the main finding in DNA aneuploid lesions, monosomy was more noted in DNA diploid neoplasms and was restricted to chromosomes 8, 11, and 17. Significant correlation between the DNA index, chromosomal aneusomy, histological grade, and tumor stage was noted. Our study indicates that (1) benign salivary gland neoplasms lack gross DNA content and numerical chromosomal abnormalities, (2) clonal chromosomal alterations are manifested in most DNA diploid and all DNA aneuploid malignant tumors, (3) chromosomal gain is the most common alteration; chromosomal loss is less frequent and restricted to certain chromosomes, and (4) DNA aneuploidy and chromosomal aneusomy characterize tumors with aggressive features.

  5. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

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

  6. A new method of representing DNA sequences which combines ease of visual analysis with machine readability.

    PubMed Central

    Cowin, J E; Jellis, C H; Rickwood, D

    1986-01-01

    A new method of representing DNA sequences has been devised which is termed stave projection. Compared with other formats for showing the base sequences of DNA, this method greatly enhances the ease of visual analysis of the sequences of bases and it is also in a machine readable form. Using this method it is possible to identify and annotate all of the functional features found in DNA sequences. PMID:3003680

  7. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    PubMed

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells. PMID:23881817

  8. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    PubMed

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.

  9. An economical and combined method for rapid and efficient isolation of fungal DNA.

    PubMed

    Lech, T; Syguła-Cholewinska, J; Szostak-Kot, J

    2014-12-18

    DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

  10. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    PubMed

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  11. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation

    PubMed Central

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi’s individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  12. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    PubMed

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  13. Personal identification of cold case remains through combined contribution from anthropological, mtDNA, and bomb-pulse dating analyses.

    PubMed

    Speller, Camilla F; Spalding, Kirsty L; Buchholz, Bruce A; Hildebrand, Dean; Moore, Jason; Mathewes, Rolf; Skinner, Mark F; Yang, Dongya Y

    2012-09-01

    In 1968, a child's cranium was recovered from the banks of a northern Canadian river and held in a trust until the "cold case" was reopened in 2005. The cranium underwent reanalysis at the Centre for Forensic Research, Simon Fraser University, using recently developed anthropological analysis, "bomb-pulse" radiocarbon analysis, and forensic DNA techniques. Craniometrics, skeletal ossification, and dental formation indicated an age-at-death of 4.4 ± 1 year. Radiocarbon analysis of enamel from two teeth indicated a year of birth between 1958 and 1962. Forensic DNA analysis indicated the child was a male, and the obtained mitochondrial profile matched a living maternal relative to the presumed missing child. These multidisciplinary analyses resulted in a legal identification 41 years after the discovery of the remains, highlighting the enormous potential of combining radiocarbon analysis with anthropological and mtDNA analyses in producing confident personal identifications for forensic cold cases dating to within the last 60 years.

  14. Radiolaria Divided into Polycystina and Spasmaria in Combined 18S and 28S rDNA Phylogeny

    PubMed Central

    Dolven, Jane K.; Ose, Randi F.; Klaveness, Dag; Kristensen, Tom; Bjørklund, Kjell R.; Shalchian-Tabrizi, Kamran

    2011-01-01

    Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis. PMID:21853146

  15. Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny.

    PubMed

    Krabberød, Anders K; Bråte, Jon; Dolven, Jane K; Ose, Randi F; Klaveness, Dag; Kristensen, Tom; Bjørklund, Kjell R; Shalchian-Tabrizi, Kamran

    2011-01-01

    Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.

  16. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  17. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    PubMed Central

    Barends, Thomas R. M.; Brosi, Richard W. W.; Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten; Seidel, Ralf; Shoeman, Robert L.; Zimmermann, Sabine; Bittl, Robert; Schlichting, Ilme; Reinstein, Jochen

    2013-01-01

    Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ. PMID:23897477

  18. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    PubMed

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  19. Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

    PubMed

    Dang, Phat M; Chen, Charles Y

    2013-02-01

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

  20. Combining RNA-DNA swapping and quantitative polymerase chain reaction for the detection of influenza A nucleoprotein.

    PubMed

    Morin, Isabelle; Schaeffer, Patrick M

    2012-01-15

    The detection of proteinaceous antigens generally relies on traditional immunoassays and, more recently, on immuno-PCR (polymerase chain reaction) assays and their derivatives, which do not take advantage of the intrinsic function or binding property of a protein. The RNA-binding nucleoprotein has been shown to be an excellent target for the development of various influenza A diagnostics due to its high antigenicity and the presence of large numbers in the virus. It binds nonspecifically to the sugar-phosphate backbone of RNA as well as to single-stranded DNA (ssDNA) in vitro. We decided to take advantage of this property to develop an ssDNA probe for the detection of nucleoprotein by quantitative PCR (qPCR). We found that recombinant influenza A nucleoprotein from avian H5N1 subtype binds strongest to a 74-base-long ssDNA. Two systems, one comprising an antibody-based nucleoprotein capture surface and the other based on direct nucleoprotein adsorption under denaturing conditions, were developed combining the replacement of RNA bound to nucleoprotein by a discrete ssDNA probe and a qPCR for the detection of nucleoprotein in the low picomolar (pM) range.

  1. Combined DNA and lipid analyses of sediments reveal changes in Holocene haptophyte and diatom populations in an Antarctic lake

    NASA Astrophysics Data System (ADS)

    Coolen, Marco J. L.; Muyzer, Gerard; Rijpstra, W. Irene C.; Schouten, Stefan; Volkman, John K.; Sinninghe Damsté, Jaap S.

    2004-06-01

    Preserved ribosomal DNA of planktonic phototrophic algae was recovered from Holocene anoxic sediments of Ace Lake (Antarctica), and the ancient community members were identified based on comparative sequence analysis. The similar concentration profiles of DNA of haptophytes and their traditional lipid biomarkers (alkenones and alkenoates) revealed that fossil rDNA also served as quantitative biomarkers in this environment. The DNA data clearly revealed the presence of six novel phylotypes related to known alkenone and alkenoate-biosynthesizing haptophytes with Isochrysis galbana UIO 102 as their closest relative. The relative abundance of these phylotypes changed as the lake chemistry, particularly salinity, evolved over time. Changes in the alkenone distributions reflect these population changes rather than a physiological response to salinity by a single haptophyte. Using this novel paleo-ecological approach of combining data from lipid biomarkers and preserved DNA, we showed that the post-glacial development of Ace Lake from freshwater basin to marine inlet and the present-day lacustrine saline system caused major qualitative and quantitative changes in the biodiversity of the planktonic populations over time.

  2. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  3. Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

    NASA Astrophysics Data System (ADS)

    Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

    2007-12-01

    Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray

  4. Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

    PubMed Central

    Barakat, Tahsin Stefan; Gribnau, Joost

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

  5. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  6. Different combinations of atomic interactions predict protein-small molecule and protein-DNA/RNA affinities with similar accuracy.

    PubMed

    Dias, Raquel; Kolazckowski, Bryan

    2015-11-01

    Interactions between proteins and other molecules play essential roles in all biological processes. Although it is widely held that a protein's ligand specificity is determined primarily by its three-dimensional structure, the general principles by which structure determines ligand binding remain poorly understood. Here we use statistical analyses of a large number of protein-ligand complexes with associated binding-affinity measurements to quantitatively characterize how combinations of atomic interactions contribute to ligand affinity. We find that there are significant differences in how atomic interactions determine ligand affinity for proteins that bind small chemical ligands, those that bind DNA/RNA and those that interact with other proteins. Although protein-small molecule and protein-DNA/RNA binding affinities can be accurately predicted from structural data, models predicting one type of interaction perform poorly on the others. Additionally, the particular combinations of atomic interactions required to predict binding affinity differed between small-molecule and DNA/RNA data sets, consistent with the conclusion that the structural bases determining ligand affinity differ among interaction types. In contrast to what we observed for small-molecule and DNA/RNA interactions, no statistical models were capable of predicting protein-protein affinity with >60% correlation. We demonstrate the potential usefulness of protein-DNA/RNA binding prediction as a possible tool for high-throughput virtual screening to guide laboratory investigations, suggesting that quantitative characterization of diverse molecular interactions may have practical applications as well as fundamentally advancing our understanding of how molecular structure translates into function.

  7. Different combinations of atomic interactions predict protein‐small molecule and protein‐DNA/RNA affinities with similar accuracy

    PubMed Central

    Dias, Raquel

    2015-01-01

    ABSTRACT Interactions between proteins and other molecules play essential roles in all biological processes. Although it is widely held that a protein's ligand specificity is determined primarily by its three‐dimensional structure, the general principles by which structure determines ligand binding remain poorly understood. Here we use statistical analyses of a large number of protein−ligand complexes with associated binding‐affinity measurements to quantitatively characterize how combinations of atomic interactions contribute to ligand affinity. We find that there are significant differences in how atomic interactions determine ligand affinity for proteins that bind small chemical ligands, those that bind DNA/RNA and those that interact with other proteins. Although protein‐small molecule and protein‐DNA/RNA binding affinities can be accurately predicted from structural data, models predicting one type of interaction perform poorly on the others. Additionally, the particular combinations of atomic interactions required to predict binding affinity differed between small‐molecule and DNA/RNA data sets, consistent with the conclusion that the structural bases determining ligand affinity differ among interaction types. In contrast to what we observed for small‐molecule and DNA/RNA interactions, no statistical models were capable of predicting protein−protein affinity with >60% correlation. We demonstrate the potential usefulness of protein‐DNA/RNA binding prediction as a possible tool for high‐throughput virtual screening to guide laboratory investigations, suggesting that quantitative characterization of diverse molecular interactions may have practical applications as well as fundamentally advancing our understanding of how molecular structure translates into function. Proteins 2015; 83:2100–2114. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:26370248

  8. The combination of gold nanorods and nanoparticles with DNA nanodevices for logic gates construction.

    PubMed

    Yao, Dongbao; Song, Tingjie; Zheng, Bin; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-10-23

    In this work, two DNA nanodevices were constructed utilizing a DNA strand displacement reaction. With the assistance of gold nanoparticles (AuNPs) and gold nanorods (AuNRs), the autonomous reactions can be reflected from the aggregation states of nanoparticles. By sequence design and the two non-overlapping double hump-like UV-vis spectral peaks of AuNPs and AuNRs, two logic gates with multiple inputs and outputs were successfully run with expected outcomes. This method not only shows how to achieve computing with multiple logic calculations but also has great potential for multiple targets detection.

  9. Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

    PubMed Central

    Chen, Pin-jia; Huang, Guo-bin; Li, Bin; Zheng, De-qing; Yu, Xiu-rong; Luo, Xiao-yong

    2014-01-01

    Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer. PMID

  10. Modified method for combined DNA and RNA isolation from peanut and other oil seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  11. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA

    NASA Astrophysics Data System (ADS)

    Kurihara, Kensuke; Tamura, Mieko; Shohda, Koh-Ichiroh; Toyota, Taro; Suzuki, Kentaro; Sugawara, Tadashi

    2011-10-01

    The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell.

  12. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.

    PubMed

    Kurihara, Kensuke; Tamura, Mieko; Shohda, Koh-Ichiroh; Toyota, Taro; Suzuki, Kentaro; Sugawara, Tadashi

    2011-10-01

    The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell. PMID:21941249

  13. Proposed Strategy for Selection Against Recessive Genetic Defects Through a Combination of Inbreeding and DNA Markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recessive genetic defects are currently on the minds of many cattle breeders. The relatively rapid development of diagnostic DNA tests for recessive defects appears to be a major recent technological advancement. However, the attitude of breeders and breed associations toward recessive defects seems...

  14. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis

    PubMed Central

    Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

    2016-01-01

    Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis. PMID:27411928

  15. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis.

    PubMed

    Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

    2016-01-01

    Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis.

  16. Combined immunofluorescence-DNA-fluorescence staining technique for enumeration of Thiobacillus ferrooxidans in a population of acidophilic bacteria

    SciTech Connect

    Muyzer, G.; De Bruyn, A.; Schmedding, D.J.M.; Bos, P.; Westbroek, P.; Kuenen, G.J.

    1987-04-01

    An antiserum raised against whole cells of Thiobacillus ferroxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferroooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.

  17. Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata.

    PubMed

    Moreira, David; von der Heyden, Sophie; Bass, David; López-García, Purificación; Chao, Ema; Cavalier-Smith, Thomas

    2007-07-01

    Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of

  18. Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations.

    PubMed

    Gilbert, S C; Schneider, J; Plebanski, M; Hannan, C M; Blanchard, T J; Smith, G L; Hill, A V

    1999-03-01

    Three types of vaccine, all expressing the same antigen from Plasmodium berghei, or a CD8+ T cell epitope from that antigen, were compared for their ability to induce CD8+ T cell responses in mice. Higher levels of lysis and numbers of IFN-gamma secreting T cells were primed with Ty virus-like particles and Modified Vaccinia Virus Ankara (MVA) than with DNA vaccines, but none of the vaccines were able to protect immunised mice from infectious challenge even after repeated doses. However, when the immune response was primed with one type of vaccine (Ty-VLPs or DNA) and boosted with another (MVA) complete protection against infection was achieved. Protection correlated with very high levels of IFN-gamma secreting T cells and lysis. This method of vaccination uses delivery systems and routes that can be used in humans and could provide a generally applicable regime for the induction of high levels of CD8+ T cells.

  19. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    PubMed

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  20. Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation

    PubMed Central

    Adolph, Elizabeth J.; Nelson, Christopher E.; Werfel, Thomas A.; Guo, Ruijing; Davidson, Jeffrey M.; Duvall, Craig L.

    2014-01-01

    Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability

  1. Mitochondrial DNA deletion in a patient with combined features of Leigh and Pearson syndromes

    SciTech Connect

    Blok, R.B.; Thorburn, D.R.; Danks, D.M.

    1994-09-01

    We describe a heteroplasmic 4237 bp mitochondrial DNA (mtDNA) deletion in an 11 year old girl who has suffered from progressive illness since birth. She has some features of Leigh syndrome (global developmental delay with regression, brainstem dysfunction and lactic acidosis), together with other features suggestive of Pearson syndrome (history of pancytopenia and failure to thrive). The deletion was present at a level greater than 50% in skeletal muscle, but barely detectable in skin fibroblasts following Southern blot analysis, and only observed in blood following PCR analysis. The deletion spanned nt 9498 to nt 13734, and was flanked by a 12 bp direct repeat. Genes for cytochrome c oxidase subunit III, NADH dehydrogenase subunits 3, 4L, 4 and 5, and tRNAs for glycine, arginine, histidine, serine({sup AGY}) and leucine({sup CUN}) were deleted. Southern blotting also revealed an altered Apa I restriction site which was shown by sequence analysis to be caused by G{r_arrow}A nucleotide substitution at nt 1462 in the 12S rRNA gene. This was presumed to be a polymorphism. No abnormalities of mitochondrial ultrastructure, distribution or of respiratory chain enzyme complexes I-IV in skeletal muscle were observed. Mitochondrial disorders with clinical features overlapping more than one syndrome have been reported previously. This case further demonstrates the difficulty in correlating observed clinical features with a specific mitochondrial DNA mutation.

  2. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells

    PubMed Central

    Vispé, Stéphane; Deroide, Arthur; Davoine, Emeline; Desjobert, Cécile; Lestienne, Fabrice; Fournier, Lucie; Novosad, Natacha; Bréand, Sophie; Besse, Jérôme; Busato, Florence; Tost, Jörg; De Vries, Luc; Cussac, Didier; Riond, Joëlle; Arimondo, Paola B.

    2015-01-01

    5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia. PMID:25948775

  3. Construction of two fluorescence-labeled non-combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population.

    PubMed

    Bai, Xue; Li, Shujin; Cong, Bin; Li, Xia; Guo, Xia; He, Lujun; Ye, Jian; Pei, Li

    2010-09-01

    MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non-combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence-labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy-Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.

  4. Combined inhibition of DNA methyltransferase and histone deacetylase restores caspase-8 expression and sensitizes SCLC cells to TRAIL.

    PubMed

    Kaminskyy, Vitaliy O; Surova, Olga V; Vaculova, Alena; Zhivotovsky, Boris

    2011-10-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising drug for the treatment of tumors; however, a number of cancer cells are resistant to this cytokine. Among the mechanisms of resistance of small cell lung carcinomas (SCLCs) to TRAIL is the lack of caspase-8 expression. Although methylation of the caspase-8 promoter has been suggested as the main mechanism of caspase-8 silencing, we showed that reduction of the enzymes involved in DNA methylation, DNA methyltransferases (DNMT) 1, 3a and 3b, was not sufficient to significantly restore caspase-8 expression in SCLC cells, signifying that other mechanisms are involved in caspase-8 silencing. We found that combination of the DNMT inhibitor decitabine with an inhibitor of histone deacetylase (HDAC) significantly increased caspase-8 expression in SCLC cells at the RNA and protein levels. Among all studied HDAC inhibitors, valproic acid (VPA) and CI-994 showed prolonged effects on histone acetylation, while combination with decitabine produced the most prominent effects on caspase-8 re-expression. Moreover, a significant reduction of survivin and cIAP-1 proteins level was observed after treatment with VPA. The combination of two drugs sensitized SCLC cells to TRAIL-induced apoptosis, involving mitochondrial apoptotic pathway and was accompanied by Bid cleavage, activation of Bax, and release of cytochrome c. Both initiator caspase-8 and -9 were required for the sensitization of SCLC cells to TRAIL. Thus, efficient restoration of caspase-8 expression in SCLC cells is achieved when a combination of DNMT and HDAC inhibitors is used, suggesting a combination of decitabine and VPA or CI-994 as a potential treatment for sensitization of SCLC cells lacking caspase-8 to TRAIL. PMID:21771726

  5. An Xpb Mouse Model for Combined Xeroderma Pigmentosum and Cockayne Syndrome Reveals Progeroid Features upon Further Attenuation of DNA Repair▿

    PubMed Central

    Andressoo, Jaan-Olle; Weeda, Geert; de Wit, Jan; Mitchell, James R.; Beems, Rudolf B.; van Steeg, Harry; van der Horst, Gijsbertus T. J.; Hoeijmakers, Jan H.

    2009-01-01

    Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPBXPCS are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background. Interestingly, the XpbXPCS Xpa double mutants display a remarkable interanimal variance, which points to stochastic DNA damage accumulation as an important determinant of clinical diversity in NER syndromes. Furthermore, mice carrying the XpbXPCS mutation together with a point mutation in the second TFIIH helicase Xpd are healthy at birth but display neonatal lethality, indicating that transcription efficiency is sufficient to permit embryonal development even when both TFIIH helicases are crippled. The double-mutant cells exhibit sensitivity to oxidative stress, suggesting a role for endogenous DNA damage in the onset of XPB-associated CS. PMID:19114557

  6. Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement.

    PubMed

    Bai, Yu-Hui; Li, Jin-Yi; Xu, Jing-Juan; Chen, Hong-Yuan

    2010-07-01

    A novel method is described for the highly effective amplifying electrochemical response of DNA based on oligonucleotides functionalized with Au/Fe(3)O(4) nanocomposites by the aid of silver (Ag) enhancement. Via electrostatic layer-by-layer (LBL) assembly, the prepared Fe(3)O(4) nanoparticles form nano-clusters coated with a bilayer composed of polystyrene sulfonate sodium salt (PSS) and poly(diallyldimethylammonium chloride) (PDDA), which are in favor of adsorbing lots of gold nanoparticles (AuNPs) on the surface. The application of magnetic Fe(3)O(4) made the procedures much more simple, convenient and feasible. The resulting composites were then used as labels via the Au-S bond for the DNA hybridization, followed by catalytic deposition of silver on the gold tags. Such an assay is then combined with a sensitive anodic stripping voltammetry (ASV) measurement of multiple silver nanoparticle tracers. A 27-mer sequence DNA target is detected at a glassy carbon (GC) electrode with a detection limit down to ca. 100 aM, which is 800 times lower than that obtained using gold nanoparticles only as labels in the control experiments. This Fe(3)O(4)/PSS/PDDA/Au composite offers a great promising future for the ultrasensitive detection of other biorecognition events.

  7. Combining peptide and DNA for protein assay: CRIP1 detection for breast cancer staging.

    PubMed

    Xie, Haona; Li, Hao; Huang, Yue; Wang, Xiaoying; Yin, Yongmei; Li, Genxi

    2014-01-01

    In this work, a novel method for a protein assay is proposed which uses the specific protein-binding peptide of the target protein and sequence-specific DNA to interact with the target as the capture and detection probe, respectively. Meanwhile, since the DNA sequence can be coupled with gold nanoparticles to amplify the signal readout, a sensitive and easily operated method for protein assay is developed. We have also employed a transcription factor named as cysteine-rich intestinal protein 1 (CRIP1), which has been identified as an ideal biomarker for staging of breast cancer, as the model protein for this study. With the proposed method, CRIP1 can be determined in a linear range from 1.25 to 10.13 ng/mL, with a detection limit of 1.25 ng/mL. Furthermore, the proposed method can be directly used to assay CRIP1 in tissue samples. Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future.

  8. Highly sensitive DNA detection and point mutation identification: an electrochemical approach based on the combined use of ligase and reverse molecular beacon.

    PubMed

    Wu, Zai-Sheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2007-06-01

    A novel strategy is described for highly sensitive DNA detection and point mutation identification based on the combination of reverse molecular beacon with DNA ligase. A 5'-phosphoryl and 3'-ferrocene terminated DNA sequence is used as detection probe, which may be ligated to capture DNA immobilized on an electrode surface in the presence of a target DNA strand that is complementary to the ends of each DNA, since this allows formation of a nicked, double-stranded DNA. The ligation product may form a hairpin structure after the removal of target DNA. By this method, target DNA can be determined in the range from 3.4 x 10(-12) to 1.4 x 10(-7) M with a detection limit of 1.0 x 10(-12) M. In contrast to existing methods based on the conformation change of redox-labeled oligonucleotides, the proposed strategy offers several substantial advantages: first, the background peak current is eliminated as the ferrocene (Fc)-tagged oligonucleotide probe is specifically ligated to capture DNA; second, a "signal-on" mechanism makes the current intensity increase with increasing target DNA concentration; third, improved current signal is obtained due to the formation of the hairpin structure of ligation products. Additionally, the present system exhibits excellent capability to discriminate mutant target sequences from fully complementary target sequences.

  9. A computer programme for estimation of genetic risk in X linked disorders, combining pedigree and DNA probe data with other conditional information.

    PubMed Central

    Sarfarazi, M; Williams, H

    1986-01-01

    A computer programme is presented for calculating the recurrence risk in X linked disorders, combining pedigree and DNA probe data with other conditional information such as carrier detection tests. The methods of computation are shown in the given examples. The programme can be used with either a single DNA probe or two 'flanking' DNA probes for both familial and isolated case pedigrees. For isolated case families the mutation rate at the disease locus can be taken into account in conjunction with the DNA probe data. PMID:3754009

  10. Autofocus using adaptive prediction approximation combined search for the fluorescence microscope in second-generation DNA sequencing system.

    PubMed

    Xu, Hancong; Liu, Jinfeng; Li, Yang; Yin, Yan; Zhu, Chenxu; Lu, Hua

    2014-07-10

    Autofocus is an important technique for high-speed image acquisition in the second-generation DNA sequencing system, and this paper studies the passive focus algorithm for the system, which consists of two parts: focus measurement (FM) and focus search (FS). Based on the properties of DNA chips' images, we choose the normalized variance as the FM algorithm and develop a new robust FS named adaptive prediction approximation combined search (APACS). APACS utilizes golden section search (GSS) to approximate the focus position and engages the curve-fitting search (CFS) to predict the position simultaneously in every step of GSS. When the difference between consecutive predictions meets the set precision, the search finishes. Otherwise, it ends as GSS. In APACS, we also propose an estimation method, named the combination of centroid estimation and overdetermined equations estimation by least squares solution, to calculate the initial vector for the nonlinear equations in APACS prediction, which reduces the iterations and accelerates the search. The simulation and measured results demonstrate that APACS not only maintains the stability but also reduces the focus time compared with GSS and CFS, which indicates APACS is a robust and fast FS for the fluorescence microscope in a sequencing system.

  11. Venom landscapes: mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach.

    PubMed

    Escoubas, Pierre; Sollod, Brianna; King, Glenn F

    2006-05-01

    The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI-TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000-5000 Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a diversity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional 'venom landscapes' derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms.

  12. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

    PubMed

    Coghlan, Megan L; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C; White, Nicole E; Byard, Roger W; Bellgard, Matthew I; Mullaney, Ian; Trengove, Robert; Allcock, Richard J N; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F; Bunce, Michael

    2015-12-10

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

  13. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

    NASA Astrophysics Data System (ADS)

    Coghlan, Megan L.; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C.; White, Nicole E.; Byard, Roger W.; Bellgard, Matthew I.; Mullaney, Ian; Trengove, Robert; Allcock, Richard J. N.; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F.; Bunce, Michael

    2015-12-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

  14. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

    PubMed Central

    Coghlan, Megan L.; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C.; White, Nicole E.; Byard, Roger W.; Bellgard, Matthew I.; Mullaney, Ian; Trengove, Robert; Allcock, Richard J. N.; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F.; Bunce, Michael

    2015-01-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines. PMID:26658160

  15. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

    PubMed

    Coghlan, Megan L; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C; White, Nicole E; Byard, Roger W; Bellgard, Matthew I; Mullaney, Ian; Trengove, Robert; Allcock, Richard J N; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F; Bunce, Michael

    2015-01-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines. PMID:26658160

  16. Delivery and processing of exogenous double-stranded DNA in mouse CD34+ hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA.

    PubMed

    Dolgova, Evgenia V; Efremov, Yaroslav R; Orishchenko, Konstantin E; Andrushkevich, Oleg M; Alyamkina, Ekaterina A; Proskurina, Anastasia S; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Chernykh, Elena R; Ostanin, Alexandr A; Taranov, Oleg S; Omigov, Vladimir V; Minkevich, Alexandra M; Rogachev, Vladimir A; Bogachev, Sergey S; Shurdov, Mikhail A

    2013-10-10

    We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~45% of the cells correspond to CD34+ hematopoietic stem cells. Taking into account that CD34+ stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34+ cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34+ bone marrow cells. When linearized plasmid DNA was used as a source of exogenous DNA, we observed that exonucleolytic processing and ligation of double-stranded DNA termini occurred in the bone marrow cells that had this DNA internalized. We also recovered "hybrid" plasmids that encompass kanamycin-resistance gene from the exogenous plasmid DNA and the fragments of plasmids from host enterobacteria, which is suggestive of recombination events taking place upon DNA internalization. CD34+ cells make up the distinctive bone marrow cell population that internalizes extracellular DNA. Cell cycle analysis of CD34+ cells treated with cyclophosphamide only or in combination with dsDNA, suggests that these cells have distinct biologic responses to these treatments. Namely, whereas upon cyclophosphamide treatment bone marrow stem cells become arrested at S-G2 phases, combined cyclophosphamide+dsDNA treatment leads to cell cycle progression without any delay. This indicates that when the genome is undergoing repair of interstrand crosslinks, injection of fragmented exogenous dsDNA results in immediate reconstitution of genome integrity. We observe that cyclophosphamide-only or a combined cyclophosphamide+dsDNA treatment of cells lead to two distinct waves of apoptosis in CD34

  17. Improved Heterosis Prediction by Combining Information on DNA- and Metabolic Markers

    PubMed Central

    Gärtner, Tanja; Steinfath, Matthias; Andorf, Sandra; Lisec, Jan; Meyer, Rhonda C.; Altmann, Thomas; Willmitzer, Lothar; Selbig, Joachim

    2009-01-01

    Background Hybrids represent a cornerstone in the success story of breeding programs. The fundamental principle underlying this success is the phenomenon of hybrid vigour, or heterosis. It describes an advantage of the offspring as compared to the two parental lines with respect to parameters such as growth and resistance against abiotic or biotic stress. Dominance, overdominance or epistasis based models are commonly used explanations. Conclusion/Significance The heterosis level is clearly a function of the combination of the parents used for offspring production. This results in a major challenge for plant breeders, as usually several thousand combinations of parents have to be tested for identifying the best combinations. Thus, any approach to reliably predict heterosis levels based on properties of the parental lines would be highly beneficial for plant breeding. Methodology/Principal Findings Recently, genetic data have been used to predict heterosis. Here we show that a combination of parental genetic and metabolic markers, identified via feature selection and minimum-description-length based regression methods, significantly improves the prediction of biomass heterosis in resulting offspring. These findings will help furthering our understanding of the molecular basis of heterosis, revealing, for instance, the presence of nonlinear genotype-phenotype relationships. In addition, we describe a possible approach for accelerated selection in plant breeding. PMID:19370148

  18. Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations.

    PubMed

    Phillips, Matthew J; McLenachan, Patricia A; Down, Christin; Gibb, Gillian C; Penny, David

    2006-02-01

    Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the "reduced-bias" mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of non-historical signal, such as from compositional non-stationarity. [Base composition; combined data; marsupial; mitochondrial genome; phylogeny.].

  19. Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations.

    PubMed

    Phillips, Matthew J; McLenachan, Patricia A; Down, Christin; Gibb, Gillian C; Penny, David

    2006-02-01

    Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the "reduced-bias" mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of non-historical signal, such as from compositional non-stationarity. [Base composition; combined data; marsupial; mitochondrial genome; phylogeny.]. PMID:16507529

  20. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

  1. Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures

    PubMed Central

    Eipel, Monika; Mayer, Felix; Arent, Tanja; Ferreira, Marcelo R. P.; Birkhofer, Carina; Gerstenmaier, Uwe; Costa, Ivan G.; Ritz-Timme, Stefanie; Wagner, Wolfgang

    2016-01-01

    Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person's cheek using buccal swabs – but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genes PDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this “Buccal-Cell-Signature” correlated with cell counts in cytological stains (R2 = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions. PMID:27249102

  2. [Combined use of irradiation and DNA tumor vaccine to treat canine oral malignant melanoma: a pilot study].

    PubMed

    Herzog, A; Buchholz, J; Ruess-Melzer, K; Lang, J; Kaser-Hotz, B

    2013-02-01

    Melanoma is the most common oral tumor in dogs, characterized by rapid growth, local invasion, and high metastatic rate. The goal of this study was to evaluate the combination of radiation therapy and DNA tumor vaccine. We hypothesized, that the concurrent use would not increase toxicity. Nine dogs with oral melanoma were treated with 4 fractions of 8 Gray at 7-day intervals. The vaccine was given 4 times every 14 days, beginning at the first radiation fraction. Local acute radiation toxicities were assessed according to the VRTOG toxicity scoring scheme over a time period of 7 weeks. In none of the evaluated dogs, mucositis, dermatitis and conjunctivitis exceeded grade 2. In 3 dogs mild fever, lethargy, and local swelling at the injection site were seen after vaccine application. In conclusion, the concurrent administration of radiation therapy and vaccine was well tolerated in all dogs. PMID:23385072

  3. A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms.

    PubMed

    Valledor, Luis; Escandón, Mónica; Meijón, Mónica; Nukarinen, Ella; Cañal, María Jesús; Weckwerth, Wolfram

    2014-07-01

    Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.

  4. Combination of PDT and a DNA demethylating agent produces anti-tumor immune response in a mouse tumor model

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Hamblin, Michael R.

    2009-06-01

    Epigenetic mechanisms, which involve DNA methylation and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. However, these changes must be actively maintained after each cell division rendering them a promising target for pharmacologic inhibition. DNA methyltransferase inhibitors like 5-aza-deoxycytidine (5-aza-dC) induce and/or up-regulate the expression of MAGE-type antigens in human and mice cancer cells. Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the additional advantage of stimulating tumor-directed immune response. We studied the effects of a new therapy that combined the demethylating agent 5-aza-dC with PDT in the breast cancer model 4T1 syngenic to immunocompetent BALB/c mice. PDT was used as a locally ablating tumor treatment that is capable of eliciting strong and tumor directed immune response while 5-aza-dC pretreatment was used promote de novo induction of the expression of P1A.protein. This is the mouse homolog of human MAGE family antigens and is reported to function as a tumor rejection antigen in certain mouse tumors. This strategy led to an increase in PDT-mediated immune response and better treatment outcome. These results strongly suggest that the MAGE family antigens are important target for PDT mediated immune response but that their expression can be silenced by epigenetic mechanisms. Therefore the possibility that PDT can be combined with epigenetic strategies to elicit anti-tumor immunity in MAGE-positive tumor models is highly clinically significant and should be studied in detail.

  5. Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene

    SciTech Connect

    Pater, A.; Pater, M.M.

    1986-05-01

    Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that the authors examined. However, cells isolated from foci were incapable of growth in soft agar. They then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.

  6. Anti-dsDNA antibodies in systemic lupus erythematosus: A combination of two quantitative methods and the ANA pattern is the most efficient strategy of detection.

    PubMed

    Almeida González, Delia; Roces Varela, Alfredo; Marcelino Rodríguez, Itahisa; González Vera, Alexander; Delgado Sánchez, Mónica; Aznar Esquivel, Antonio; Casañas Rodríguez, Carlos; Cabrera de León, Antonio

    2015-12-01

    Several methods have been used to measure anti-double-stranded DNA auto-antibody (anti-dsDNA). Our aim was to determine the most efficient strategy to test anti-dsDNA in systemic lupus erythematosus (SLE). In this study, anti-dsDNA and anti-nuclear antibody (ANA) tests were requested for 644 patients. Anti-dsDNA was tested by RIA, ELISA and CLIA in all patients. The results indicated that 78 patients had a positive anti-dsDNA test according to at least one of the methods. After a 3-year follow-up period only 26 patients were diagnosed with SLE. We evaluated each method and combination of methods. Specificity and positive predictive value (PPV) increased with the number of assay methods used (p=0.002 for trend), and PPV was 100% in patients whose results were positive by all three anti-dsDNA assay methods. The proportion of anti-dsDNA-positive patients who had SLE was highest (82%; p b 0.001) among those with a homogeneous pattern of ANA staining, followed by those with a speckled pattern. In ANA positive patients, when only RIA was considered, 59% of anti-dsDNA-positive patients had SLE, but when RIA and CLIA were both considered, all patients with positive results on both tests had SLE. The combination of RIA+CLIA in patients with homogeneous and speckled ANA staining showed a similar cost and higher sensitivity than RIA alone in ANA positive patients (p b 0.001). We conclude that the most efficient strategy was to combine simultaneously two quantitative and sensitive methods but only in patients with a homogeneous or speckled pattern of ANA staining. This approach maximized specificity and PPV, and reduced costs.

  7. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    PubMed

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  8. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions

    PubMed Central

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-01-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 109 and 2.6 × 1010 cm−2. The p-doping change on WSe2 by Ln-DNA is adjusted between −1.0 × 1010 and −2.4 × 1010 cm−2. In Eu3+ or Gd3+-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~1010 cm−2). However, in the devices doped by Tb3+ or Er3+-Co-DNA, a light n-doping (~1010 cm−2) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb3+- and Gd3+-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb3+ or Er3+-Co-DNA (n-doping) and the Eu3+ or Gd3+-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb3+ ions on the TMD photodetectors. PMID:26838524

  9. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions

    NASA Astrophysics Data System (ADS)

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-02-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 109 and 2.6 × 1010 cm-2. The p-doping change on WSe2 by Ln-DNA is adjusted between -1.0 × 1010 and -2.4 × 1010 cm-2. In Eu3+ or Gd3+-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~1010 cm-2). However, in the devices doped by Tb3+ or Er3+-Co-DNA, a light n-doping (~1010 cm-2) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb3+- and Gd3+-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb3+ or Er3+-Co-DNA (n-doping) and the Eu3+ or Gd3+-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb3+ ions on the TMD photodetectors.

  10. Combining morphology, DNA sequences, and morphometrics: revising closely related species in the orb-weaving spider genus Araniella (Araneae, Araneidae).

    PubMed

    Spasojevic, Tamara; Kropf, Christian; Nentwig, Wolfgang; Lasut, Liana

    2016-01-01

    The integration of independent data sets could solve problems in both traditional and DNA-based taxonomy. The aim of this study is to investigate the power of CO1 sequences and of morphometrics to distinguish closely related species in the spider genus Araniella. We put special emphasis on the species pair A. cucurbitina (Clerck, 1757) and A. opisthographa (Kulczyński, 1905) since the females are morphologically difficult to distinguish and often misidentified. A total of 216 sequences of eight Araniella species from seven European countries, North America and Asia were included in the molecular analysis. The results from both maximum likelihood and Bayesian phylogenetic inference indicate successful separation of six out of eight Araniella species, including A. cucurbitina and A. opisthographa. For the same six species, we detect no overlap of intra- and interspecific genetic divergence, leading to successful species identification with a threshold approach. In addition, morphometric analysis of the epigyna of A. cucurbitina and A. opisthographa supports species separation by two best explanatory ratios: receptaculum length and distance between receptaculum and copulatory duct. Although a small overlap in the ratios exists, the species identification rate increases when combining morphometric and molecular data, which demonstrates the efficiency of integrative approaches for distinguishing closely related species. However, none of the molecular approaches was able to separate closely related A. alpica (L. Koch, 1869) and A. inconspicua (Simon, 1874) due to shared CO1 haplotypes. Considering the clear morphological separation of the males and different habitat preferences, incomplete lineage sorting or introgressive hybridization could have led to identical CO1 sequences. Therefore, DNA-barcoding must be thoroughly tested even within small homogenous genera of spiders. PMID:27395098

  11. Combined immunization using DNA-Sm14 and DNA-Hsp65 increases CD8+ memory T cells, reduces chronic pathology and decreases egg viability during Schistosoma mansoni infection

    PubMed Central

    2014-01-01

    Background Schistosomiasis is one of the most important neglected diseases found in developing countries and affects 249 million people worldwide. The development of an efficient vaccination strategy is essential for the control of this disease. Previous work showed partial protection induced by DNA-Sm14 against Schistosoma mansoni infection, whereas DNA-Hsp65 showed immunostimulatory properties against infectious diseases, autoimmune diseases, cancer and antifibrotic properties in an egg-induced granuloma model. Methods C57BL/6 mice received 4 doses of DNA-Sm14 (100 μg/dose) and DNA-Hsp65 (100 μg/dose), simultaneously administrated, or DNA-Sm14 alone, once a week, during four weeks. Three groups were included: 1- Control (no immunization); 2- DNA-Sm14; 3- DNA-Sm14/DNA-Hsp65. Two weeks following last immunization, animals were challenged subcutaneously with 30 cercariae. Fifteen, 48 and 69 days after infection splenocytes were collected to evaluate the number of CD8+ memory T cells (CD44highCD62low) using flow cytometry. Forty-eight days after challenge adult worms were collected by portal veins perfusion and intestines were collected to analyze the intestinal egg viability. Histological, immunohistochemical and soluble quantification of collagen and α-SMA accumulation were performed on the liver. Results In the current work, we tested a new vaccination strategy using DNA-Sm14 with DNA-Hsp65 to potentiate the protection against schistosomiasis. Combined vaccination increased the number of CD8+ memory T cells and decreased egg viability on the intestinal wall of infected mice. In addition, simultaneous vaccination with DNA-Sm14/DNA-Hsp65 reduced collagen and α-SMA accumulation during the chronic phase of granuloma formation. Conclusion Simultaneous vaccination with DNA-Sm14/DNA-Hsp65 showed an immunostimulatory potential and antifibrotic property that is associated with the reduction of tissue damage on Schistosoma mansoni experimental infection. PMID

  12. Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

    PubMed

    McClements, W L; Armstrong, M E; Keys, R D; Liu, M A

    1996-10-15

    DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

  13. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation.

    PubMed

    Lavertu, Marc; Méthot, Stephane; Tran-Khanh, Nicolas; Buschmann, Michael D

    2006-09-01

    Chitosan is a biodegradable natural polysaccharide that has shown potential for gene delivery, although the ideal molecular weight (MW) and degree of deacetylation (DDA) for this application have not been elucidated. To examine the influence of these parameters on gene transfer, we produced chitosans with different DDAs (98%, 92%, 80% and 72%) and depolymerized them with nitrous acid to obtain different MWs (150, 80, 40 and 10 kDa). We produced 64 formulations of chitosan/pDNA complexes (16 chitosans, 2 amine-to-phosphate (N:P) ratios of 5:1 and 10:1 and 2 transfection media pH of 6.5 and 7.1), characterized them for size and surface charge, and tested them for gene transfection in HEK 293 cells in vitro. Several formulations produced high levels of transgene expression while two conditions, 92-10-5 and 80-10-10 [DDA-MW-N:P ratio] at pH 6.5, showed equivalence to our best positive control. The results also revealed an important coupling between DDA and MW of chitosan in determining transgene expression. Maximum expression was obtained with a certain combination of DDA and MW that depended on N:P ratio and the pH, but similar expression levels could be achieved by simultaneously lowering MW and increasing DDA or lowering DDA and increasing MW, suggesting a predominant role of particle stability, through co-operative electrostatic binding, in determining transfection efficiency.

  14. Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.

    PubMed

    Hovhannisyan, G; Rapp, A; Arutyunyan, R; Greulich, K O; Gebhart, E

    2005-03-01

    The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future. PMID:15702234

  15. Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA.

    PubMed

    Mao, Pingdao; Ning, Yi; Li, Wenkai; Peng, Zhihui; Chen, Yongzhe; Deng, Le

    2014-01-10

    A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples. PMID:24267562

  16. Synergistic anti-breast cancer effect of a combined treatment with the methyl donor S-adenosyl methionine and the DNA methylation inhibitor 5-aza-2'-deoxycytidine.

    PubMed

    Chik, Flora; Machnes, Ziv; Szyf, Moshe

    2014-01-01

    DNA-demethylating agents activate tumor suppressor genes that are silenced by DNA methylation in cancer and are therefore emerging as a novel approach to cancer therapy. 5-azacytidine (VIDAZA), the first representative of this class of drugs was approved for treatment of myelodysplastic syndromes and is currently being tested on other cancers including solid tumors. However, 5-azacytidine or its deoxy-analog, 5-aza-2'-deoxycytidine (5-azaCdR) could also induce methylated prometastatic genes by DNA demethylation and induce cancer cell invasiveness. Since 5-azacytidine is a potent cancer growth inhibitor, we tested whether combining it with a DNA-methylating agent, the methyl donor S-adenosyl methionine (SAM), would block the adverse demethylating activity of 5-azaCdR while maintaining its growth suppression effects. We show here using several invasive and non-invasive breast cancer cell lines that SAM inhibits global- and gene-specific demethylation induced by 5-azaCdR, prevents 5-azaCdR activation of prometastatic genes uPA and MMP2, resulting in inhibition of cell invasiveness while augmenting the growth inhibitory effects of 5-azaCdR and its effects on tumor suppressor genes. Combination of drugs acting on the DNA methylation machinery at different levels is proposed as a new strategy for epigenetic therapy of cancer.

  17. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    PubMed

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  18. Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells.

    PubMed

    Lu, Chun-Feng; Yuan, Xiao-Yan; Li, Li-Zhong; Zhou, Wei; Zhao, Jun; Wang, Yi-Mei; Peng, Shuang-Qing

    2015-12-01

    Growing evidence has confirmed that exposure to ambient particulate matters (PM) is associated with increased morbidity and mortality of cardiovascular and pulmonary diseases. Ambient PM is a complex mixture of particles and air pollutants. Harmful effects of PM are specifically associated with ultrafine particles (UFPs) that can adsorb high concentrations of toxic air pollutants and are easily inhaled into the lungs. However, combined effects of UFPs and air pollutants on human health remain unclear. In the present study, we elucidated the combined toxicity of silica nanoparticles (nano-SiO2), a typical UFP, and lead acetate (Pb), a typical air pollutant. Lung adenocarcinoma A549 cells were exposed to nano-SiO2 and Pb alone or their combination, and their combined toxicity was investigated by focusing on cellular oxidative stress and DNA damage. Factorial analyses were performed to determine the potential interactions between nano-SiO2 and Pb. Our results showed that exposure of A549 cells to a modest cytotoxic concentration of Pb alone induced oxidative stress, as evidenced by elevated reactive oxygen species generation and lipid peroxidation, and reduced glutathione content and superoxide dismutase and glutathione peroxidase activities. In addition, exposure of A549 cells to Pb alone induced DNA damage, as evaluated by alkaline comet assay. Exposure of A549 cells to non-cytotoxic concentration of nano-SiO2 did not induce cellular oxidative stress and DNA damage. However, exposure to the combination of nano-SiO2 and Pb potentiated oxidative stress and DNA damage in A549 cells. Factorial analyses indicated that the potentiation of combined toxicity of nano-SiO2 and Pb was induced by additive or synergistic interactions.

  19. Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones.

    PubMed

    Ozdemir-Kaynak, Elif; Yesil-Celiktas, Ozlem

    2015-10-01

    A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol-chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2min instead of 24h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8±0.2°C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40ng/μl and proved to be a superior alternative to the phenol-chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.

  20. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    PubMed Central

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H.; Bignami, Margherita; Mazzei, Filomena

    2016-01-01

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  1. Combining combing and secondary ion mass spectrometry to study DNA on chips using (13)C and (15)N labeling.

    PubMed

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA - the combing, imaging by SIMS or CIS method - has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  2. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  3. Utility of the trnH–psbA Intergenic Spacer Region and Its Combinations as Plant DNA Barcodes: A Meta-Analysis

    PubMed Central

    Liu, Rui; Liang, Dong; Li, Huan; Cherny, Stacey S.; Chen, Shilin

    2012-01-01

    Background The trnH–psbA intergenic spacer region has been used in many DNA barcoding studies. However, a comprehensive evaluation with rigorous sequence preprocessing and statistical testing on the utility of trnH–psbA and its combinations as DNA barcodes is lacking. Methodology/Principal Findings Sequences were searched from GenBank for a meta-analysis on the usefulness of trnH–psbA and its combinations as DNA barcodes. After preprocessing, we constructed full and matching data sets that contained 17 983 trnH–psbA sequences and 2190 sets of trnH–psbA, matK, rbcL, and ITS2 sequences from the same sample, repectively. These datasets were used to analyze the ability of trnH–psbA and its combinations to discriminate species by the BLAST and BLAST+P methods. The Fisher's exact test was used to evaluate the significance of performance differences. For the full data set, the identification success rates of trnH–psbA exceeded 70% in 18 families and 12 genera, respectively. For the matching data set, the identification rates of trnH–psbA were significantly higher than those of the other loci in two families and four genera. Similarly, the identification rates of trnH–psbA+ITS2 were significantly higher than those of matK+rbcL in 18 families and 21 genera. Conclusion/Significane This study provides valuable information on the higher utility of trnH–psbA and its combinations. We found that trnH–psbA+ITS2 combination performs better or equally well compared with other combinations in most taxonomic groups investigated. This information will guide the optimal usage of trnH–psbA and its combinations for species identification. PMID:23155412

  4. Identification of invasive fungal diseases in immunocompromised patients by combining an Aspergillus specific PCR with a multifungal DNA-microarray from primary clinical samples.

    PubMed

    Boch, T; Reinwald, M; Postina, P; Cornely, O A; Vehreschild, J J; Heußel, C P; Heinz, W J; Hoenigl, M; Eigl, S; Lehrnbecher, T; Hahn, J; Claus, B; Lauten, M; Egerer, G; Müller, M C; Will, S; Merker, N; Hofmann, W-K; Buchheidt, D; Spiess, B

    2015-12-01

    The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy.

  5. Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing

    PubMed Central

    2010-01-01

    Background Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized. Results Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation. Conclusions This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes. PMID:20181289

  6. Ibuprofen causes photocleavage through ROS generation and intercalates with DNA: a combined biophysical and molecular docking approach.

    PubMed

    Husain, Mohammed Amir; Sarwar, Tarique; Rehman, Sayeed Ur; Ishqi, Hassan Mubarak; Tabish, Mohammad

    2015-06-01

    Ibuprofen is an important nonsteroidal anti-inflammatory drug endowed with various pharmacological and biological activities. In the present study, the photochemical properties of ibuprofen were evaluated by assaying the generation of various reactive oxygen species (ROS) such as superoxide, singlet oxygen and the hydroxyl radical. ROS generated by ibuprofen in the presence of white light causes DNA strand scission as observed by plasmid nicking assay. Ibuprofen induced ROS generation is also capable of causing DNA degradation in lymphocytes as observed by photocomet assay. ROS generation properties of ibuprofen were further strengthened by the formation of carbonyl groups in BSA and TBARS in linoleic acid as observed by carbonyl assay and lipid peroxidation assay respectively. We have also investigated the mode of interaction of ibuprofen with calf thymus DNA through a series of in vitro experiments. UV-visible spectroscopy established the formation of a complex between ibuprofen and Ct DNA. The steady state fluorescence experiments at different temperatures revealed a binding constant of ∼10(4) L mol(-1), which is indicative of intercalative binding between ibuprofen and the DNA helix. Analysis of the various thermodynamic parameters ΔG, ΔH and ΔS calculated at different temperatures indicated that the hydrogen bonds played a major role in the interaction. The intercalative binding mode is further confirmed by competitive displacement assays, urea denaturation, iodide quenching, viscosity measurements and CD analysis. In silico molecular docking revealed the binding of ibuprofen within the GC base pairs of DNA, confirming the intercalative binding mode.

  7. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

  8. The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

    PubMed Central

    2012-01-01

    Background Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics. PMID:23217215

  9. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

  10. Correlation of in Situ Oxazolidine Formation with Highly Synergistic Cytotoxicity and DNA Cross-Linking in Cancer Cells from Combinations of Doxorubicin and Formaldehyde.

    PubMed

    Barthel, Benjamin L; Mooz, Erin L; Wiener, Laura Elizabeth; Koch, Gary G; Koch, Tad H

    2016-03-10

    Anthracyclines are a class of antitumor compounds that are successful and widely used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA cross-linker. Contrary to current dogma, we show that both efficient DNA cross-linking and potent synergy in combination with formaldehyde correlate with the anthracycline's ability to form cyclic formaldehyde conjugates as oxazolidine moieties and that the cyclic conjugates are better cross-linking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development.

  11. Arsenic-Induced Antioxidant Depletion, Oxidative DNA Breakage, and Tissue Damages are Prevented by the Combined Action of Folate and Vitamin B12.

    PubMed

    Acharyya, Nirmallya; Deb, Bimal; Chattopadhyay, Sandip; Maiti, Smarajit

    2015-11-01

    Arsenic is a grade I human carcinogen. It acts by disrupting one-carbon (1C) metabolism and cellular methyl (-CH3) pool. The -CH3 group helps in arsenic disposition and detoxification of the biological systems. Vitamin B12 and folate, the key promoters of 1C metabolism were tested recently (daily 0.07 and 4.0 μg, respectively/100 g b.w. of rat for 28 days) to evaluate their combined efficacy in the protection from mutagenic DNA-breakage and tissue damages. The selected tissues like intestine (first-pass site), liver (major xenobiotic metabolizer) and lung (major arsenic accumulator) were collected from arsenic-ingested (0.6 ppm/same schedule) female rats. The hemo-toxicity and liver and kidney functions were monitored. Our earlier studies on arsenic-exposed humans can correlate carcinogenesis with DNA damage. Here, we demonstrate that the supplementation of physiological/therapeutic dose of vitamin B12 and folate protected the rodents significantly from arsenic-induced DNA damage (DNA fragmentation and comet assay) and hepatic and renal tissue degeneration (histo-architecture, HE staining). The level of arsenic-induced free-radical products (TBARS and conjugated diene) was significantly declined by the restored actions of several antioxidants viz. urate, thiol, catalase, xanthine oxidase, lactoperoxidase, and superoxide dismutase in the tissues of vitamin-supplemented group. The alkaline phosphatase, transaminases, urea and creatinine (hepatic and kidney toxicity marker), and lactate dehydrogenase (tissue degeneration marker) were significantly impaired in the arsenic-fed group. But a significant protection was evident in the vitamin-supplemented group. In conclusion, the combined action of folate and B12 results in the restitution in the 1C metabolic pathway and cellular methyl pool. The cumulative outcome from the enhanced arsenic methylation and antioxidative capacity was protective against arsenic induced mutagenic DNA breakages and tissue damages.

  12. Arsenic-Induced Antioxidant Depletion, Oxidative DNA Breakage, and Tissue Damages are Prevented by the Combined Action of Folate and Vitamin B12.

    PubMed

    Acharyya, Nirmallya; Deb, Bimal; Chattopadhyay, Sandip; Maiti, Smarajit

    2015-11-01

    Arsenic is a grade I human carcinogen. It acts by disrupting one-carbon (1C) metabolism and cellular methyl (-CH3) pool. The -CH3 group helps in arsenic disposition and detoxification of the biological systems. Vitamin B12 and folate, the key promoters of 1C metabolism were tested recently (daily 0.07 and 4.0 μg, respectively/100 g b.w. of rat for 28 days) to evaluate their combined efficacy in the protection from mutagenic DNA-breakage and tissue damages. The selected tissues like intestine (first-pass site), liver (major xenobiotic metabolizer) and lung (major arsenic accumulator) were collected from arsenic-ingested (0.6 ppm/same schedule) female rats. The hemo-toxicity and liver and kidney functions were monitored. Our earlier studies on arsenic-exposed humans can correlate carcinogenesis with DNA damage. Here, we demonstrate that the supplementation of physiological/therapeutic dose of vitamin B12 and folate protected the rodents significantly from arsenic-induced DNA damage (DNA fragmentation and comet assay) and hepatic and renal tissue degeneration (histo-architecture, HE staining). The level of arsenic-induced free-radical products (TBARS and conjugated diene) was significantly declined by the restored actions of several antioxidants viz. urate, thiol, catalase, xanthine oxidase, lactoperoxidase, and superoxide dismutase in the tissues of vitamin-supplemented group. The alkaline phosphatase, transaminases, urea and creatinine (hepatic and kidney toxicity marker), and lactate dehydrogenase (tissue degeneration marker) were significantly impaired in the arsenic-fed group. But a significant protection was evident in the vitamin-supplemented group. In conclusion, the combined action of folate and B12 results in the restitution in the 1C metabolic pathway and cellular methyl pool. The cumulative outcome from the enhanced arsenic methylation and antioxidative capacity was protective against arsenic induced mutagenic DNA breakages and tissue damages. PMID

  13. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    PubMed

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.

  14. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    PubMed

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  15. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    PubMed Central

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-01-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  16. Combined treatment with vitamin B12b and ascorbic acid causes in vitro DNA degradation in tumor cells.

    PubMed

    Medvedev, A I; Akatov, V S; Kreshchenko, N D; Solov'eva, M E; Leshchenko, V V; Lezhnev, E I; Yakubovskaya, R I

    2001-04-01

    Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

  17. The combined effects of BDE47 and BaP on oxidatively generated DNA damage in L02 cells and the possible molecular mechanism.

    PubMed

    An, Jing; Yin, Lingling; Shang, Yu; Zhong, Yufang; Zhang, Xinyu; Wu, Minghong; Yu, Zhiqiang; Sheng, Guoying; Fu, Jiamo; Huang, Yuecheng

    2011-04-01

    Polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) coexist widely in the environment and have generated adverse effects on the environment and human health. The purpose of this study was to investigate the combined toxic effects of these chemicals and the related mechanism. L02 cells were exposed to BDE47 (5, 10μmol/L) or/and BaP (50μmol/L) in different administration order. The cell growth and survival, DNA strand breaks, oxidative stress index (ROS, SOD, GSH, and MDA), LDH release and the expression level of CYP1 family members were measured. The result showed that BDE47 or/and BaP had no effect on the cell growth and survival under the present conditions. However, compared with the groups treated with BDE47 or BaP alone, the combined-treated groups induced significantly elevated DNA strand breaks, ROS production, and MDA level. Especially, pretreatment with BDE47 followed by BaP led to the strongest effects. Addition of the antioxidant N-acetyl-l-cysteine (NAC) markedly reduced the ROS level and partly suppressed the DNA strand breaks induced by BDE47 or/and BaP. Meanwhile, the combined treatment groups also markedly increased the SOD activity, GSH content, and LDH release level compared with the control group. The real-time PCR results showed that BaP could significantly induce the expression of CYP1A1 and CYP1B1, however, the pre-treatment with BDE47 appeared to attenuate the BaP-induced CYP1 expression. All of above findings indicated that BDE47 and BaP had a synergistic effect on oxidatively generated DNA damage in L02 cells via regulation on the oxidative stress response and the expression of CYP1 metabolism enzymes.

  18. Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

    PubMed

    Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

    2016-08-17

    Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus. PMID:27420472

  19. Development of a 24-locus multiplex system to incorporate the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS).

    PubMed

    Guo, Fei; Shen, Hongying; Tian, Huaizhou; Jin, Ping; Jiang, Xianhua

    2014-01-01

    The 24-locus multiplex system allows co-amplification and fluorescent detection of 24 loci (23 STR loci and Amelogenin), including STR loci in the Combined DNA Index System (CODIS) and the ESS (European Standard Set) as well as five additional loci (D2S1338, D6S1043, D19S433, Penta D and Penta E) commonly used in commercial kits. It facilitates data sharing and minimizes adventitious matches within national or between international DNA databases. Additionally, the system can amplify directly from blood and buccal samples spotted on filter paper and swabs and reduce the cycling time to less than one hour and a half. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house with a design strategy to work in this multiplex. Developmental validation experiments followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The system was evaluated by species specificity, sensitivity, stability, precision and accuracy, case-type samples, population, mixture and PCR-based studies. The results demonstrate that the 24-locus multiplex system is a robust and reliable identification assay as required for forensic DNA typing and databasing.

  20. Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

    PubMed

    Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

    2016-08-17

    Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.

  1. Visual recovery in a man with the rare combination of mtDNA 11778 LHON mutation and a MS-like disease after mitoxantrone therapy.

    PubMed

    Buhmann, C; Gbadamosi, J; Heesen, C

    2002-10-01

    We describe a young man with prognostic unfavourable homoplasmatic mitochondrial DNA(mt DNA) 11778 Leber's hereditary optic neuropathy (LHON) point mutation and confirmed multiple sclerosis (MS). This combination of LHON and MS-like disease is rare in both sexes, and in men has been described in only a few case reports. In a 4-year follow-up during immunosuppressive therapy with mitoxantrone, we found a remarkable time delayed visual recovery 12 months after acute onset of rapid sequential bilateral subtotal visual loss followed by episodes of isolated acute demyelinative optic neuropathy. Visual recovery to such extent after this latency is uncommon in both mtDNA 11778 LHON mutation and optic neuritis (ON) in MS. Relapses in visual deterioration must be considered as extremely rare in LHON. This case might support the hypothesis of an immunological pathogenetic factor in combined LHON and MS, and possibly in LHON alone. We suggest a search for the LHON mutation in MS patients with predominant visual impairment, independent of patients' gender.

  2. Combination of 768-well microplate array diagonal gel electrophoresis with duplex PCR of X and Y chromosome markers for quality control of epidemiological DNA banks.

    PubMed

    Huang, Shuwen; Chen, Xiao-he; Day, Ian N M

    2006-08-01

    Large DNA banks for human epidemiological studies have become an increasingly important research tool. The power of genotype-phenotype studies is dependent both on the quality of phenotyping and of genotyping and of correct linking of phenotypes to genotypes. Samples must be tracked through numerous steps between subject or patient and post-genotypic data. Only one phenotype, sex, has a perfect and binary correlation with genotype. In mixed sex studies, it may be advantageous for purposes of quality control to keep sexes mixed during the steps from acquisition to DNA bank, in order to be able to check later for sample swaps. We have designed a duplex PCR combining an amplicon from MAOA marking the X chromosome and an amplicon from DDX3Y marking the Y chromosome. We combined this with a simple economical palmtop sized 768-well microplate compatible electrophoresis system developed in-house for examination of duplex PCR products. We applied this quality control test in the validation of two DNA banks.

  3. Induction of cytokine production in cholesteatoma keratinocytes by extracellular high-mobility group box chromosomal protein 1 combined with DNA released by apoptotic cholesteatoma keratinocytes.

    PubMed

    Chi, Zhangcai; Wang, Zhengmin; Liang, Qiong; Zhu, Yaying; du, Qiang

    2015-02-01

    High-mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to determine HMGB-1 expression in vivo and to identify the effect of extracellular HMGB-1 in inflammatory process associated with bone destruction in cholesteatoma. We investigated the expression and location of HMGB-1 in the cholesteatoma and healthy skin using an immunofluorescence assay. We also detected apoptosis and DNA fragments in the cholesteatoma by TUNEL staining. HMGB-1 concentration in apoptotic supernatants from UV light-treated cells, culture supernatants and its translocation in cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells were measured by immunoblot analysis and immunofluorescence assay. Cultures of human cholesteatoma keratinocytes were exposed to CpG-DNA, HMGB-1, or CpG-DNA complexed to HMGB-1 for 24 h. Cytokines in the culture supernatant were measured by ELISA. In addition, levels of proinflammatory cytokines released by cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells with or without anti-HMGB-1 antibodies and supernatants from UV light-treated cells with DNase 1 were measured by enzyme-linked immunosorbent assay. The expression of HMGB-1 in cholesteatoma increased and it translocated both to the cytoplasm and extracellular space. Furthermore, the HMGB-1 concentration in supernatants increased significantly after addition of supernatants from UV light-treated cells. TNF-α and IL-1β can be induced by purified HMGB-1 combined with CpG-DNA in the cholesteatoma keratinocytes. In addition, supernatants of apoptotic cells containing HMGB-1-DNA were effective in inducing TNF-α and IL-1β secretion. This study suggested that persistent expression of extracellular HMGB-1 and DNA fragments in cholesteatoma leads to TNF-α and IL-1β production, causing bone resorption and destruction. Thus, we have

  4. Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

    PubMed Central

    Kosinska, Anna D.; Zhang, Ejuan; Johrden, Lena; Liu, Jia; Seiz, Pia L.; Zhang, Xiaoyong; Ma, Zhiyong; Kemper, Thekla; Fiedler, Melanie; Glebe, Dieter; Wildner, Oliver; Dittmer, Ulf; Lu, Mengji; Roggendorf, Michael

    2013-01-01

    A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients. PMID:23785279

  5. Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots.

    PubMed

    Andreadou, Margarita; Liandris, Emmanouil; Gazouli, Maria; Mataragka, Antonia; Tachtsidis, Ilias; Goutas, Nikolaοs; Vlachodimitropoulos, Dimitrios; Ikonomopoulos, John

    2016-04-01

    Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/μl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings. PMID:26658854

  6. Structure of Low-Lying Excited States of Guanine in DNA and Solution: Combined Molecular Mechanics and High-Level Coupled Cluster Studies

    DOE PAGES

    Kowalski, Karol; Valiev, Marat

    2007-01-01

    High-level ab-initio equation-of-motion coupled-cluster methods with singles, doubles, and noniterative triples are used, in conjunction with the combined quantum mechanical molecular mechanics approach, to investigate the structure of low-lying excited states of the guanine base in DNA and solvated environments. Our results indicate that while the excitation energy of the first excited state is barely changed compared to its gas-phase counterpart, the excitation energy of the second excited state is blue-shifted by 0.24 eV.

  7. Inhibition of DNA Topoisomerase Type IIα (TOP2A) by Mitoxantrone and Its Halogenated Derivatives: A Combined Density Functional and Molecular Docking Study

    PubMed Central

    Abu Saleh, Md.; Solayman, Md.; Hoque, Mohammad Mazharol; Khan, Mohammad A. K.; Sarwar, Mohammed G.; Halim, Mohammad A.

    2016-01-01

    In this study, mitoxantrone and its halogenated derivatives have been designed by density functional theory (DFT) to explore their structural and thermodynamical properties. The performance of these drugs was also evaluated to inhibit DNA topoisomerase type IIα (TOP2A) by molecular docking calculation. Noncovalent interactions play significant role in improving the performance of halogenated drugs. The combined quantum and molecular mechanics calculations revealed that CF3 containing drug shows better preference in inhibiting the TOP2A compared to other modified drugs. PMID:27088089

  8. Personal identification of cold case remains through combined contribution from anthropological, mtDNA and bomb–pulse dating analyses*†

    PubMed Central

    Speller, Camilla F.; Spalding, Kirsty L.; Buchholz, Bruce A.; Hildebrand, Dean; Moore, Jason; Mathewes, Rolf; Skinner, Mark F.; Yang, Dongya Y.

    2013-01-01

    In 1968, a child’s cranium was recovered from the banks of a northern Canadian river, and held in trust until the ‘cold case’ was re-opened in 2005. The cranium underwent re-analysis at the Centre for Forensic Research, Simon Fraser University, using recently developed anthropological, ‘bomb-pulse’ radiocarbon analysis and forensic DNA techniques. Craniometrics, skeletal ossification and dental formation indicated an age-at-death of 4.4 ±1 years. Radiocarbon analysis of enamel from two teeth indicated a year of birth between 1958–1962. Forensic DNA analysis indicated the child was male, and the obtained mitochondrial profile matched a living maternal relative of the presumed missing child. These multi-disciplinary analyses resulted in a legal identification 41 years after the discovery of the remains, highlighting the enormous potential of combining radiocarbon analysis with anthropological and mtDNA analyses in producing confident personal identifications for forensic cold cases dating to within the last 60 years. PMID:22804335

  9. Reproductive morphology and DNA sequences of the brown alga Platysiphon verticillatus support the new combination Platysiphon glacialis.

    PubMed

    Kawai, Hiroshi; Hanyuda, Takeaki; Yamagishi, Takahiro; Kai, Atsushi; Lane, Chris E; McDevit, Dan; Küpper, Frithjof C; Saunders, Gary W

    2015-10-01

    Platysiphon verticillatus, a brown alga endemic to the Arctic, was described based on vegetative specimens collected at Inglefield Bay, West Greenland. The species is distinctive in having a lanceolate blade-like thallus terminated by a terete portion, both covered with hair-like assimilatory filaments. Punctaria glacialis was described from Eastern Greenland, and the species differs from other Punctaria species in lacking hairs and plurilocular zoidangia. Unilocular zoidangia were reported, but instead of zoids being released they formed cell walls in situ developing the appearance of plurilocular zoidangia. However, the fate of the zoids, as well as the walled cells was not traced, and the life history of the alga has remained unclear. By comparing DNA sequences (cox1, cox3, and rDNA ITS2) of specimens morphologically referable to Platysiphon verticillatus and Punctaria glacialis collected at Baffin Island, as well as re-examining morphology and studying crude cultures, we concluded that they are the same taxonomic entity. Furthermore, their cox3 sequence and vegetative morphology agreed with those of the type specimen of Punctaria glacialis. Consequently, we propose Platysiphon glacialis comb. nov. The life cycle could not be completed in culture, but we hypothesize that in situ germination of the unizoids produces reduced gametophytes housed in peripheral tissue of erect sporophytic thalli. PMID:26986887

  10. Response of arsenic-induced oxidative stress, DNA damage, and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats.

    PubMed

    Bhadauria, S; Flora, S J S

    2007-03-01

    Arsenic and its compounds cause adverse health effects in humans. Current treatment employs administration of thiol chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic

  11. CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging.

    PubMed

    Parviainen, S; Ahonen, M; Diaconu, I; Hirvinen, M; Karttunen, Å; Vähä-Koskela, M; Hemminki, A; Cerullo, V

    2014-02-01

    Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

  12. Combination of ADH1B*2/ALDH2*2 polymorphisms alters acetaldehyde-derived DNA damage in the blood of Japanese alcoholics.

    PubMed

    Yukawa, Yoshiyuki; Muto, Manabu; Hori, Kimiko; Nagayoshi, Haruna; Yokoyama, Akira; Chiba, Tsutomu; Matsuda, Tomonari

    2012-09-01

    The acetaldehyde associated with alcoholic beverages is an evident carcinogen for the esophagus. Genetic polymorphisms of the alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes are associated with the risk of esophageal cancer. However, the exact mechanism via which these genetic polymorphisms affect esophageal carcinogenesis has not been elucidated. ADH1B*2 is involved in overproduction of acetaldehyde due to increased ethanol metabolism into acetaldehyde, and ALDH2*2 is involved in accumulation of acetaldehyde due to the deficiency of acetaldehyde metabolism. Acetaldehyde can interact with DNA and form DNA adducts, resulting in DNA damage. N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) is the most abundant DNA adduct derived from acetaldehyde. Therefore, we quantified N(2)-ethylidene-dG levels in blood samples from 66 Japanese alcoholic patients using liquid chromatography/electrospray tandem mass spectrometry, and investigated the relationship between N(2)-ethylidene-dG levels and ADH1B and ALDH2 genotypes. The median N(2)-ethylidene-dG levels (25th percentile, 75th percentile) in patients with ADH1B*1/*1 plus ALDH2*1/*1, ADH1B*2 carrier plus ALDH2*1/*1, ADH1B*1/*1 plus ALDH2*1/*2, and ADH1B*2 carrier plus ALDH2*1/*2 were 2.14 (0.97, 2.37)/10(7) bases, 2.38 (1.18, 2.98)/10(7) bases, 5.38 (3.19, 6.52)/10(7) bases, and 21.04 (12.75, 34.80)/10(7) bases, respectively. In the ALDH2*1/*2 group, N(2)-ethylidene-dG levels were significantly higher in ADH1B*2 carriers than in the ADH1B*1/*1 group (P < 0.01). N(2)-ethylidene-dG levels were significantly higher in the ALDH2*1/*2 group than in the ALDH2*1/*1 group, regardless of ADH1B genotype (ADH1B*1/*1, P < 0.05; ADH1B*2 carriers, P < 0.01) N(2)-ethylidene-dG levels in blood DNA of the alcoholics was remarkably higher in individuals with a combination of the ADH1B*2 and ALDH2*2 alleles. These results provide a new perspective on the carcinogenicity of the acetaldehyde associated with

  13. Correlation of in Situ Oxazolidine Formation with Highly Synergistic Cytotoxicity and DNA Cross-Linking in Cancer Cells from Combinations of Doxorubicin and Formaldehyde.

    PubMed

    Barthel, Benjamin L; Mooz, Erin L; Wiener, Laura Elizabeth; Koch, Gary G; Koch, Tad H

    2016-03-10

    Anthracyclines are a class of antitumor compounds that are successful and widely used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA cross-linker. Contrary to current dogma, we show that both efficient DNA cross-linking and potent synergy in combination with formaldehyde correlate with the anthracycline's ability to form cyclic formaldehyde conjugates as oxazolidine moieties and that the cyclic conjugates are better cross-linking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development. PMID:26881291

  14. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    SciTech Connect

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-08-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: (/sup 3/H)thymidine incorporation decreased by 80% and (/sup 14/C)leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of (/sup 3/H)histidine/(/sup 14/C)leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

  15. Combination of running exercise and high dose of anabolic androgenic steroid, nandrolone decanoate, increases protamine deficiency and DNA damage in rat spermatozoa.

    PubMed

    Shokri, S; Hemadi, M; Bayat, G; Bahmanzadeh, M; Jafari-Anarkooli, I; Mashkani, Beta

    2014-03-01

    High doses of anabolic-androgenic steroids (AAS) are used by some athletes to increase muscle mass, that is often associated with male infertility. The aim of this study was to investigate the possible cause/s of male infertility using a rat model by analysing sperm quality, including its protamine content and DNA integrity, as well as pregnancy rate. Five groups of male Wistar rats were treated for 10 weeks as follows: nandrolone decanoate (10 mg kg(-1) per week) (ND); running exercise (50 min per day, 5 days a week) (EX); Combination of ND and exercise (ND-EX); nandrolone decanoate solvent (Sham); and control without any injection or exercise (CO). Deterioration in sperm quantity was observed in all test groups (P ≤ 0.01). The frequency of fertile rats was decreased in the ND-EX and ND groups (P ≤ 0.05). Chromomycin-A3 staining showed a protamine deficiency in the epididymal spermatozoa in the ND-EX rats (P ≤ 0.05). Chromatin analysis indicated an abnormal maturation of the sperm nuclei in all test groups compared with the controls (P ≤ 0.05). TUNEL analyses showed a highly significant increase in apoptosis in the EX, ND, and ND-EX groups (P ≤ 0.01). Our data show that a combination of exercise and high doses of nandrolone decanoate negatively influences the DNA integrity and protamine content resulting in lower sperm quality and reduced pregnancy rate.

  16. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

  17. Photophysics and photochemistry of a DNA-protein cross-linking model: a synergistic approach combining experiments and theory.

    PubMed

    Micciarelli, Marco; Valadan, Mohammadhassan; Della Ventura, Bartolomeo; Di Fabio, Giovanni; De Napoli, Lorenzo; Bonella, Sara; Röthlisberger, Ursula; Tavernelli, Ivano; Altucci, Carlo; Velotta, Raffaele

    2014-05-15

    The photophysical and photochemical properties of 5-benzyluracil and 5,6-benzyluracil, the latter produced by photocyclization of the former through irradiation with femtosecond UV laser pulses, are investigated both experimentally and theoretically. The absorption spectra of the two molecules are compared, and the principal electronic transitions involved are discussed, with particular emphasis on the perturbation induced on the two chromophore species (uracil and benzene) by their proximity. The photoproduct formation for different irradiation times was verified with high-performance liquid chromatography and nuclear magnetic resonance measurements. The steady-state fluorescence demonstrates that the fluorescence is a distinctive physical observable for detection and selective identification of 5- and 5,6-benzyluracil. The principal electronic decay paths of the two molecules, obtained through TDDFT calculations, explain the features observed in the emission spectra and the photoreactivity of 5-benzyluracil. The order of magnitude of the lifetime of the excited states is derived with steady-state fluorescence anisotropy measurements and rationalized with the help of the computational findings. Finally, the spectroscopic data collected are used to derive the photocyclization and fluorescence quantum yields. In obtaining a global picture of the photophysical and photochemical properties of the two molecules, our findings demonstrates that the use of 5-benzyluracil as a model system to study the proximity relations of a DNA base with a close-lying aromatic amino acid is valid at a local level since the main characteristics of the decay processes from the excited states of the uracil/thymine molecules remain almost unchanged in 5-benzyluracil, the main perturbation arising from the presence of the close-lying aromatic group.

  18. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    NASA Astrophysics Data System (ADS)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p < 0.005, all R > 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure

  19. Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic anti-tumor effects.

    PubMed

    Tseng, Chih Wen; Monie, Archana; Trimble, Cornelia; Alvarez, Ronald D; Huh, Warner K; Buchsbaum, Donald J; Straughn, J Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T-C

    2008-08-12

    There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as anti-tumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation.

  20. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China*

    PubMed Central

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-01-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population’s genetic background, for individual identification, and for paternity testing in forensic practice. PMID:23733431

  1. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China.

    PubMed

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-06-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population's genetic background, for individual identification, and for paternity testing in forensic practice.

  2. Folic acid or combination of folic acid and vitamin B(12) prevents short-term arsenic trioxide-induced systemic and mitochondrial dysfunction and DNA damage.

    PubMed

    Majumdar, Sangita; Mukherjee, Sandip; Maiti, Anasuya; Karmakar, Subhra; Das, Asankur Sekhar; Mukherjee, Maitrayee; Nanda, Arunabha; Mitra, Chandan

    2009-08-01

    The effect of folic acid and folic acid + vitamin B(12) supplementation upon short-term arsenic-induced systemic and pancreatic islet cell mitochondria oxidative stress was investigated in male rats. Arsenic trioxide was administered orally at a dose of 3 mg kg body weight(-1) day(-1) for 30 days, and folic acid and vitamin B(12) were administered at a dose of 36 and 0.63 microg kg body weight(-1) day(-1), respectively, for 30 days. Compared to control, arsenic-treated group showed a significant increase in the levels of systemic oxidative markers, malondialdehyde (MDA), nitric oxide (NO), and hydroxyl radical (OH(-)) formation, which were found decreased significantly after supplementation either with folic acid or a combination of folic acid + vitamin B(12). Similar supplementations were found effective against arsenic-induced oxidative marker changes (MDA, NO, and OH(-)) in pancreatic islet cell mitochondria. Also, low activities of antioxidant defense enzymes such as superoxide dismutase and catalase, and level of antioxidant glutathione, all could regain significantly on supplementations both against systemic and islet cell mitochondria oxidative stress. Results of agarose-gel electrophoresis of DNA from lymphocytes and islet cells of arsenic-exposed rats showed DNA smearing, which could be reduced with simultaneous administration either with folic acid or a combination of folic acid + vitamin B(12). Significantly, similar supplementations were found effective in increasing the urinary clearance of arsenic. Together, these results indicate that folic acid and vitamin B(12) may be effective to reduce the arsenic-induced damage at molecular target level.

  3. Integration of Known DNA, RNA and Protein Biomarkers Provides Prediction of Anti-TNF Response in Rheumatoid Arthritis: Results from the COMBINE Study

    PubMed Central

    Folkersen, Lasse; Brynedal, Boel; Diaz-Gallo, Lina Marcela; Ramsköld, Daniel; Shchetynsky, Klementy; Westerlind, Helga; Sundström, Yvonne; Schepis, Danika; Hensvold, Aase; Vivar, Nancy; Eloranta, Maija-Leena; Rönnblom, Lars; Brunak, Søren; Malmström, Vivianne; Catrina, Anca; Moerch, Ulrik GW; Klareskog, Lars; Padyukov, Leonid; Berg, Louise

    2016-01-01

    OBJECTIVE: In rheumatoid arthritis (RA) several recent efforts have sought to discover means of predicting which patients would benefit from treatment. However, results have been discrepant with few successful replications. Our objective was to build a biobank with DNA, RNA and protein measurements to test the claim that the current state-of-the-art precision medicine will benefit RA patients. METHODS: We collected 451 blood samples from 61 healthy individuals and 185 RA patients initiating treatment, before treatment initiation and at a 3 month follow-up time. All samples were subjected to high-throughput RNA sequencing, DNA genotyping, extensive proteomics and flow cytometry measurements, as well as comprehensive clinical phenotyping. Literature review identified 2 proteins, 52 single-nucleotide polymorphisms (SNPs) and 72 gene-expression biomarkers that had previously been proposed as predictors of Tumor Necrosis Factor (TNF) inhibitor response (ΔDAS28-CRP). RESULTS: From these published TNFi biomarkers we found that 2 protein, 2 SNP and 8 mRNA biomarkers could be replicated in the 59 TNF initiating patients. Combining these replicated biomarkers into a single signature we found that we could explain 51% of the variation in ΔDAS28-CRP. This corresponds to a sensitivity of 0.73 and specificity of 0.78 for the prediction of three month ΔDAS28-CRP better than –1.2. CONCLUSIONS: The COMBINE biobank is currently the largest collection of multi-omics data from RA patients with high potential for discovery and replication. Taking advantage of this we surveyed the current state-of-the-art of drug-response stratification in RA, and identified a small set of previously published biomarkers available in peripheral blood which predicts clinical response to TNF blockade in this independent cohort. PMID:27532898

  4. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    PubMed

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  5. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    PubMed

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  6. Phylogenetic relationships of Middle American cichlids (Cichlidae, Heroini) based on combined evidence from nuclear genes, mtDNA, and morphology.

    PubMed

    Rícan, Oldrich; Zardoya, Rafael; Doadrio, Ignacio

    2008-12-01

    Heroine cichlids are the second largest and very diverse tribe of Neotropical cichlids, and the only cichlid group that inhabits Mesoamerica. The taxonomy of heroines is complex because monophyly of most genera has never been demonstrated, and many species groups are without applicable generic names after their removal from the catch-all genus Cichlasoma (sensu Regan, 1905). Hence, a robust phylogeny for the group is largely wanting. A rather complete heroine phylogeny based on cytb sequence data is available [Concheiro Pérez, G.A., Rícan O., Ortí G., Bermingham, E., Doadrio, I., Zardoya, R. 2007. Phylogeny and biogeography of 91 species of heroine cichlids (Teleostei: Cichlidae) based on sequences of the cytochrome b gene. Mol. Phylogenet. Evol. 43, 91-110], and in the present study, we have added and analyzed independent data sets (nuclear and morphological) to further confirm and strengthen the cytb-phylogenetic hypothesis. We have analyzed a combined cytb-nuclear (RAG1 and two S7 introns) data set of 48 species representing main heroine lineages to achieve further resolution of heroine higher taxonomic levels and a combined cytb-morphological data set of 92 species to stabilize generic taxonomy. The recovered phylogenies supported the circumamazonian--CAM--Heroini (sensu Concheiro Peréz et al., 2007) as a monophyletic group, that could be divided into six main clades: (1) australoheroines (the southernmost heroine genus Australoheros), (2) nandopsines (the Antillean genus Nandopsis), (3) caquetaines (including the north western Amazonian genera Caquetaia and Heroina), (4) astatheroines (including Astatheros, Herotilapia and Rocio), (5) amphilophines (including Amphilophus and related genera), and (6) herichthyines (including Herichthyis and related genera). Nuclear and mitochondrial data partitions arrived at highly congruent topologies. Suprageneric relationships were influenced mainly by the nuclear signal, as well as the most basal phylogenetic position

  7. Evolution of the assassin’s arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea)

    PubMed Central

    Zhang, Junxia; Gordon, Eric R. L.; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R.; Johnson, Kevin P.; Meier, Rudolf; Weirauch, Christiane

    2016-01-01

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators. PMID:26916580

  8. Evolution of the assassin's arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea).

    PubMed

    Zhang, Junxia; Gordon, Eric R L; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R; Johnson, Kevin P; Meier, Rudolf; Weirauch, Christiane

    2016-01-01

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators. PMID:26916580

  9. Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation, hydrogen peroxide, or c-Myc overexpression.

    PubMed

    Jin, Yeung Bae; Choi, Seo-Hyun; Lee, Jae Seon; Kim, Jae-Kyung; Lee, Ju-Woon; Hong, Seung-Cheol; Myung, Sung Ho; Lee, Yun-Sil

    2014-03-01

    The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H(2)O(2) (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H(2)O(2), or c-Myc activation.

  10. CCL17 combined with CCL19 as a nasal adjuvant enhances the immunogenicity of an anti-caries DNA vaccine in rodents

    PubMed Central

    Yan, Yan-hong; Yu, Fei; Zeng, Chang; Cao, Li-hua; Zhang, Zhou; Xu, Qing-an

    2016-01-01

    Aim: CCL19 and its receptor CCR7 are essential molecules for facilitating the trafficking of mature dendritic cells (DCs) and helping to establish a microenvironment in lymphoid tissues to initiate primary immune responses, whereas CCL17 is required in the CCR7-CCL19-dependent migration of DCs. In this study we examined whether co-administration of CCL17 and CCL19 could enhance the immunogenicity of an anti-caries DNA vaccine, pCIA-P, in rodents. Methods: Plasmids encoding CCL17 (pCCL17/VAX) and CCL19 (pCCL19/VAX) were constructed. BALB/c mice were intranasally administered pCCL17/VAX, pCCL19/VAX, or pCCL17/VAX plus pCCL19/VAX, the migration of DCs to the spleen and draining lymph nodes (DLNs) was assessed with flow cytometry. The mice were co-administered pCIA-P; and the anti-PAc antibodies in the serum and saliva were detected with ELISA. Wistar rats were orally challenged with Streptococcus mutans and then administered pCIA-P in combination with pCCL17/VAX, pCCL19/VAX, or pCCL17/VAX plus pCCL19/VAX. The amount of S mutans sustained on rat molar surfaces was assessed using a colony forming assay. Caries activity was scored with the Keyes method. Results: Co-administration of the CCL17 and CCL19 genes in mice caused a greater increase in the number of mature DCs in the spleen and DLNs compared with administration of CCL17 or CCL19 genes alone. CCL17 and CCL19 double-adjuvant plus pCIA-P induced significantly higher levels of anti-PAc salivary IgA and anti-PAc serum IgG antibody in mice, and strengthened the ability of pCIA-P in inhibiting the colonization of S mutans on rat tooth surfaces. The caries activity of the combined adjuvant group was significantly lower than that of the pCCL17/VAX or the pCCL19/VAX group. Conclusion: A nasal adjuvant consisting of a combination of CCL17 and CCL19 attracts more mature DCs to secondary lymphoid tissues, inducing enhanced antibody responses against the anti-caries DNA vaccine pCIA-P and reducing S mutans infection in

  11. Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

    PubMed

    Foti, M; Marshalko, S; Schurter, E; Kumar, S; Beardsley, G P; Schweitzer, B I

    1997-05-01

    The nucleoside analog 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, DHPG) is an antiviral drug that is used in the treatment of a variety of herpes viruses in immunocompromised patients and in a gene therapy protocol that has shown promising activity for the treatment of cancer. To probe the structural effects of ganciclovir when incorporated into DNA, we determined and compared the solution structure of a modified ganciclovir-containing decamer duplex [d(CTG)(ganciclovir)d(ATCCAG)]2 and a control duplex d[(CTGGATCCAG)]2 using nuclear magnetic resonance techniques. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H and 31P NMR experiments. Proton-proton distances determined from NOESY data and dihedral angles determined from DQF-COSY data were used in restrained molecular dynamics simulations starting from canonical A- and B-form DNA models. Both the control and ganciclovir sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures that were in excellent agreement with the observed NOE intensities. Examination of the final ganciclovir-containing structures reveals that the base of the ganciclovir residue is hydrogen bonded to its complementary dC and is stacked in the helix; in fact, the base of ganciclovir exhibits increased stacking with the 5' base relative to the control. Interestingly, some of the most significant distortions in the structures occur 3' to the lesion site, including a noticeable kink in the sugar-phosphate backbone at this position. Further examination reveals that the backbone conformation, sugar pucker, and glycosidic torsion angle of the residue 3' to the lesion site all indicate an A-type conformation at this position. A possible correlation of these structural findings with results obtained from earlier biochemical studies will be discussed.

  12. Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

    PubMed

    Foti, M; Marshalko, S; Schurter, E; Kumar, S; Beardsley, G P; Schweitzer, B I

    1997-05-01

    The nucleoside analog 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, DHPG) is an antiviral drug that is used in the treatment of a variety of herpes viruses in immunocompromised patients and in a gene therapy protocol that has shown promising activity for the treatment of cancer. To probe the structural effects of ganciclovir when incorporated into DNA, we determined and compared the solution structure of a modified ganciclovir-containing decamer duplex [d(CTG)(ganciclovir)d(ATCCAG)]2 and a control duplex d[(CTGGATCCAG)]2 using nuclear magnetic resonance techniques. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H and 31P NMR experiments. Proton-proton distances determined from NOESY data and dihedral angles determined from DQF-COSY data were used in restrained molecular dynamics simulations starting from canonical A- and B-form DNA models. Both the control and ganciclovir sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures that were in excellent agreement with the observed NOE intensities. Examination of the final ganciclovir-containing structures reveals that the base of the ganciclovir residue is hydrogen bonded to its complementary dC and is stacked in the helix; in fact, the base of ganciclovir exhibits increased stacking with the 5' base relative to the control. Interestingly, some of the most significant distortions in the structures occur 3' to the lesion site, including a noticeable kink in the sugar-phosphate backbone at this position. Further examination reveals that the backbone conformation, sugar pucker, and glycosidic torsion angle of the residue 3' to the lesion site all indicate an A-type conformation at this position. A possible correlation of these structural findings with results obtained from earlier biochemical studies will be discussed. PMID:9154915

  13. Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus

    PubMed Central

    Dvorak, Katerina; Payne, Claire M; Chavarria, Melissa; Ramsey, Lois; Dvorakova, Barbora; Bernstein, Harris; Holubec, Hana; Sampliner, Richard E; Guy, Naihsuan; Condon, Amanda; Bernstein, Carol; Green, Sylvan B; Prasad, Anil; Garewal, Harinder S

    2007-01-01

    Background Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. Objective To test the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. Methods Oxidative stress was evaluated by staining Barrett's oesophagus tissues with different degrees of dysplasia with 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) antibody. The levels of 8‐OH‐dG were also evaluated ex vivo in Barrett's oesophagus tissues incubated for 10 min with control medium and medium acidified to pH 4 and supplemented with 0.5 mM bile acid cocktail. Furthermore, three oesophageal cell lines (Seg‐1 cells, Barrett's oesophagus cells and HET‐1A cells) were exposed to control media, media containing 0.1 mM bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1 mM bile acid cocktail, and evaluated for induction of reactive oxygen species (ROS). Results Immunohistochemical analysis showed that 8‐OH‐dG is formed mainly in the epithelial cells in dysplastic Barrett's oesophagus. Importantly, incubation of Barrett's oesophagus tissues with the combination of bile acid cocktail and acid leads to increased formation of 8‐OH‐dG. An increase in ROS in oesophageal cells was detected after exposure to pH 4 and bile acid cocktail. Conclusions Oxidative stress and oxidative DNA damage can be induced in oesophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of Barrett's oesophagus and tumour progression. PMID:17145738

  14. Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences.

    PubMed

    Zhao, Guang-Hui; Li, Hong-Mei; Ryan, Una M; Cong, Mei-Mei; Hu, Bing; Gao, Man; Ren, Wan-Xin; Wang, Xing-Ye; Zhang, Shui-Ping; Lin, Qing; Zhu, Xing-Quan; Yu, San-Ke

    2012-09-01

    The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.

  15. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

    PubMed

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  16. γ-H2AX responds to DNA damage induced by long-term exposure to combined low-dose-rate neutron and γ-ray radiation.

    PubMed

    Zhang, Junlin; He, Ying; Shen, Xianrong; Jiang, Dingwen; Wang, Qingrong; Liu, Qiong; Fang, Wen

    2016-01-01

    Risk estimates for low-dose radiation (LDR) remain controversial. The possible involvement of DNA repair-related genes in long-term low-dose-rate neutron-gamma radiation exposure is poorly understood. In this study, 60 rats were divided into control groups and irradiated groups, which were exposed to low-dose-rate n-γ combined radiation (LDCR) for 15, 30, or 60 days. The effects of different cumulative radiation doses on peripheral blood cell (PBC), subsets of T cells of peripheral blood lymphocytes (PBL) and DNA damage repair were investigated. Real-time PCR and immunoblot analyses were used to detect expression of DNA DSB-repair-related genes involved in the NHEJ pathway, such as Ku70 and Ku80, in PBL. The mRNA level of H2AX and the expression level of γ-H2AX were detected by real-time PCR, immunoblot, and flow cytometry. White blood cells (WBC) and platelets (PLT) of all ionizing radiation (IR) groups decreased significantly, while no difference was seen between the 30 day and 60 day exposure groups. The numbers of CD3(+), CD4(+) T cells and CD4(+)/CD8(+) in the PBL of IR groups were lower than in the control group. In the 30 day and 60 day exposure groups, CD8(+) T cells decreased significantly. Real-time PCR and immunoblot results showed no significant difference in the mRNA and protein expression of Ku70 and Ku80 between the control groups and IR groups. However, the mRNA of H2AX increased significantly, and there was a positive correlation with dose. There was no difference in the protein expression of γ-H2AX between 30 day and 60 day groups, which may help to explain the damage to PBL. In conclusion, PBL damage increased with cumulative dose, suggesting that γ-H2AX, but neither Ku70 nor Ku80, plays an important role in PBL impairment induced by LDCR.

  17. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  18. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

    PubMed

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  19. Deciphering transcriptional networks that govern Coffea arabica seed development using combined cDNA array and real-time RT-PCR approaches.

    PubMed

    Salmona, Jordi; Dussert, Stéphane; Descroix, Frédéric; de Kochko, Alexandre; Bertrand, Benoît; Joët, Thierry

    2008-01-01

    Due to its economic importance, Coffea arabica is becoming the subject of increasing genomic research and, in particular, the genes involved in the final chemical composition of the bean and the sensorial quality of the coffee beverage. The aim of the present study was to decipher the transcriptional networks that govern the development of the C. arabica seed, a model for non-orthodox albuminous seeds of tropical origin. For this purpose, we developed a transcriptomic approach combining two techniques: targeted cDNA arrays, containing 266 selected candidate gene sequences, and real-time RT-PCR on a large subset of 111 genes. The combination of the two techniques allowed us to limit detection of false positives and to reveal the advantages of using large real-time RT-PCR screening. Multivariate analysis was conducted on both datasets and results were broadly convergent. First, principle component analysis (PCA) revealed a dramatic re-programming of the transcriptional machinery between early cell division and elongation, storage and maturation phases. Second, hierarchical clustering analysis (HCA) led to the identification of 11 distinct patterns of gene expression during seed development as well as to the detection of genes expressed at specific developmental stages that can be used as functional markers of phenological changes. In addition, this study led to the description of gene expression profiles for quality-related genes, most of them formerly uncharacterised in Coffea. Their involvement in storage compound synthesis and accumulation during endosperm development and final metabolic re-adjustments during maturation is discussed. PMID:18026845

  20. Superior Protective Immunity against Murine Listeriosis by Combined Vaccination with CpG DNA and Recombinant Salmonella enterica Serovar Typhimurium▿ †

    PubMed Central

    Berchtold, Christina; Panthel, Klaus; Jellbauer, Stefan; Köhn, Brigitte; Roider, Elisabeth; Partilla, Miriam; Heesemann, Jürgen; Endres, Stefan; Bourquin, Carole; Rüssmann, Holger

    2009-01-01

    Preexisting antivector immunity can severely compromise the ability of Salmonella enterica serovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60217-225 peptide from Listeria monocytogenes subcutaneously into BALB/c mice and coadministered a p60-translocating Salmonella strain by the orogastric route. The distribution of tetramer-positive p60217-225-specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinant Salmonella or single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection with L. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellular Listeria. In conclusion, we provide compelling evidence that the combination of Salmonella type III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities. PMID:19797070

  1. Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

    PubMed

    Yang, Dazuo; Zhou, Yibing; Zhao, Huan; Zhou, Xiaoxiao; Sun, Na; Wang, Bin; Yuan, Xiutang

    2012-11-01

    We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

  2. Antisense vimentin cDNA combined with chondroitinase ABC promotes axon regeneration and functional recovery following spinal cord injury in rats.

    PubMed

    Xia, Yongzhi; Yan, Yi; Xia, Haijian; Zhao, Tianzhi; Chu, Weihua; Hu, Shengli; Feng, Hua; Lin, Jiangkai

    2015-03-17

    The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.

  3. Allele frequencies of combined DNA index system (CODIS) and non-CODIS short tandem repeat loci in Goiás, Central Brazil.

    PubMed

    Rodovalho, R G; Santos, G S; Cavalcanti, L M; Moura, B F S M; Rodrigues, E L; Lima, P R; Gigonzac, M A D; Vieira, T C

    2015-07-03

    In studies of human identification, obtaining a high standard of outcomes and satisfactory conclusions are directly related to the use of highly polymorphic molecular markers. In addition to the combined DNA index system (CODIS) group, it is also important to implement non-CODIS markers into the analysis, as they increase the power of discrimination. During the identification process, it is essential to consider the genetic variation among distinct groups of populations, as the allele frequencies are directly associated with the power of discrimination. However, the population of Goiás, a State located in Central Brazil, is characterized by a highly mixed population due to its diverse ethnic origins. In this study, a survey of the allelic frequencies in the Goiás population was carried out using a molecular assembly composed of 21 autosomal loci both from and external to the CODIS group. The new data, for some of the markers used, were statistically similar to those from previous studies. This consistency means that the use of these markers might serve as a parameter for future population comparisons. The results from these analyses further our knowledge of the study of human identification.

  4. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease.

    PubMed

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-07-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. PMID:27123586

  5. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells.

    PubMed

    Soo, Jaslyn Sian-Siu; Ng, Char-Hong; Tan, Si Hoey; Malik, Rozita Abdul; Teh, Yew-Ching; Tan, Boon-Shing; Ho, Gwo-Fuang; See, Mee-Hoong; Taib, Nur Aishah Mohd; Yip, Cheng-Har; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Teo, Soo-Hwang; Leong, Chee-Onn

    2015-10-01

    Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers. PMID:26276035

  6. Combination of electron microscopic in situ hybridization and anti-DNA antibody labelling reveals a peculiar arrangement of ribosomal DNA in the fibrillar centres of the plant cell nucleolus.

    PubMed

    Yano, Hiroyuki; Sato, Seiichi

    2002-01-01

    The fibrillar centres (FCs) in the nucleoli of Allium cepa usually contained compact dense chromatin, which was always surrounded with light fibrous material (LFM). Distribution of 18S ribosomal DNA (rDNA) in the FCs was examined by in situ hybridization at the light and electron microscopic levels and the results were compared with those obtained by immunogold labelling with anti-DNA antibodies. Anti-DNA antibodies heavily labelled the dense chromatin of the FCs but scarcely labelled the LFM. However, electron microscopic in situ hybridization using the 18S rDNA probe showed that the label in the dense chromatin was extremely weak compared with that obtained by the anti-DNA antibody labelling: the specific label with anti-DNA antibodies of the dense chromatin was about 15 times as much as that of the LFM, whereas the specific label with in situ hybridization in the dense chromatin was only about 1.7 times higher than in the LFM. These results suggest that the rDNA encoding rRNA is preferentially released from the dense chromatin and that non-transcribed intergenic spacers remain in the dense chromatin as the anchoring sites of rDNA. PMID:12227553

  7. A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

    PubMed

    Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

    2015-10-01

    Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. PMID:26196248

  8. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  9. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    PubMed

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements.

  10. Combined effects of DNA methyltransferase 1 and 3A polymorphisms and urinary total arsenic levels on the risk for clear cell renal cell carcinoma.

    PubMed

    Yang, Shu-Mei; Huang, Chao-Yuan; Shiue, Horng-Sheng; Pu, Yeong-Shiau; Hsieh, Yi-Hsun; Chen, Wei-Jen; Lin, Ying-Chin; Hsueh, Yu-Mei

    2016-08-15

    Our previous study showed that high urinary total arsenic levels were associated with higher odds ratio (OR) for renal cell carcinoma (RCC). Single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) might influence DNMT enzyme activity associated with tumorigenesis. In this study, we investigated the association of five SNPs from DNMT1 (rs8101626 and rs2228611), DNMT3A (rs34048824 and rs1550117), and DNMT3B (rs1569686) with the risk of clear cell renal cell carcinoma (ccRCC). We also examined the combined effects of DNMT genotypes and urinary arsenic levels on ccRCC risk. We conducted a hospital-based case-control study, which included 293 subjects with ccRCC and 293 age- and gender-matched controls. The urinary arsenic species were determined by a high performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Genotypes were investigated using polymerase chain reaction and restriction fragment length polymorphism analyses. We observed that the DNMT1 rs8101626 G/G genotype was significantly associated with reduced odds ratio (OR) of ccRCC [OR=0.38, 95% confidence interval (CI) 0.14-0.99]. Subjects with concurrent DNMT1 rs8101626 A/A+A/G and DNMT3A rs34048824 T/T+T/C genotypes had significantly higher OR for ccRCC [OR=2.88, 95% CI 1.44-5.77]. Participants with the high-risk genotype of DNMT1 rs8101626 and DNMT3A rs34048824 with concurrently high urinary total arsenic levels had even higher OR of ccRCC in a dose-response manner. This is the first study to evaluate variant DNMT1 rs8101626 and DNMT3A rs34048824 genotypes that modify the arsenic-related ccRCC risk in a geographic area without significant arsenic exposure in Taiwan. PMID:27292127

  11. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    PubMed

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements. PMID:27410247

  12. Titration of varicella-zoster virus DNA in throat swabs from varicella patients by combined use of PCR and microplate hybridization.

    PubMed

    Hondo, R; Ito, S; Inouye, S

    1995-01-01

    We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.

  13. National decline in invasive prenatal diagnostic procedures in association with uptake of combined first trimester and cell-free DNA aneuploidy screening.

    PubMed

    Robson, Stephen J; Hui, Lisa

    2015-10-01

    In late 2012, a new screening test for fetal aneuploidy based on circulating cell-free DNA (cfDNA) became available to Australian women. The introduction of this technology in the United States has led to a reduction in invasive diagnostic procedures. Analysis of the number of amniocentesis and chorionic villus sampling (CVS) procedures performed in Australia from 1994 to 2014 shows that the introduction of cfDNA testing has been associated with the most rapid decline in invasive procedures in the last 20 years. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS. PMID:26259499

  14. National decline in invasive prenatal diagnostic procedures in association with uptake of combined first trimester and cell-free DNA aneuploidy screening.

    PubMed

    Robson, Stephen J; Hui, Lisa

    2015-10-01

    In late 2012, a new screening test for fetal aneuploidy based on circulating cell-free DNA (cfDNA) became available to Australian women. The introduction of this technology in the United States has led to a reduction in invasive diagnostic procedures. Analysis of the number of amniocentesis and chorionic villus sampling (CVS) procedures performed in Australia from 1994 to 2014 shows that the introduction of cfDNA testing has been associated with the most rapid decline in invasive procedures in the last 20 years. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS.

  15. Titration of human cytomegalovirus (HCMV) DNA in urine by combined use of PCR and microplate hybridization in a renal transplant patient with HCMV pneumonitis.

    PubMed

    Meigata, K; Hondo, R; Fujima, A; Shinkai-Shibata, M; Itoh, S; Kikuchi, K; Ando, Y; Ichikawa, N; Nomura, Y; Watanabe, K; Degawa, H; Beck, Y; Tomikawa, S; Nagao, T; Uchida, H

    1996-06-01

    We titrated human cytomegalovirus (HCMV) DNA in urine specimens obtained from 14 healthy individuals and a renal transplant patient with HCMV pneumonitis by modifying the method for titration of varicella-zoster virus DNA previously described (1,2). Of 14 HCMV seropositive healthy individuals, 13 had HCMV DNA under the detection limit of 10(2.0) copies/ml, whereas one person had 10(2.0) copies/ml. The viral DNA in urine samples was at a low level in healthy individuals with latent infection. In a case with HCMV pneumonitis after renal transplantation, the amount of HCMV DNA in urine gradually increased from the level under 10(2.0) copies/ml and reached a peak of 10(4.7) copies/ml one month prior to the manifestation of pneumonitis. It, thereafter, decreased with the course of clinical remission, and finally settled at under 10(2.0) copies/ml. Serial titrations of HCMV DNA in urine specimens proved to be useful in identifying recipients at risk of developing active HCMV infection after renal transplantation and as a guide for treatment of patients.

  16. Single and Combined Effects of Deoxynivalenol Mycotoxin and a Microbial Feed Additive on Lymphocyte DNA Damage and Oxidative Stress in Broiler Chickens

    PubMed Central

    Awad, Wageha A.; Ghareeb, Khaled; Dadak, Agnes; Hess, Michael; Böhm, Josef

    2014-01-01

    The immune and intestinal epithelial cells are particularly sensitive to the toxic effects of deoxynivalenol (DON). The aim of this experiment was to study the effects of DON and/or a microbial feed additive on the DNA damage of blood lymphocytes and on the level of thiobarbituric acid reactive substance (TBARS) as an indicator of lipid peroxidation and oxidative stress in broilers. A total of forty 1-d-old broiler chicks were randomly assigned to 1 of 4 dietary treatments (10 birds per group) for 5 wk. The dietary treatments were 1) basal diet; 2) basal diet contaminated with 10 mg DON/kg feed; 3) basal diet contaminated with 10 mg DON/kg feed and supplemented with 2.5 kg/ton of feed of Mycofix Select; 4) basal diet supplemented with Mycofix Select (2.5 kg/ton of feed). At the end of the feeding trial, blood were collected for measuring the level of lymphocyte DNA damage of blood and the TBARS level was measured in plasma, heart, kidney, duodenum and jejunum. The dietary exposure of DON caused a significant increase (P = 0.001) of DNA damage in blood lymphocytes (31.99±0.89%) as indicated in the tail of comet assay. Interestingly addition of Mycofix Select to DON contaminated diet decreased (P = 0.001) the DNA damage (19.82±1.75%) induced by DON. In order to clarify the involvement of lipid peroxidation in the DNA damage of DON, TBARS levels was measured. A significant increase (P = 0.001) in the level of TBARS (23±2 nmol/mg) was observed in the jejunal tissue suggesting that the lipid peroxidation might be involved in the DNA damage. The results indicate that DON is cytotoxic and genotoxic to the chicken intestinal and immune cells and the feed additive have potential ability to prevent DNA damage induced by DON. PMID:24498242

  17. An Enhanced Synthetic Multiclade DNA Prime Induces Improved Cross-Clade-Reactive Functional Antibodies when Combined with an Adjuvanted Protein Boost in Nonhuman Primates

    PubMed Central

    Wise, Megan C.; Hutnick, Natalie A.; Pollara, Justin; Myles, Devin J. F.; Williams, Constance; Yan, Jian; LaBranche, Celia C.; Khan, Amir S.; Sardesai, Niranjan Y.; Montefiori, David; Barnett, Susan W.; Zolla-Pazner, Susan; Ferrari, Guido

    2015-01-01

    ABSTRACT The search for an efficacious human immunodeficiency virus type 1 (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1-specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small-animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4+ and CD8+ T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA-only or protein-only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding and neutralizing and antibody-dependent cellular cytotoxicity (ADCC) activity after immunization with the DNA prime-protein boost regimen, with the multiple-Env formulation inducing a more robust and broader response than the single-Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce, which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses. IMPORTANCE Even with effective antiretroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine

  18. SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

    PubMed

    Nawattanapaiboon, Kawin; Kiatpathomchai, Wansika; Santanirand, Pitak; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Srikhirin, Toemsak

    2015-12-15

    In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.

  19. Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes.

    PubMed

    Rand, Keith N; Young, Graeme P; Ho, Thu; Molloy, Peter L

    2013-01-01

    We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed 'helper' oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called 'helper-dependent chain reaction'. The process uses disabled primers called 'drivers' that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.

  20. Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.

    PubMed

    Nakanishi, Hideyuki; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2014-09-30

    PhiC31 integrase-based vectors can integrate therapeutic genes selectively into attP or pseudo-attP sites in genomes, but considerable numbers of pseudo-attP sites in human genomes exist inside endogenous gene-coding regions. To avoid endogenous gene disruptions, we aimed to enhance the integration site-specificity of the phiC31 integrase-based vector using a sequence-specific DNA-binding protein containing Gal4 and LexA DNA-binding motifs. The dual DNA-binding protein was designed to tether the UAS-containing donor vector to the target sequence, the LexA operator, and restrict integration to sites close to the LexA operator. To analyze the site-specificity in chromosomal integration, a human cell line having LexA operators on the genome was established, and the cell line was transfected with donor vectors expressing the DNA-binding protein and the phiC31 integrase expression vector (helper vector). Quantitative PCR indicated that integration around the LexA operator was 26-fold higher with the UAS-containing donor vector than with the control. Sequence analysis confirmed that the integration occurred around the LexA operator. The dual DNA-binding protein-based targeted integration strategy developed herein would allow safer and more reliable genetic manipulations for various applications, including gene and cell therapies.

  1. A combined computational and experimental study on DNA-photocleavage of Ru(II) polypyridyl complexes [Ru(bpy)2(L)]2+ (L = pip, o-mopip and p-mopip).

    PubMed

    Xu, Lian-Cai; Shi, Shuo; Li, Jun; Liao, Si-Yan; Zheng, Kang-Cheng; Ji, Liang-Nian

    2008-01-14

    A combined computational and experimental study on DNA-photocleavage by Ru(II) polypyridyl complexes [Ru(bpy)2(L)]2+ 1-3 (bpy = 2,2-bipyridine; L: pip = 2-phenylimidazo[4,5-f]1,10-phenanthroline, o-mopip = 2-(2-methoxyphenyl)imidazo[4,5-f]1,10-phenanthroline and p-mopip = 2-(4-methoxyphenyl)imidazo[4,5-f]1,10-phenanthroline) has been carried out. The DNA-photocleavage behavior of these complexes was comparably measured by the gel electrophoresis experiments. The experimental results show that they can induce considerable DNA-photocleavage, and have different DNA-photocleavage efficiencies (phi) following the order phi (1) < phi (2) < phi (3). In order to understand their DNA-photocleavage mechanism and trend, the theoretical studies on the geometric and electronic structures of these complexes in the ground state (S0), the first singlet excited state (S1) and triplet excited states (T1), have been carried out using the density functional theory (DFT/TD-DFT), Hartree-Fock (HF) and configuration interaction singles (CIS) methods. In particular, the reduction potentials (E*red) of the excited complexes in aqueous solution, which seem to be closely responsible for the DNA-photocleavage behavior, were calculated to be 0.966 V (vs. SCE) for complex , 1.024 V (vs. SCE) for complex and 1.030 V (vs. SCE) for complex , respectively. Such computational results show that the reduction potentials of the excited complexes reach the theoretical range for oxidizing some DNA-bases, and follow the order E*red (1) < E*red (2) < E*red (3). Therefore, here, in addition to the general theoretical explanation of their DNA-photocleavage mechanism according to our recent report, a further explanation on the trend of their DNA-photocleavage efficiencies, i.e., phi (1) < phi (2) < phi (3), was reasonably carried out, on the basis of the calculated electrochemical properties in the excited states as well as general photochemical insights. PMID:18097496

  2. Solid-phase based on-chip DNA purification through a valve-free stepwise injection of multiple reagents employing centrifugal force combined with a hydrophobic capillary barrier pressure.

    PubMed

    Zhang, Hainan; Tran, Hong Hanh; Chung, Bong Hyun; Lee, Nae Yoon

    2013-03-21

    In this paper, we demonstrate a simple technique for sequentially introducing multiple sample liquids into microchannels driven by centrifugal force combined with a hydrophobic barrier pressure and apply the technique for performing solid-phase based on-chip DNA purification. Three microchannels with varying widths, all equipped with independent sample reservoirs at the inlets, were fabricated on a hydrophobic elastomer, poly(dimethylsiloxane) (PDMS). First, glass beads were packed inside the reaction chamber, and a whole cell containing the DNA extract was introduced into the widest channel by applying centrifugal force for physical adsorption of the DNA onto the glass beads. Next, washing and elution solutions were sequentially introduced into the intermediate and narrowest microchannels, respectively, by gradually increasing the amount of centrifugal force. Through a precise manipulation of the centrifugal force, the DNA adsorbed onto the glass beads was successfully washed and eluted in a continuous manner without the need to introduce each solution manually. A stepwise injection of liquids was successfully demonstrated using multiple ink solutions, the results of which corresponded well with the theoretical analyses. As a practical application, the D1S80 locus of human genomic DNA, which is widely used for forensic purposes, was successfully purified using the microdevice introduced in this study, as demonstrated through successful target amplification. This will pave the way for the construction of a control-free valve system for realizing on-chip DNA purification, which is one of the most labor-intensive and hard-to-miniaturize components, on a greatly simplified and miniaturized platform employing hydrophobic PDMS.

  3. Genetic variants in DNA repair pathways and risk of upper aerodigestive tract cancers: combined analysis of data from two genome-wide association studies in European populations.

    PubMed

    Babron, Marie-Claude; Kazma, Rémi; Gaborieau, Valérie; McKay, James; Brennan, Paul; Sarasin, Alain; Benhamou, Simone

    2014-07-01

    DNA repair pathways are good candidates for upper aerodigestive tract cancer susceptibility because of their critical role in maintaining genome integrity. We have selected 13 pathways involved in DNA repair representing 212 autosomal genes. To assess the role of these pathways and their associated genes, two European data sets from the International Head and Neck Cancer Epidemiology consortium were pooled, totaling 1954 cases and 3121 controls, with documented demographic, lifetime alcohol and tobacco consumption information. We applied an innovative approach that tests single nucleotide polymorphism (SNP)-sets within DNA repair pathways and then within genes belonging to the significant pathways. We showed an association between the polymerase pathway and oral cavity/pharynx cancers (P-corrected = 4.45 × 10(-) (2)), explained entirely by the association with one SNP, rs1494961 (P = 2.65 × 10(-) (4)), a missense mutation V306I in the second exon of HELQ gene. We also found an association between the cell cycle regulation pathway and esophagus cancer (P-corrected = 1.48 × 10(-) (2)), explained by three SNPs located within or near CSNK1E gene: rs1534891 (P = 1.27 × 10(-) (4)), rs7289981 (P = 3.37 × 10(-) (3)) and rs13054361 (P = 4.09 × 10(-) (3)). As a first attempt to investigate pathway-level associations, our results suggest a role of specific DNA repair genes/pathways in specific upper aerodigestive tract cancer sites. PMID:24658182

  4. Using the ubiquitous pH meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1.

    PubMed

    Xie, Shunbi; Yuan, Yali; Song, Yue; Zhuo, Ying; Li, Tian; Chai, Yaqin; Yuan, Ruo

    2014-12-28

    Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time.

  5. Forensic DNA profiling and database.

    PubMed

    Panneerchelvam, S; Norazmi, M N

    2003-07-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection.

  6. Archaeal DNA polymerases in biotechnology.

    PubMed

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Huang, Yanchao

    2015-08-01

    DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

  7. Simultaneous RNA-DNA FISH.

    PubMed

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

  8. Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

    PubMed Central

    Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

    2001-01-01

    The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

  9. Combined exercise and insulin-like growth factor-1 supplementation induces neurogenesis in old rats, but do not attenuate age-associated DNA damage.

    PubMed

    Koltai, Erika; Zhao, Zhongfu; Lacza, Zsombor; Cselenyak, Attila; Vacz, Gabriella; Nyakas, Csaba; Boldogh, Istvan; Ichinoseki-Sekine, Noriko; Radak, Zsolt

    2011-12-01

    We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 μg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO₂ max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.

  10. Combined QM/MM investigation on the light-driven electron-induced repair of the (6-4) thymine dimer catalyzed by DNA photolyase.

    PubMed

    Faraji, Shirin; Groenhof, Gerrit; Dreuw, Andreas

    2013-09-01

    The (6-4) photolyases are blue-light-activated enzymes that selectively bind to DNA and initiate splitting of mutagenic thymine (6-4) thymine photoproducts (T(6-4)T-PP) via photoinduced electron transfer from flavin adenine dinucleotide anion (FADH(-)) to the lesion triggering repair. In the present work, the repair mechanism after the initial electron transfer and the effect of the protein/DNA environment are investigated theoretically by means of hybrid quantum mechanical/molecular mechanical (QM/MM) simulations using X-ray structure of the enzyme-DNA complex. By comparison of three previously proposed repair mechanisms, we found that the lowest activation free energy is required for the pathway in which the key step governing the repair photocycle is electron transfer coupled with the proton transfer from the protonated histidine, His365, to the N3' nitrogen of the pyrimidone thymine. The transfer simultaneously occurs with concerted intramolecular OH transfer without formation of an oxetane or isolated water molecule intermediate. In contrast to previously suggested mechanisms, this newly identified pathway requires neither a subsequent two-photon process nor electronic excitation of the photolesion.

  11. Complex kinetics of DNA condensation revealed through DNA twist tracing.

    PubMed

    Li, Wei; Wong, Wei Juan; Lim, Ci Ji; Ju, Hai-Peng; Li, Ming; Yan, Jie; Wang, Peng-Ye

    2015-08-01

    Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

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

    PubMed Central

    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. PMID:25692233

  13. Combination of carbon ion beam and gemcitabine causes irreparable DNA damage and death of radioresistant pancreatic cancer stem-like cells in vitro and in vivo

    PubMed Central

    Sai, Sei; Wakai, Toshifumi; Vares, Guillaume; Yamada, Shigeru; Kamijo, Takehiko; Kamada, Tadashi; Shirai, Toshiyuki

    2015-01-01

    We try to elucidate whether a carbon ion beam alone or in combination with gemcitabine has advantages over X-ray in targeting putative pancreatic cancer stem-like cells (CSCs) in vitro and in vivo. Colony, spheroid formation and tumorigenicity assays confirmed that CD44+/ESA+ cells sorted from PANC1 and PK45 cells have more CSC properties than CD44−/ESA− cells. The number of colonies and spheroids formed from CSCs after carbon ion beam irradiation was significantly reduced compared to after X-ray irradiation, and they were extremely highly suppressed when carbon ion beam combined with gemcitabine. The relative biological effectiveness (RBE) values for the carbon ion beam relative to X-ray at the D10 levels for CSCs were 2.23-2.66. Expressions of multiple cell death-related genes were remarkably highly induced, and large numbers of γH2AX foci in CSCs were formed after carbon ion beam combined with gemcitabine. The highly expressed CSC markers were significantly inhibited after 30 Gy of carbon ion beam and almost lost after 25 Gy carbon ion beam combined with 50 mg/kg gemcitabine. In conclusion, a carbon ion beam combined with gemcitabine has superior potential to kill pancreatic CSCs via irreparable clustered DSB compared to a carbon ion alone or X-rays combined with gemcitabine. PMID:25849939

  14. Enhancement of Blood–Brain Barrier Permeability and Delivery of Antisense Oligonucleotides or Plasmid DNA to the Brain by the Combination of Bubble Liposomes and High-Intensity Focused Ultrasound

    PubMed Central

    Negishi, Yoichi; Yamane, Masaya; Kurihara, Naho; Endo-Takahashi, Yoko; Sashida, Sanae; Takagi, Norio; Suzuki, Ryo; Maruyama, Kazuo

    2015-01-01

    The blood–brain barrier (BBB) is a major obstacle that prevents therapeutic drugs or genes from being delivered to the central nervous system. Therefore, it is important to develop methods to enhance the permeability of the BBB. We have developed echo-contrast gas (C3F8) entrapping liposomes (Bubble liposomes, BLs) that can work as a gene delivery tool in combination with ultrasound (US) exposure. Here, we studied whether the permeability of the BBB can be enhanced by the combination of BLs and high-intensity focused ultrasound (HIFU). Mice were intravenously injected with Evans blue (EB). BLs were subsequently injected, and the right hemispheres were exposed to HIFU. As a result, the accumulation of EB in the HIFU-exposed brain hemispheres was increased over that observed in the non-HIFU-exposed hemispheres, depending on the intensity and the duration of the HIFU. Similarly, the combination of BLs and HIFU allowed fluorescent-labeled antisense oligonucleotides to be delivered into the HIFU-exposed left hemispheres of the treated mice. Furthermore, a firefly luciferase-expressing plasmid DNA was delivered to the brain by the combination method of BLs and HIFU, which resulted in the increased gene expression in the brain at the focused-US exposure site. These results suggest that the method of combining BLs and HIFU together serves as a useful means for accelerating the permeability of BBB and thereby enabling antisense oligonucleotides or genes to be delivered to the focused brain site. PMID:26402694

  15. DNA origami nanopores.

    PubMed

    Bell, Nicholas A W; Engst, Christian R; Ablay, Marc; Divitini, Giorgio; Ducati, Caterina; Liedl, Tim; Keyser, Ulrich F

    2012-01-11

    We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.

  16. Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction.

    PubMed

    Kajiyama, Tomoharu; Fujii, Akihiko; Arikawa, Kouji; Habu, Toru; Mochizuki, Nobuyoshi; Nagatani, Akira; Kambara, Hideki

    2015-07-01

    Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts.

  17. Isolation and identification of spoilage microorganisms using food-based media combined with rDNA sequencing: ranch dressing as a model food.

    PubMed

    Waite, Joy G; Jones, Joseph M; Yousef, Ahmed E

    2009-05-01

    Investigating microbial spoilage of food is hampered by the lack of suitable growth media and protocols to characterize the causative agents. Microbial spoilage of salad dressing is sporadic and relatively unpredictable, thus processors struggle to develop strategies to minimize or prevent spoilage of this product. The objectives of this study were to (i) induce and characterize spoilage events in ranch-style dressing as a model food, and (ii) isolate and identify the causative microorganisms using traditional and food-based media, coupled with rDNA sequence analysis. Ranch dressing (pH 4.4) was prepared and stored at 25 degrees C for 14 d and microbial populations were recovered on MRS agar and ranch dressing agar (RDA), a newly formulated food-based medium. When isolates suspected as the spoilage agents were inoculated into ranch dressing and held at 25 degrees C for 9-10 d, three unique spoilage events were characterized. Using rDNA sequence comparisons, spoilage organisms were identified as Lactobacillus brevis, Pediococcus acidilactici, and Torulaspora delbrueckii. P. acidilactici produced flat-sour spoilage, whereas Lb. brevis resulted in product acidification and moderate gas production. The RDA medium allowed for optimum recovery of the excessive gas-producing spoilage yeast, T. delbrueckii. The isolation and identification strategy utilized in this work should assist in the characterization of spoilage organisms in other food systems.

  18. Improved Immunogenicity of a Vaccination Regimen Combining a DNA Vaccine Encoding Brucella melitensis Outer Membrane Protein 31 (Omp31) and Recombinant Omp31 Boosting▿

    PubMed Central

    Cassataro, Juliana; Velikovsky, Carlos A.; Bruno, Laura; Estein, Silvia M.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2007-01-01

    In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis. PMID:17428946

  19. Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction.

    PubMed

    Kajiyama, Tomoharu; Fujii, Akihiko; Arikawa, Kouji; Habu, Toru; Mochizuki, Nobuyoshi; Nagatani, Akira; Kambara, Hideki

    2015-07-01

    Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts. PMID:26092972

  20. Combined analysis of gene expression, DNA copy number, and mutation profiling data to display biological process anomalies in individual breast cancers.

    PubMed

    Shi, Weiwei; Balazs, Balint; Györffy, Balazs; Jiang, Tingting; Symmans, W Fraser; Hatzis, Christos; Pusztai, Lajos

    2014-04-01

    The goal of this analysis was to develop a computational tool that integrates the totality of gene expression, DNA copy number, and sequence abnormalities in individual cancers in the framework of biological processes. We used the hierarchical structure of the gene ontology (GO) database to create a reference network and projected mRNA expression, DNA copy number and mutation anomalies detected in single samples into this space. We applied our method to 59 breast cancers where all three types of molecular data were available. Each cancer had a large number of disturbed biological processes. Locomotion, multicellular organismal process, and signal transduction pathways were the most commonly affected GO terms, but the individual molecular events were different from case-to-case. Estrogen receptor-positive and -negative cancers had different repertoire of anomalies. We tested the functional impact of 27 mRNAs that had overexpression in cancer with variable frequency (<2-42 %) using an siRNA screen. Each of these genes inhibited cell growth in at least some of 18 breast cancer cell lines. We developed a free, on-line software tool ( http://netgoplot.org ) to display the complex genomic abnormalities in individual cancers in the biological framework of the GO biological processes. Each cancer harbored a variable number of pathway anomalies and the individual molecular events that caused an anomaly varied from case-to-case. Our in vitro experiments indicate that rare case-specific molecular abnormalities can play a functional role and driver events may vary from case-to-case depending on the constellation of other molecular anomalies.

  1. Evaluation of the Combined Effect of 2ME2 and 60Co on the Inducement of DNA Damage by IUdR in a Spheroid Model of the U87MG Glioblastoma Cancer Cell Line Using Alkaline Comet Assay

    PubMed Central

    Khoei, Samideh; Delfan, Sara; Neshasteh-Riz, Ali; Mahdavi, Seyed Rabi

    2011-01-01

    Objective: In this study, we investigated the combined effect of 2-Methoxyestradiol (2ME2) and 60Co on the cytogenetic damage of iododeoxyuridine (IUdR) in the spheroid model of U87MG glioblastoma cancer cell lines by alkaline comet assay. Materials and Methods: U87MG cells were cultured as spheroids with diameters of 350 µm. As control, the spheroids of one plate were not treated. Other cultures were pretreated with 2ME2 (250 µM) for one volume doubling time (1 VDT). After this time, the subsequent treatments were performed according to the following groups: Vehicle (this sample was not treated in the 2nd VDT) Treated with 2ME2 (250 µM) for 1 VDT Treated simultaneously with 2ME2 (250 µM) and IUdR (1 µM) for 1 VDT Treated with 2ME2 (250 µM) for 1 VDT then irradiated with 60Co (2 Gy) Treated simultaneously with 2ME2 (250 µM) and IUdR (1 µM) for 1 VDT then irradiated with 60Co (2 Gy) Then the DNA damage was evaluated using the alkaline comet assay method. Results: The results showed that 2ME2 in combination with gamma irradiation of 60Co significantly (p<0.001) increased the DNA damage by IUdR as compared to the control group. Thus the combination of these two agents increased the cytogenetic effects of IUdR in the spheroid culture model of U87MG glioblastoma cell lines. Conclusion: By inhibiting the HIF-1α protein and preventing the G0 phase arrest, 2ME2 causes an increased progression into S phase and increases the IUdR absorption. Then the DNA damage in the spheroid cells increases as the uptake of IUdR is increased. These results suggest that the combined use of 2ME2 and 60Co can increase the radiosensitization effect of IUdR. PMID:23508289

  2. Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

    PubMed

    Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

    2016-05-15

    In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future. PMID:26686922

  3. A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

    PubMed

    Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

    2013-01-14

    In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

  4. A contribution to the taxonomy of the genus Rinodina (Physciaceae, lichenized Ascomycotina) using combined ITS and mtSSU rDNA data

    PubMed Central

    NADYEINA, Olga; GRUBE, Martin; MAYRHOFER, Helmut

    2011-01-01

    To test the phylogenetic position of phenotypically peculiar species in the Physciaceae we generated 47 new sequences (26 of nrITS region and 21 of mtSSU rDNA) from 19 crustose taxa of Physciaceae mainly from the genus Rinodina. Phylogenetic analysis confirmed the Buellia and Physcia groups. The analysis revealed a considerable variability of characters traditionally used for classification, especially in the delimitation of the genera Buellia and Rinodina. While ascus types agree well with the distinction of the Buellia and Physcia groups, none of the other traditional characters, including excipulum type and ascospore thickening, were consistent within subclades of the Physcia group. We suggest that both excipulum type and ascospore characters are rather dynamic in the evolution of Rinodina species and only appear consistent in morphologically more complex foliose and fruticose groups, which are characterized by thallus characters not present in the crustose groups. Two recent taxonomic changes are supported by molecular characters: Endohyalina insularis (syn. ‘Rinodina’ insularis) and Rinodina lindingeri (syn. ‘Buellia’ lindingeri). In addition Rinodina parvula (syn. ‘Buellia’ parvula) is reinstated. New records for Endohyalina brandii, E. diederichii, E. insularis and Rinodina albana are presented. PMID:22121298

  5. Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae): the first eukaryotic species description combining transcriptomic, DNA barcoding and micro-CT imaging data.

    PubMed

    Stoev, Pavel; Komerički, Ana; Akkari, Nesrine; Liu, Shanlin; Zhou, Xin; Weigand, Alexander M; Hostens, Jeroen; Hunter, Christopher I; Edmunds, Scott C; Porco, David; Zapparoli, Marzio; Georgiev, Teodor; Mietchen, Daniel; Roberts, David; Faulwetter, Sarah; Smith, Vincent; Penev, Lyubomir

    2013-01-01

    We demonstrate how a classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. A cave-dwelling centipede, Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae), found in a remote karst region in Knin, Croatia, is the first eukaryotic species for which, in addition to the traditional morphological description, we provide a fully sequenced transcriptome, a DNA barcode, detailed anatomical X-ray microtomography (micro-CT) scans, and a movie of the living specimen to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a new species for the first time, we create a high-resolution morphological and anatomical dataset that allows virtual reconstructions of the specimen and subsequent interactive manipulation to test the recently introduced 'cybertype' notion. In addition, the transcriptome was recorded with a total of 67,785 scaffolds, having an average length of 812 bp and N50 of 1,448 bp (see GigaDB). Subsequent annotation of 22,866 scaffolds was conducted by tracing homologs against current available databases, including Nr, SwissProt and COG. This pilot project illustrates a workflow of producing, storing, publishing and disseminating large data sets associated with a description of a new taxon. All data have been deposited in publicly accessible repositories, such as GigaScience GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability. PMID:24723752

  6. A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods.

    PubMed

    Zhang, Ai-bing; Feng, Jie; Ward, Robert D; Wan, Ping; Gao, Qiang; Wu, Jun; Zhao, Wei-zhong

    2012-01-01

    Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.

  7. A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola, the septoria tritici leaf blotch pathogen of wheat.

    PubMed Central

    Kema, Gert H J; Goodwin, Stephen B; Hamza, Sonia; Verstappen, Els C P; Cavaletto, Jessica R; Van der Lee, Theo A J; de Weerdt, Marjanne; Bonants, Peter J M; Waalwijk, Cees

    2002-01-01

    An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae. PMID:12196395

  8. Comparison and combination of blood DNA methylation at smoking-associated genes and at lung cancer-related genes in prediction of lung cancer mortality.

    PubMed

    Zhang, Yan; Breitling, Lutz P; Balavarca, Yesilda; Holleczek, Bernd; Schöttker, Ben; Brenner, Hermann

    2016-12-01

    Epigenome-wide association studies have established methylation patterns related to smoking, the major risk factor of lung cancer (LC), which are distinct from methylation profiles disclosed in LC patients. This study simultaneously investigated associations of smoking-associated and LC-related methylation markers with LC mortality. DNA methylation was determined by HM450K assay in baseline blood samples of 1,565 older adults in a population-based case-cohort study. The associations of 151 smoking-associated CpGs (smoCpGs) and 3,806 LC-related CpGs (caCpGs) with LC mortality were assessed by weighted Cox regression models, controlling for potential confounders. Multi-loci methylation scores were separately constructed based on smoCpGs and caCpGs. During a median follow-up of 13.8 years, 60 participants who had a first diagnosis of LC died from LC. The average time between sample collection and LC diagnosis was 5.8 years. Hypomethylation at 77 smoCpGs and 121 caCpGs, and hypermethylation at 4 smoCpGs and 66 caCpGs were associated with LC mortality. The associations were much stronger for smoCpGs than for caCpGs. Hazard ratios (95% CI) were 7.82 (2.91-21.00) and 2.27 (0.75-6.85), respectively, for participants in highest quartile of Score I (based on 81 smoCpGs) and Score II (based on 187 caCpGs), compared with participants in the corresponding lower three quartiles. Score I outperformed Score II, with an optimism-corrected C-index of 0.87 vs. 0.77. In conclusion, although methylation changes of both smoking-associated and LC-related genes are associated with LC mortality, only smoking-associated methylation markers predict LC mortality with high accuracy, and may thus serve as promising candidates to identify high risk populations for LC screening. PMID:27503000

  9. Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles

    PubMed Central

    BAI, WEN-KUN; ZHANG, WEI; HU, BING; YING, TAO

    2016-01-01

    Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. PMID:27313702

  10. Close linkage of the locus for X chromosome-linked severe combined immunodeficiency to polymorphic DNA markers in Xq11-q13

    SciTech Connect

    de Saint Basile, G.; Arveiler, B.; Oberle, I.; Malcolm, S.; Levinsky, R.J.; Lau, Y.L.; Hofker, M.; Debre, M.; Fischer, A.; Griscelli, C.; Mandel, J.L.

    1987-11-01

    The gene for X chromosome-linked severe combined immunodeficiency (SCID), a disease characterized by a block in early T-cell differentiation, has been mapped to the region Xq11-q13 by linkage analysis with restriction fragment length polymorphisms. High logarithm of odds (lod) scores were obtained with the marker 19.2 (DXS3) and with the marker cpX73 (DXS159) that showed complete cosegregation with the disease locus in the informative families analyzed. Other significant linkages were obtained with several markers from Xq11 to q22. With the help of a recently developed genetic map of the region, it was possible to perform multipoint linkage analysis, and the most likely genetic order is DXS1-(SCID, DXS159)-DXYS1-DXYS12-DXS3, with a maximum multipoint logarithm of odds score of 11.0. The results demonstrate that the SCID locus (gene symbol IMD4) is not closely linked to the locus of Bruton's agammaglobulinemia (a defect in B-cell maturation). They also provide a way for a better estimation of risk for carrier and antenatal diagnosis.

  11. Combining Push Pull Tracer Tests and Microbial DNA and mRNA Analysis to Assess In-Situ Groundwater Nitrate Transformations

    NASA Astrophysics Data System (ADS)

    Henson, W.; Graham, W. D.; Huang, L.; Ogram, A.

    2015-12-01

    Nitrogen transformation mechanisms in the Upper Floridan Aquifer (UFA) are still poorly understood because of karst aquifer complexity and spatiotemporal variability in nitrate and carbon loading. Transformation rates have not been directly measured in the aquifer. This study quantifies nitrate-nitrogen transformation potential in the UFA using single well push-pull tracer injection (PPT) experiments combined with microbial characterization of extracted water via qPCR and RT-qPCR of selected nitrate reduction genes. Tracer tests with chloride and nitrate ± carbon were executed in two wells representing anoxic and oxic geochemical end members in a spring groundwater contributing area. A significant increase in number of microbes with carbon addition suggests stimulated growth. Increases in the activities of denitrification genes (nirK and nirS) as measured by RT-qPCR were not observed. However, only microbes suspended in the tracer were obtained, ignoring effects of aquifer material biofilms. Increases in nrfA mRNA and ammonia concentrations were observed, supporting Dissimilatory Reduction of Nitrate to Ammonia (DNRA) as a reduction mechanism. In the oxic aquifer, zero order nitrate loss rates ranged from 32 to 89 nmol /L*hr with no added carbon and 90 to 240 nmol /L*hr with carbon. In the anoxic aquifer, rates ranged from 18 to 95 nmol /L*hr with no added carbon and 34 to 207 nmol /L*hr with carbon. These loss rates are low; 13 orders of magnitude less than the loads applied in the contributing area each year, however they do indicate that losses can occur in oxic and anoxic aquifers with and without carbon. These rates may include, ammonia adsorption, uptake, or denitrification in aquifer material biofilms. Rates with and without carbon addition for both aquifers were similar, suggesting aquifer redox state and carbon availability alone are insufficient to predict response to nutrient additions without characterization of microbial response. Surprisingly, these

  12. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    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)

  13. COMBINES AND COMBINING.

    ERIC Educational Resources Information Center

    RIDENOUR, HARLAN E.

    THROUGH THE USE OF THIS MANUAL, VOCATIONAL AGRICULTURE STUDENTS WITH OCCUPATIONAL INTEREST IN GRAIN FARMING AND CUSTOM COMBINE OPERATION MAY GAIN KNOWLEDGE ABOUT THE BASIC DESIGN AND OPERATION OF COMBINES. DEVELOPMENT BY A STATE CURRICULUM MATERIALS DIRECTOR INCLUDED CONSULTATION WITH ENGINEERS, TRIAL, AND REVISION. OBJECTIVES ARE STATED IN TERMS…

  14. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  15. Dna Sequencing

    DOEpatents

    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.

  16. Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution

    PubMed Central

    Morgan, Hugh; Incorvaia, Elisabetta; Rangam, Gopinath; Dean, Wendy; Santos, Fatima; Reik, Wolf; Petersen-Mahrt, Svend K.

    2014-01-01

    Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated. PMID:25025377

  17. Biosensors for DNA sequence detection

    NASA Technical Reports Server (NTRS)

    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.

  18. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review.

  19. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review

  20. Effects of oxaliplatin on DNA condensation

    NASA Astrophysics Data System (ADS)

    Ju, HaiPeng; Zhang, HongYan; Li, Wei; Wang, PengYe

    2014-11-01

    In this paper the interactions between DNA and anti-cancer drug oxaliplatin were investigated by using magnetic tweezers. The dynamics of DNA condensation due to oxaliplatin was traced under various forces. It is found that torsion constraint in DNA enhances the ability of oxaliplatin for shortening DNA. The transplatin helps oxaliplatin combine to DNA and increase the rate of DNA condensation. All these results are consistent to the previously proposed model and are helpful for further investigation of interaction between DNA and oxaliplatin.

  1. Individual and Combined Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predict Shorter Survival of Soft Tissue Sarcoma Patients

    PubMed Central

    Park, See-Hyoung; Park, Hye Jeong; Wang, Sung Il; Park, Ho Sung; Lee, Ho; Kwon, Keun Sang; Moon, Woo Sung; Lee, Dong Geun; Kim, Jung Ryul; Jang, Kyu Yun

    2016-01-01

    DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, γH2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1, γH2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and γH2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, γH2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P < 0.001) and EFS (P = 0.016). The ten-year DSS rate of the CSddrm-low, CSddrm-intermediate, and CSddrm-high subgroups were 81%, 26%, and 0%, respectively. In conclusion, this study demonstrates that the individual and combined expression patterns of the DDR molecules PARP1, γH2AX, BRCA1, and BRCA2 could be predictive of the prognosis of STS patients and suggests that controlling the activity of these DDR molecules could be employed in new therapeutic stratagems for the treatment of STS. PMID:27643881

  2. Individual and Combined Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predict Shorter Survival of Soft Tissue Sarcoma Patients.

    PubMed

    Kim, Kyoung Min; Moon, Young Jae; Park, See-Hyoung; Park, Hye Jeong; Wang, Sung Il; Park, Ho Sung; Lee, Ho; Kwon, Keun Sang; Moon, Woo Sung; Lee, Dong Geun; Kim, Jung Ryul; Jang, Kyu Yun

    2016-01-01

    DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, γH2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1, γH2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and γH2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, γH2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P < 0.001) and EFS (P = 0.016). The ten-year DSS rate of the CSddrm-low, CSddrm-intermediate, and CSddrm-high subgroups were 81%, 26%, and 0%, respectively. In conclusion, this study demonstrates that the individual and combined expression patterns of the DDR molecules PARP1, γH2AX, BRCA1, and BRCA2 could be predictive of the prognosis of STS patients and suggests that controlling the activity of these DDR molecules could be employed in new therapeutic stratagems for the treatment of STS. PMID:27643881

  3. Hypomethylation and up-regulation of PD-1 in T cells by azacytidine in MDS/AML patients: A rationale for combined targeting of PD-1 and DNA methylation.

    PubMed

    Ørskov, Andreas D; Treppendahl, Marianne B; Skovbo, Anni; Holm, Mette S; Friis, Lone S; Hokland, Marianne; Grønbæk, Kirsten

    2015-04-20

    The hypomethylating agents (HMAs) are standard therapy for patients with higher-risk myelodysplastic syndrome (MDS); however, the majority of the patients will lose their response to HMAs over time due to unknown mechanisms. It has recently been shown that T cell expression of the immunoinhibitory receptor PD-1 is regulated by DNA methylation. In 12 of 27 patients (44%) PD-1 promoter demethylation was observed in sorted peripheral blood T cells isolated over consecutive cycles of treatment with 5-azacytidine (5-aza). The PD-1 promoter demethylation correlated with an increase in PD-1 expression. Moreover, demethylation of the PD-1 promoter correlated with a significantly worse overall response rate (8% vs. 60%, p = 0.014), and a trend towards a shorter overall survival (p = 0.11) was observed. A significantly higher baseline methylation level of the PD-1 promoter was observed in T cells of non-responding patients compared to healthy controls (p = 0.023). Accordingly, in addition to their beneficial function, HMAs induce PD-1 expression on T cells in the MDS microenvironment, thereby likely hampering the immune response against the MDS blasts. Thus, we suggest that activation of the PD-1 checkpoint during HMA treatment can be a possible resistance mechanism, which may be overcome by combination therapy with a PD-1 pathway inhibitor. PMID:25823822

  4. DNA-PK assay

    DOEpatents

    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.

  5. Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-{kappa}B-STAT3-directed gene expression

    SciTech Connect

    Ivanov, Vladimir N. Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

    2011-07-01

    Mitochondrial DNA depleted ({rho}{sup 0}) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-{kappa}B and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental {rho}{sup +} HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in {rho}{sup 0} cells compared to {rho}{sup +} HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, {Oota}L17{Beta}, {Oota}L18, {Oota}L19, and {Oota}L28{Beta}) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-{kappa}B and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-{kappa}B/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in {rho}{sup +} HSF, but this response was substantially decreased in {rho}{sup 0} HSF. Suppression of the IKK-NF-{kappa}B pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated {rho}{sup +} HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-{kappa}B activation was partially lost in {rho}{sup 0} HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-{kappa}B targets, further suppressing IL6

  6. DNA nanomachines.

    PubMed

    Bath, Jonathan; Turberfield, Andrew J

    2007-05-01

    We are learning to build synthetic molecular machinery from DNA. This research is inspired by biological systems in which individual molecules act, singly and in concert, as specialized machines: our ambition is to create new technologies to perform tasks that are currently beyond our reach. DNA nanomachines are made by self-assembly, using techniques that rely on the sequence-specific interactions that bind complementary oligonucleotides together in a double helix. They can be activated by interactions with specific signalling molecules or by changes in their environment. Devices that change state in response to an external trigger might be used for molecular sensing, intelligent drug delivery or programmable chemical synthesis. Biological molecular motors that carry cargoes within cells have inspired the construction of rudimentary DNA walkers that run along self-assembled tracks. It has even proved possible to create DNA motors that move autonomously, obtaining energy by catalysing the reaction of DNA or RNA fuels.

  7. Cryptography with DNA binary strands.

    PubMed

    Leier, A; Richter, C; Banzhaf, W; Rauhe, H

    2000-06-01

    Biotechnological methods can be used for cryptography. Here two different cryptographic approaches based on DNA binary strands are shown. The first approach shows how DNA binary strands can be used for steganography, a technique of encryption by information hiding, to provide rapid encryption and decryption. It is shown that DNA steganography based on DNA binary strands is secure under the assumption that an interceptor has the same technological capabilities as sender and receiver of encrypted messages. The second approach shown here is based on steganography and a method of graphical subtraction of binary gel-images. It can be used to constitute a molecular checksum and can be combined with the first approach to support encryption. DNA cryptography might become of practical relevance in the context of labelling organic and inorganic materials with DNA 'barcodes'.

  8. DNA extraction, preservation, and amplification.

    PubMed

    Knebelsberger, Thomas; Stöger, Isabella

    2012-01-01

    The effectiveness of DNA barcoding as a routine practice in biodiversity research is strongly dependent on the quality of the source material, DNA extraction method, and selection of adequate primers in combination with optimized polymerase chain reaction (PCR) conditions. For the isolation of nucleic acids, silica-gel membrane methods are to be favored because they are easy to handle, applicable for high sample throughput, relatively inexpensive, and provide high DNA quality, quantity, and purity which are pre-requisites for successful PCR amplification and long-term storage of nucleic acids in biorepositories, such as DNA banks. In this section, standard protocols and workflow schemes for sample preparation, DNA isolation, DNA storage, PCR amplification, PCR product quality control, and PCR product cleanup are proposed and described in detail. A PCR troubleshooting and primer design section may help to solve problems that hinder successful amplification of the desired barcoding gene region.

  9. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  10. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    SciTech Connect

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  11. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  12. DNA Dynamics.

    ERIC Educational Resources Information Center

    Warren, Michael D.

    1997-01-01

    Explains a method to enable students to understand DNA and protein synthesis using model-building and role-playing. Acquaints students with the triplet code and transcription. Includes copies of the charts used in this technique. (DDR)

  13. DNA Adductomics

    PubMed Central

    2015-01-01

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the 32P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC–MSn), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC–MSn instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology. PMID:24437709

  14. Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma.

    PubMed

    Azzariti, Amalia; Brunetti, Oronzo; Porcelli, Letizia; Graziano, Giusi; Iacobazzi, Rosa Maria; Signorile, Michele; Scarpa, Aldo; Lorusso, Vito; Silvestris, Nicola

    2016-01-01

    Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40-sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients) or gemcitabine plus nab-paclitaxel combination (six patients). The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients), and at time to progression (18 patients). The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre-post treatment sCD40L levels with respect to clinical response, while Pearson's correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal-Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05±7,097.13 pg/mL vs 4,689.42±5,409.96 pg/mL; P<0.01). Conversely, in patients with progressive disease, the biomarker statistically increased in the same time (9,351.51±7,356.91 pg/mL vs 22,282.92±11,629.35 pg/mL; P<0.01). This trend of sCD40L was confirmed in 18 patients at time to progression after the first evaluation. No differences were recorded within the stable disease group. Moreover, there was a positive correlation between the sCD40L and CA19.9 pre-post treatment variation percentage (Pearson's correlation coefficient =0.52; P<0.05). Our data suggest a possible predictive role of sCD40L in pancreatic cancer patients, similar to CA19.9. PMID:27555786

  15. Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma

    PubMed Central

    Azzariti, Amalia; Brunetti, Oronzo; Porcelli, Letizia; Graziano, Giusi; Iacobazzi, Rosa Maria; Signorile, Michele; Scarpa, Aldo; Lorusso, Vito; Silvestris, Nicola

    2016-01-01

    Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40–sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients) or gemcitabine plus nab-paclitaxel combination (six patients). The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients), and at time to progression (18 patients). The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre–post treatment sCD40L levels with respect to clinical response, while Pearson’s correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal–Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05±7,097.13 pg/mL vs 4,689.42±5,409.96 pg/mL; P<0.01). Conversely, in patients with progressive disease, the biomarker statistically increased in the same time (9,351.51±7,356.91 pg/mL vs 22,282.92±11,629.35 pg/mL; P<0.01). This trend of sCD40L was confirmed in 18 patients at time to progression after the first evaluation. No differences were recorded within the stable disease group. Moreover, there was a positive correlation between the sCD40L and CA19.9 pre–post treatment variation percentage (Pearson’s correlation coefficient =0.52; P<0.05). Our data suggest a possible predictive role of sCD40L in pancreatic cancer patients, similar to CA19.9. PMID:27555786

  16. Slowing DNA Transport Using Graphene–DNA Interactions

    PubMed Central

    Banerjee, Shouvik; Wilson, James; Shim, Jiwook; Shankla, Manish; Corbin, Elise A.

    2015-01-01

    Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases. PMID:26167144

  17. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  18. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

    SciTech Connect

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L.F.; Kaminski, Norbert E.

    2013-11-15

    We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

  19. DNA-based watermarks using the DNA-Crypt algorithm

    PubMed Central

    Heider, Dominik; Barnekow, Angelika

    2007-01-01

    Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

  20. Making the Bend: DNA Tertiary Structure and Protein-DNA Interactions

    PubMed Central

    Harteis, Sabrina; Schneider, Sabine

    2014-01-01

    DNA structure functions as an overlapping code to the DNA sequence. Rapid progress in understanding the role of DNA structure in gene regulation, DNA damage recognition and genome stability has been made. The three dimensional structure of both proteins and DNA plays a crucial role for their specific interaction, and proteins can recognise the chemical signature of DNA sequence (“base readout”) as well as the intrinsic DNA structure (“shape recognition”). These recognition mechanisms do not exist in isolation but, depending on the individual interaction partners, are combined to various extents. Driving force for the interaction between protein and DNA remain the unique thermodynamics of each individual DNA-protein pair. In this review we focus on the structures and conformations adopted by DNA, both influenced by and influencing the specific interaction with the corresponding protein binding partner, as well as their underlying thermodynamics. PMID:25026169

  1. What Is Mitochondrial DNA?

    MedlinePlus

    ... 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 ...

  2. Single-molecule DNA conductance in water solutions: Role of DNA low-frequency dynamics

    NASA Astrophysics Data System (ADS)

    Starikov, E. B.; Quintilla, A.; Nganou, C.; Lee, K. H.; Cuniberti, G.; Wenzel, W.

    2009-01-01

    Dependence of charge transmission through several experimentally studied DNA duplexes on their lowest-frequency acoustic modes, combined with the molecular dynamics in picosecond characteristic time range, has been studied. Based on this analysis we were able to identify the specific acoustic modes responsible for the noticeable increase in DNA charge transmission. Other factors influencing electric properties of DNA duplexes are discussed.

  3. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    PubMed

    Bu, Nan-Nan; Tang, Chun-Xia; He, Xi-Wen; Yin, Xue-Bo

    2011-07-21

    Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

  4. Ancient DNA.

    PubMed

    Willerslev, Eske; Cooper, Alan

    2005-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets.

  5. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  6. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  7. DNA topoisomerases.

    PubMed

    Wang, J C

    1996-01-01

    The various problems of disentangling DNA strands or duplexes in a cell are all rooted in the double-helical structure of DNA. Three distinct subfamilies of enzymes, known as the DNA topoisomerases, have evolved to solve these problems. This review focuses on work in the past decade on the mechanisms and cellular functions of these enzymes. Newly discovered members and recent biochemical and structural results are reviewed, and mechanistic implications of these results are summarized. The primary cellular functions of these enzymes, including their roles in replication, transcription, chromosome condensation, and the maintenance of genome stability, are then discussed. The review ends with a summary of the regulation of the cellular levels of these enzymes and a discussion of their association with other cellular proteins.

  8. Structural Organization of DNA in Chlorella Viruses

    PubMed Central

    Wulfmeyer, Timo; Polzer, Christian; Hiepler, Gregor; Hamacher, Kay; Shoeman, Robert; Dunigan, David D.; Van Etten, James L.; Lolicato, Marco; Moroni, Anna; Thiel, Gerhard; Meckel, Tobias

    2012-01-01

    Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes. PMID:22359540

  9. DNA: Polymer and molecular code

    NASA Astrophysics Data System (ADS)

    Shivashankar, G. V.

    1999-10-01

    The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes

  10. Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

    PubMed Central

    Lamothe, P; Baril, B; Chi, A; Lee, L; Baril, E

    1981-01-01

    Three forms of DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] were partially purified from the combined nuclear extract and postmicrosomal supernatant solution of synchronized HeLa cells. These enzymes, designated DNA polymerases alpha 1, alpha 2, and alpha 3, on the basis of their order of elution from DEAE-Bio-Gel, differ in their abilities to utilize single-strand DNA templates. DNA polymerase alpha 2 has equal catalytic activities with activated and single-strand DNAs as template-primers. DNA polymerase alpha 1 has only partial catalytic activity with single-strand DNA templates, and DNA polymerase alpha 3 is essentially inactive with this template. Successive steps of hydrophobic affinity chromatography and phosphocellulose chromatography of DNA polymerase alpha 2 resolved the polymerase alpha activity and two protein factors (C1 and C2) that are required for its catalytic activity with a DNA template-primer that contains extended single-strand regions. In the absence of the factors, DNA polymerase alpha activity is measurable with activated but not single-strand DNA templates. In the presence of the C1 and C2 factors DNA polymerase alpha activity with single-strand DNA templates is restored to about 75% of the catalytic activity of DNA polymerase alpha 2 with this template. Images PMID:6946421

  11. Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC.

    PubMed

    Zhao, Xiaobei; Wang, Anyou; Walter, Vonn; Patel, Nirali M; Eberhard, David A; Hayward, Michele C; Salazar, Ashley H; Jo, Heejoon; Soloway, Matthew G; Wilkerson, Matthew D; Parker, Joel S; Yin, Xiaoying; Zhang, Guosheng; Siegel, Marni B; Rosson, Gary B; Earp, H Shelton; Sharpless, Norman E; Gulley, Margaret L; Weck, Karen E; Hayes, D Neil; Moschos, Stergios J

    2015-01-01

    The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

  12. Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

    PubMed Central

    Walter, Vonn; Patel, Nirali M.; Eberhard, David A.; Hayward, Michele C.; Salazar, Ashley H.; Jo, Heejoon; Soloway, Matthew G.; Wilkerson, Matthew D.; Parker, Joel S.; Yin, Xiaoying; Zhang, Guosheng; Siegel, Marni B.; Rosson, Gary B.; Earp, H. Shelton; Sharpless, Norman E.; Gulley, Margaret L.; Weck, Karen E.

    2015-01-01

    The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion. PMID:26076459

  13. DNA repair of oxidative DNA damage in human carcinogenesis

    PubMed Central

    Paz-Elizur, Tamar; Sevilya, Ziv; Leitner-Dagan, Yael; Elinger, Dalia; Roisman, Laila; Livneh, Zvi

    2008-01-01

    Efficient DNA repair mechanisms comprise a critical component in the protection against human cancer, as indicated by the high predisposition to cancer of individuals with germ-line mutations in DNA repair genes. This includes biallelic germ-line mutations in the MUYH gene, encoding a DNA glycosylase that is involved in the repair of oxidative DNA damage, which strongly predispose humans to a rare hereditary form of colorectal cancer. Extensive research efforts including biochemical, enzymological and genetic studies in model organisms established that the oxidative DNA lesion 8-oxoguanine is mutagenic, and that several DNA repair mechanisms operate to prevent its potentially mutagenic and carcinogenic outcome. Epidemiological studies on the association with sporadic cancers of single nucleotide polymorphisms in genes such as OGG1, involved in the repair of 8-oxoguanine yielded conflicting results, and suggest a minor effect at best. A new approach based on the functional analysis of DNA repair enzymatic activity showed that reduced activity of 8-oxoguanine DNA glycosylase (OGG) is a risk factor in lung and head and neck cancer. Moreover, the combination of smoking and low OGG activity was associated with a higher risk, suggesting a potential strategy for risk assessment and prevention of lung cancer, as well as other types of cancer. PMID:18374480

  14. Crystal structure of DnaT84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode

    PubMed Central

    Liu, Zheng; Chen, Peng; Wang, Xuejuan; Cai, Gang; Niu, Liwen; Teng, Maikun; Li, Xu

    2014-01-01

    DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA. PMID:25053836

  15. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L

    PubMed Central

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer. PMID:25375372

  16. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L.

    PubMed

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer.

  17. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  18. DNA Investigations.

    ERIC Educational Resources Information Center

    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)

  19. 1st Baby Born with DNA from 3 Parents

    MedlinePlus

    ... news/fullstory_161176.html 1st Baby Born With DNA From 3 Parents Technique designed to help couples ... be born using a controversial technique that combines DNA from three people -- two women and a man. ...

  20. HIV DNA Vaccine: Stepwise Improvements Make a Difference

    PubMed Central

    Felber, Barbara K.; Valentin, Antonio; Rosati, Margherita; Bergamaschi, Cristina; Pavlakis, George N.

    2014-01-01

    Inefficient DNA delivery methods and low expression of plasmid DNA have been major obstacles for the use of plasmid DNA as vaccine for HIV/AIDS. This review describes successful efforts to improve DNA vaccine methodology over the past ~30 years. DNA vaccination, either alone or in combination with other methods, has the potential to be a rapid, safe, and effective vaccine platform against AIDS. Recent clinical trials suggest the feasibility of its translation to the clinic. PMID:26344623

  1. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    SciTech Connect

    Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

    2013-06-25

    A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

  2. Superstructure of linear duplex DNA.

    PubMed Central

    Vollenweider, H J; Koller, T; Parello, J; Sogo, J M

    1976-01-01

    The superstructure of a covalently closed circular DNA (of bacteriophage PM 2) was compared by electron microscopy with that of a linear duplex DNA (of bacteriophage T7) when ionic strength and benzyldimethylalkylammonium chloride concentration were varied. In parallel studies the sedimentation behavior of these DNAs was studied by analytical ultracentrifugation, but for technical reasons these had to be without benzyldimethylalkylammonium chloride. By combining the information from the two methods one has to conclude that with increasing ionic strength the linear duplex T7 DNA spontaneously forms a structure similar to that of the superhelical structure of closed circular PM 2 DNA. The superstructure is destroyed under premelting conditions and in the presence of an excess of ethidium bromide. Images PMID:1069302

  3. Swabbing firearms for handler's DNA.

    PubMed

    Richert, Nicholas J

    2011-07-01

    Obtaining quality DNA profiles from firearms can be instrumental in assisting criminal investigations. Typically, swabbings of firearms for handler's DNA are conducted on specific regions of the firearm prior to submission to the laboratory for analysis. This review examines and compares 32 cases whose gun swabbings were either analyzed individually according to the specific region which was swabbed, or analyzed collectively by combining the swabbings from all the individual areas. Those firearms whose swabs were analyzed separately exhibited lower DNA yields and consequently fewer loci exhibiting genotypes. Those cases whose swabs were analyzed collectively exhibited higher DNA yields and consequently greater numbers of loci exhibiting genotypes. These findings demonstrate that collective swabbings result in more complete profiles and lead to the recommendation that a firearm be swabbed in its entirety using no more than two swabs.

  4. Sorting fluorescent nanocrystals with DNA

    SciTech Connect

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  5. Does the tautomeric status of the adenine bases change upon the dissociation of the A*·A(syn) Topal-Fresco DNA mismatch? A combined QM and QTAIM atomistic insight.

    PubMed

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M

    2014-02-28

    We have scrupulously explored the tautomerisation mechanism via the double proton transfer of the A*·A(syn) Topal-Fresco base mispair (C(s) symmetry), formed by the imino and amino tautomers of the adenine DNA base in the anti- and syn-conformations, respectively, bridging quantum-mechanical calculations with Bader's quantum theory of atoms in molecules. It was found that the A*·A(syn) ↔ A·A*(syn) tautomerisation is the asynchronous concerted process. It was established that the A*·A(syn) DNA mismatch is stabilized by the N6H···N6 (6.35) and N1H···N7 (6.17) hydrogen (H) bonds, whereas the A·A*(syn) base mispair (Cs) by the N6H···N6 (8.82) and N7H···N1 (9.78) H-bonds and the C8H···HC2 HH-bond (0.30 kcal mol(-1)). Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···N6 and N1H···N7/N7H···N1 H-bonds are anti-cooperative and mutually weaken each other in the A*·A(syn) and A·A*(syn) mispairs. It was revealed that the A·A*(syn) DNA mismatch is a dynamically unstable structure with a short lifetime of 1.12 × 10(-13) s and any of its 6 low-frequency intermolecular vibrations can develop during this period of time. This observation makes it impossible to change the tautomeric status of the A bases upon the dissociation of the A*·A(syn) base mispair into the monomers during DNA replication.

  6. Does the tautomeric status of the adenine bases change upon the dissociation of the A*·A(syn) Topal-Fresco DNA mismatch? A combined QM and QTAIM atomistic insight.

    PubMed

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M

    2014-02-28

    We have scrupulously explored the tautomerisation mechanism via the double proton transfer of the A*·A(syn) Topal-Fresco base mispair (C(s) symmetry), formed by the imino and amino tautomers of the adenine DNA base in the anti- and syn-conformations, respectively, bridging quantum-mechanical calculations with Bader's quantum theory of atoms in molecules. It was found that the A*·A(syn) ↔ A·A*(syn) tautomerisation is the asynchronous concerted process. It was established that the A*·A(syn) DNA mismatch is stabilized by the N6H···N6 (6.35) and N1H···N7 (6.17) hydrogen (H) bonds, whereas the A·A*(syn) base mispair (Cs) by the N6H···N6 (8.82) and N7H···N1 (9.78) H-bonds and the C8H···HC2 HH-bond (0.30 kcal mol(-1)). Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···N6 and N1H···N7/N7H···N1 H-bonds are anti-cooperative and mutually weaken each other in the A*·A(syn) and A·A*(syn) mispairs. It was revealed that the A·A*(syn) DNA mismatch is a dynamically unstable structure with a short lifetime of 1.12 × 10(-13) s and any of its 6 low-frequency intermolecular vibrations can develop during this period of time. This observation makes it impossible to change the tautomeric status of the A bases upon the dissociation of the A*·A(syn) base mispair into the monomers during DNA replication. PMID:24418908

  7. Optimizing Phytoplasma DNA purification for genome analysis.

    PubMed

    Tran-Nguyen, L T T; Gibb, K S

    2007-04-01

    Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.

  8. Diatom DNA as historical information

    NASA Astrophysics Data System (ADS)

    Stoof, K. R.; Epp, L. S.; Trauth, M. H.; Tiedemann, R.

    2009-04-01

    This survey is about the reconstruction of paleo environmental conditions in East Africa using recent and historic lakes sediments of shallow lakes in Kenya. This interdisciplinary approach combines the molecular analysis of ancient diatom DNA and the morphological analysis of diatom assemblages of short sediment cores to reveal the potential of molecular genetics in geosciences. This study comprises the investigation of two short sediment cores from Lake Naivasha (Kenya) taken in 2007, covering a stretch of ca. 80 years. Several sediment slices were analysed by molecular methods that concludes in species identification based on DNA fragments. Therefore total DNA was extracted and applied to a diatom DNA specific PCR amplifying a gene fragment that is frequently used for species identification. After cloning PCR products, clones were sequenced, sequences from different diatom species were analysed. As a comparison permanent slides were prepared for each sediment sample for counting diatom valves. The results suggest that DNA damage, perhaps species depended, and specific factors of PCR contribute to an overhang of two dominant diatom species (Aulacoseira ambigua and Aulacoseira granulata) represented by the molecular approach whereas rare species seen by light microscopy are not yet identifiable with molecular methods. Moreover changes in diatom assemblages and the reconstructed conductivity indicate a decrease of lake level around 1940. This shift seems to be also represented in the molecular approach as a decrease in the amount of diverse diatom DNA sequences found, what is probably caused by worse DNA preservation conditions at that time.

  9. Association Between Chloroplast DNA and Mitochondrial DNA Haplotypes in Prunus spinosa L. (Rosaceae) Populations across Europe

    PubMed Central

    MOHANTY, APARAJITA; MARTÍN, JUAN PEDRO; GONZÁLEZ, LUIS MIGUEL; AGUINAGALDE, ITZIAR

    2003-01-01

    Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum‐length spanning tree. PMID:14534199

  10. Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.

    PubMed

    Von Beust, B R; Brown, W C; Estes, D M; Zarlenga, D S; McElwain, T F; Palmer, G H

    1999-01-28

    Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion

  11. Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.

    PubMed

    Fumoto, Shintaro; Nishimura, Koyo; Nishida, Koyo; Kawakami, Shigeru

    2016-01-01

    Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues. PMID:26824850

  12. Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.

    PubMed

    Fumoto, Shintaro; Nishimura, Koyo; Nishida, Koyo; Kawakami, Shigeru

    2016-01-01

    Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.

  13. Combination Vaccines

    PubMed Central

    Skibinski, David AG; Baudner, Barbara C; Singh, Manmohan; O’Hagan, Derek T

    2011-01-01

    The combination of diphtheria, tetanus, and pertussis vaccines into a single product has been central to the protection of the pediatric population over the past 50 years. The addition of inactivated polio, Haemophilus influenzae, and hepatitis B vaccines into the combination has facilitated the introduction of these vaccines into recommended immunization schedules by reducing the number of injections required and has therefore increased immunization compliance. However, the development of these combinations encountered numerous challenges, including the reduced response to Haemophilus influenzae vaccine when given in combination; the need to consolidate the differences in the immunization schedule (hepatitis B); and the need to improve the safety profile of the diphtheria, tetanus, and pertussis combination. Here, we review these challenges and also discuss future prospects for combination vaccines. PMID:21572611

  14. Power combiner

    SciTech Connect

    Arnold, Mobius; Ives, Robert Lawrence

    2006-09-05

    A power combiner for the combining of symmetric and asymmetric traveling wave energy comprises a feed waveguide having an input port and a launching port, a reflector for reflecting launched wave energy, and a final waveguide for the collection and transport of launched wave energy. The power combiner has a launching port for symmetrical waves which comprises a cylindrical section coaxial to the feed waveguide, and a launching port for asymmetric waves which comprises a sawtooth rotated about a central axis.

  15. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    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.

  16. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  17. Epigenetics of Ancient DNA

    PubMed Central

    Zhenilo, S. V.; Sokolov, A.S.; Prokhortchouk, E. B.

    2016-01-01

    Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences. PMID:27795845

  18. Synthesis and structure elucidation of new μ-oxamido-bridged dicopper(II) complex with in vitro anticancer activity: A combined study from experiment verification and docking calculation on DNA/protein-binding property.

    PubMed

    Zhu, Ling; Zheng, Kang; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2016-02-01

    A new oxamido-bridged dicopper(II) complex with formula of [Cu2(deap)(pic)2], where H2deap and pic represent N,N'-bis[3-(diethylamino)propyl]oxamide and picrate, respectively, was synthesized and characterized by elemental analyses, molar conductance measurements, IR and electronic spectral study, and single-crystal X-ray diffraction. The crystal structure analyses revealed that the two copper(II) atoms in the dicopper(II) complex are bridged by the trans-deap(2-) ligand with the distances of 5.2116(17)Å, and the coordination environment around the copper(II) atoms can be described as a square-planar geometry. Hydrogen bonding and π-π stacking interactions link the dicopper(II) complex into a three-dimensional infinite network. The DNA/protein-binding properties of the complex are investigated by molecular docking and experimental assays. The results indicate that the dicopper(II) complex can interact with HS-DNA in the mode of intercalation and effectively quench the intrinsic fluorescence of protein BSA by 1:1 binding with the most possible binding site in the proximity of Trp134. The in vitro anticancer activities suggest that the complex is active against the selected tumor cell lines, and IC50 values for SMMC-7721 and HepG2 are lower than cisplatin. The effects of the electron density distribution of the terminal ligand and the chelate ring arrangement around copper(II) ions bridged by symmetric N,N'-bis(substituted)oxamides on DNA/BSA-binding ability and in vitro anticancer activity are preliminarily discussed.

  19. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    SciTech Connect

    Verebová, Valéria; Adamcik, Jozef; Danko, Patrik; Podhradský, Dušan; Miškovský, Pavol; Staničová, Jana

    2014-01-31

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

  20. Chemoenzymatic synthesis and antibody detection of DNA glycoconjugates.

    PubMed

    Wang, Yingli; Sheppard, Terry L

    2003-01-01

    A chemoenzymatic approach for the efficient synthesis of DNA-carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5'-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc-DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc-DNA with beta-1,4-galactosyl transferase (GalT) and UDP-Gal produced N-acetyllactosamine-modified DNA (LacNAc-DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)-DNA conjugate by alpha-1,3-fucosyltransferase VI (FucT) and GDP-Fuc. The facile enzymatic synthesis of LeX-DNA from GlcNAc-DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5'-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX-DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol ("Southwestern"): membrane-immobilized LeX-DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies. PMID:14624649

  1. Chemoenzymatic synthesis and antibody detection of DNA glycoconjugates.

    PubMed

    Wang, Yingli; Sheppard, Terry L

    2003-01-01

    A chemoenzymatic approach for the efficient synthesis of DNA-carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5'-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc-DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc-DNA with beta-1,4-galactosyl transferase (GalT) and UDP-Gal produced N-acetyllactosamine-modified DNA (LacNAc-DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)-DNA conjugate by alpha-1,3-fucosyltransferase VI (FucT) and GDP-Fuc. The facile enzymatic synthesis of LeX-DNA from GlcNAc-DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5'-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX-DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol ("Southwestern"): membrane-immobilized LeX-DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies.

  2. Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA

    PubMed Central

    Richie, Thomas L.; Charoenvit, Yupin; Wang, Ruobing; Epstein, Judith E.; Hedstrom, Richard C.; Kumar, Sanjai; Luke, Thomas C.; Freilich, Daniel A.; Aguiar, Joao C.; Sacci, Jr., John B.; Sedegah, Martha; Nosek, Jr., Ronald A.; De La Vega, Patricia; Berzins, Mara P.; Majam, Victoria F.; Abot, Esteban N.; Ganeshan, Harini; Richie, Nancy O.; Banania, Jo Glenna; Baraceros, Maria Fe B.; Geter, Tanya G.; Mere, Robin; Bebris, Lolita; Limbach, Keith; Hickey, Bradley W.; Lanar, David E.; Ng, Jennifer; Shi, Meng; Hobart, Peter M.; Norman, Jon A.; Soisson, Lorraine A.; Hollingdale, Michael R.; Rogers, William O.; Doolan, Denise L.; Hoffman, Stephen L.

    2012-01-01

    When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997−1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000–2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines. PMID:23151451

  3. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  4. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults

    PubMed Central

    Ingabire, Rosine; Nanvubya, Annet; Anzala, Omu; Karita, Etienne; Hayes, Peter; Kopycinski, Jakub; Dally, Len; Hannaman, Drew; Egan, Michael A.; Eldridge, John H.; Syvertsen, Kristen; Lehrman, Jennifer; Rasmussen, Beth; Gilmour, Jill; Cox, Josephine H.; Fast, Patricia E.; Schmidt, Claudia

    2015-01-01

    Background Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study. Methods Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM. Results All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime—Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected. Conclusion The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected. Trial Registration ClinicalTrials.gov NCT01496989 PMID:26252526

  5. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  6. Combinations Therapies.

    PubMed

    Reinmuth, Niels; Reck, Martin

    2015-01-01

    Immunotherapy of cancer encompasses different strategies that elicit or enhance the immune response against tumors. The first results from clinical studies have provided promising data for the treatment of lung cancer patients with immunomodulating monotherapies. To improve the potential benefit of cancer immunotherapy, synergistic combinations of the various immunotherapy approaches or of different elements within each of the immunotherapy approaches are being explored. The rationale typically involves different but complementary mechanisms of action, eventually impinging on more than one immune system mechanism. As a prominent example, the simultaneous blockade of PD-1 and CTLA-4 is giving rise to therapeutic synergy, while still offering room for efficacy improvement. Moreover, combinations of immunomodulating agents with chemotherapy or targeted molecules are being tested. Animal models suggest that immunotherapies in combination with these various options offer evidence for synergistic effects and are likely to radically change cancer treatment paradigms. However, data obtained so far indicate that toxic side effects are also potentiated, which may even restrict the selection of patients that are suitable for these combinational approaches. Advancing the field of combinatorial immunotherapy will require changes in the way investigational agents are clinically developed as well as novel experimental end-points for efficacy evaluation. However, this combined therapeutic manipulation of both tumor and stromal cells may lead to a dramatic change in the therapeutic options of lung cancer patients in any disease stage that can only grossly be appreciated by the current studies. PMID:26384009

  7. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  8. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity

    PubMed Central

    Ferguson, Brian J; Mansur, Daniel S; Peters, Nicholas E; Ren, Hongwei; Smith, Geoffrey L

    2012-01-01

    Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity. DOI: http://dx.doi.org/10.7554/eLife.00047.001 PMID:23251783

  9. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm.

  10. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  11. Synthesis of DNA

    DOEpatents

    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.

  12. Trapping and manipulating single molecules of DNA

    NASA Astrophysics Data System (ADS)

    Shon, Min Ju

    This thesis presents the development and application of nanoscale techniques to trap and manipulate biomolecules, with a focus on DNA. These methods combine single-molecule microscopy and nano- and micro-fabrication to study biophysical properties of DNA and proteins. The Dimple Machine is a lab-on-a-chip device that can isolate and confine a small number of molecules from a bulk solution. It traps molecules in nanofabricated chambers, or "dimples", and the trapped molecules are then studied on a fluorescence microscope at the single-molecule level. The sampling of bulk solution by dimples is representative, reproducible, and automated, enabling highthroughput single-molecule experiments. The device was applied to study hybridization of oligonucleotides, particularly in the context of reaction thermodynamics and kinetics in nanoconfinement. The DNA Pulley is a system to study protein binding and the local mechanical properties of DNA. A molecule of DNA is tethered to a surface on one end, and a superparamagnetic bead is attached to the other. A magnet pulls the DNA taut, and a silicon nitride knife with a nanoscale blade scans the DNA along its contour. Information on the local properties of the DNA is extracted by tracking the bead with nanometer precision in a white-light microscope. The system can detect proteins bound to DNA and localize their recognition sites, as shown with a model protein, EcoRI restriction enzyme. Progress on the measurements of nano-mechanical properties of DNA is included.

  13. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  14. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  15. DNA systematics. Volume II

    SciTech Connect

    Dutta, S.K.

    1986-01-01

    This book discusses the following topics: PLANTS: PLANT DNA: Contents and Systematics. Repeated DNA Sequences and Polyploidy in Cereal Crops. Homology of Nonrepeated DNA Sequences in Phylogeny of Fungal Species. Chloropast DNA and Phylogenetic Relationships. rDNA: Evolution Over a Billion Years. 23S rRNA-derived Small Ribosomal RNAs: Their Structure and Evolution with Reference to Plant Phylogeny. Molecular Analysis of Plant DNA Genomes: Conserved and Diverged DNA Sequences. A Critical Review of Some Terminologies Used for Additional DNA in Plant Chromosomes and Index.

  16. Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination.

    PubMed

    Sutton, M D; Walker, G C

    2001-07-17

    Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

  17. Noninvasive monitoring of the genetic evolution of EGFR-mutant non-small-cell lung cancer by analyzing circulating tumor DNA during combination chemotherapy with gefitinib and pemetrexed or S-1

    PubMed Central

    Nakahara, Yoshiro; Takagi, Yusuke; Hosomi, Yukio; Kagei, Akiko; Yamamoto, Tomohiro; Sawada, Takeshi; Yomota, Makiko; Okuma, Yusuke; Mikura, Shinichiro; Okamura, Tatsuru

    2016-01-01

    Background Repetitive genotyping is useful to assess the genetic evolution of non-small-cell lung cancer (NSCLC) during treatment, but the need for sampling by biopsy is a major obstacle. Digital polymerase chain reaction (PCR) is a promising procedure for the detection of mutant alleles in plasma of cancer patients. Methods This prospective study enrolled patients with NSCLC and known epidermal growth factor receptor (EGFR) mutations and who had experienced disease progression during ongoing EGFR-tyrosine kinase inhibitor (TKI) therapy. Eligible patients received daily gefitinib and either pemetrexed or S-1 every 3 weeks until disease progression or the development of unacceptable toxicity. Peripheral blood was collected before and after the combination therapy for digital PCR and hepatocyte growth factor measurement. Results From May 2012 to January 2014, nine patients with a median age of 67 (range 52–80) years were enrolled. Patterns of disease progression during adjacent EGFR-TKI therapy were acquired resistance, observed in seven patients, and primary resistance, observed in two patients. Known EGFR mutations were detected in plasma samples of six (67%) patients at study enrollment. Of these, T790M mutation was concurrently detected in three (50%) patients. Four patients underwent gefitinib plus pemetrexed therapy, and five patients underwent gefitinib and S-1 therapy. The median number of cycles delivered was five, and the median progression-free survival was 5.7 months. Efficacy outcomes did not differ between treatments. After the combination therapy, plasma T790M status changed to positive in two patients. Hepatocyte growth factor level did not significantly change through the combination therapy. Conclusion The usefulness of monitoring the genetic evolution of EGFR-driven tumors using noninvasive procedures was demonstrated. Since continuation of EGFR-TKI therapy with cytotoxic agents has an acceptable tolerability and a possibility of inducing T790M

  18. Noninvasive monitoring of the genetic evolution of EGFR-mutant non-small-cell lung cancer by analyzing circulating tumor DNA during combination chemotherapy with gefitinib and pemetrexed or S-1

    PubMed Central

    Nakahara, Yoshiro; Takagi, Yusuke; Hosomi, Yukio; Kagei, Akiko; Yamamoto, Tomohiro; Sawada, Takeshi; Yomota, Makiko; Okuma, Yusuke; Mikura, Shinichiro; Okamura, Tatsuru

    2016-01-01

    Background Repetitive genotyping is useful to assess the genetic evolution of non-small-cell lung cancer (NSCLC) during treatment, but the need for sampling by biopsy is a major obstacle. Digital polymerase chain reaction (PCR) is a promising procedure for the detection of mutant alleles in plasma of cancer patients. Methods This prospective study enrolled patients with NSCLC and known epidermal growth factor receptor (EGFR) mutations and who had experienced disease progression during ongoing EGFR-tyrosine kinase inhibitor (TKI) therapy. Eligible patients received daily gefitinib and either pemetrexed or S-1 every 3 weeks until disease progression or the development of unacceptable toxicity. Peripheral blood was collected before and after the combination therapy for digital PCR and hepatocyte growth factor measurement. Results From May 2012 to January 2014, nine patients with a median age of 67 (range 52–80) years were enrolled. Patterns of disease progression during adjacent EGFR-TKI therapy were acquired resistance, observed in seven patients, and primary resistance, observed in two patients. Known EGFR mutations were detected in plasma samples of six (67%) patients at study enrollment. Of these, T790M mutation was concurrently detected in three (50%) patients. Four patients underwent gefitinib plus pemetrexed therapy, and five patients underwent gefitinib and S-1 therapy. The median number of cycles delivered was five, and the median progression-free survival was 5.7 months. Efficacy outcomes did not differ between treatments. After the combination therapy, plasma T790M status changed to positive in two patients. Hepatocyte growth factor level did not significantly change through the combination therapy. Conclusion The usefulness of monitoring the genetic evolution of EGFR-driven tumors using noninvasive procedures was demonstrated. Since continuation of EGFR-TKI therapy with cytotoxic agents has an acceptable tolerability and a possibility of inducing T790M

  19. The improvement of liposome-mediated transfection of pEGFP DNA into human prostate cancer cells by combining low-frequency and low-energy ultrasound with microbubbles.

    PubMed

    Bai, Wen-Kun; Wu, Zuo-Hui; Shen, E; Zhang, Ji-Zhen; Hu, Bing

    2012-02-01

    The aim of this study was to explore the use of a contrast agent to study the effects of exposure to ultrasound, in combination with microbubbles, on liposome-mediated transfection of genes into human prostate cancer cells. A contrast agent was used to study the effects of ultrasound exposure in combination with microbubbles on liposomes, which transfect genes into human prostate cancer cells. The human prostate cancer cell line PC-3 in suspension was exposed to ultrasound with a 20% duty cycle (i.e., 2 sec 'on' time and 8 sec 'off' time) lasting 5 min, with and without ultrasound contrast agent (SonoVue™) using a digital sonifier at a frequency of 21 kHz and an intensity of 4.6 mW/cm2. Immediately after exposure to ultrasound, cell viability and membrane damage were measured. After exposure to ultrasound, the cell suspensions were put into 12‑well plates and cultured for 24 h. Fluorescence microscopy and flow cytometry were used to detect pEGFP transfection efficiency. Exposure to ultrasound alone and ultrasound combined with microbubbles resulted in minimal cell death and induced negligible cell membrane damage. Ultrasound combined with microbubbles had a greater effect on cell membrane damage in all groups: the average cell membrane damage was 41.87%, and it was approximately 42‑fold greater than in the control group. The average transfection efficiency of PC-3 cells was 20.30% for the liposome (Lipofectamine™)+pEGFP+ultrasound+ultrasound contrast agent (SonoVue) group; this was the highest rate of all groups measured and was approximately 81‑fold greater than that of the control group. The use of low-frequency and low-energy ultrasound, in combination with microbubbles, could be a potent physical method for increasing liposome gene delivery efficiency. This technique is a promising non-viral approach that can be used in prostate cancer gene therapy.

  20. Microarray-based resonance light scattering assay for detecting DNA methylation and human DNA methyltransferase simultaneously with high sensitivity.

    PubMed

    Ma, Lan; Su, Min; Li, Tao; Wang, Zhenxin

    2014-07-21

    A microarray-based resonance light scattering assay, with the combination of methylation-sensitive endonuclease and gold nanoparticle (GNP) probes, has been proposed to sensitively distinguish the DNA methylation level as low as 0.01% (10 pM methylated DNA in 100 nM total DNA) and detect human DNA methyltransferase 1 (Dnmt1) down to 0.1 U mL(-1).

  1. The gene expression profile of psoralen plus UVA-induced premature senescence in skin fibroblasts resembles a combined DNA-damage and stress-induced cellular senescence response phenotype.

    PubMed

    Borlon, Céline; Debacq-Chainiaux, Florence; Hinrichs, Christina; Scharffetter-Kochanek, Karin; Toussaint, Olivier; Wlaschek, Meinhard

    2007-09-01

    After a finite number of population doublings, normal human cells undergo replicative senescence accompanied by growth arrest. We previously described a model of stress-induced premature senescence by treatment of dermal fibroblasts with psoralen plus UVA, a common photodermatological therapy. Psoralen photoactivation has long been used as a therapy for hyperproliferative skin disorders. The repetitive therapeutical treatment is accompanied by premature aging of the skin. Treatment of fibroblasts in vitro with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation results in growth arrest with morphological and functional changes reminiscent of replicative senescence. For gene expression profiling in two strains of human skin fibroblasts after PUVA treatment, we used a low-density DNA array representing 240 genes involved in senescence and stress response. Twenty-nine genes were differentially expressed after PUVA treatment in the two strains of human skin fibroblasts. These genes are involved in growth arrest, stress response, modification of the extracellular matrix and senescence. This study contributes further to the elucidation of the PUVA model and its validation as a useful stress-induced premature senescence model aiming to characterize the premature senescence of fibroblasts and to identify biomarkers that could be applied in vivo.

  2. DNA Nanotechnology-- Architectures Designed with DNA

    NASA Astrophysics Data System (ADS)

    Han, Dongran

    As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.

  3. DNA vaccines: a simple DNA sensing matter?

    PubMed

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J

    2013-10-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA plasmid can be expressed in stromal cells (i.e., muscle cells) as well as DCs, where these antigens are processed and presented to naïve CD4 or CD8 T cells either by direct or cross presentation, respectively; and (2) the transfected DNA plasmid itself may bind to an un-identified cytosolic DNA sensor and activate the TBK1-STING pathway and the production of type I interferons (IFNs) which function as an adjuvant. Recent studies investigating double-stranded cytosolic DNA sensor(s) have highlighted new mechanisms in which cytosolic DNA may release secondary metabolites, which are in turn recognized by a novel DNA sensing machinery. Here, we discuss these new metabolites and the possibilities of translating this knowledge into improved immunogenicity for DNA vaccines.

  4. Combination Light

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The Rayovac TANDEM is an advanced technology combination work light and general purpose flashlight that incorporates several NASA technologies. The TANDEM functions as two lights in one. It features a long range spotlight and wide angle floodlight; simple one-hand electrical switching changes the beam from spot to flood. TANDEM developers made particular use of NASA's extensive research in ergonomics in the TANDEM's angled handle, convenient shape and different orientations. The shatterproof, water resistant plastic casing also draws on NASA technology, as does the shape and beam distance of the square diffused flood. TANDEM's heavy duty magnet that permits the light to be affixed to any metal object borrows from NASA research on rare earth magnets that combine strong magnetic capability with low cost. Developers used a NASA-developed ultrasonic welding technique in the light's interior.

  5. The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics

    PubMed Central

    Biebricher, Andreas S.; Heller, Iddo; Roijmans, Roel F. H.; Hoekstra, Tjalle P.; Peterman, Erwin J. G.; Wuite, Gijs J. L.

    2015-01-01

    DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy. PMID:26084388

  6. Dynamics of DNA bending/unbending in complex with DNA-bending protein IHF

    NASA Astrophysics Data System (ADS)

    Ansari, Anjum; Vivas, Paula; Kuznetsov, Serguei

    2007-03-01

    Kinetics of conformational changes in proteins and DNA that lead to precise recognition of specific DNA binding sites are difficult to observe with the limited time-resolution of stop-flow and single-molecule techniques. Here we use a ˜10 ns laser T-jump apparatus to probe the kinetics of a ˜35-bp DNA substrate bound to E. coli Integration Host Factor (IHF) and end-labeled with a FRET pair. These T-jump measurements, in combination with stop-flow, provide the first direct observation of the DNA bending/unbending kinetics in a protein-DNA complex (Sugimura and Crothers, PNAS, in press; Kuznetsov et al., PNAS, in press). The rates and activation energy of DNA bending are similar to that of a single A:T base pair opening inside uncomplexed DNA, suggesting that spontaneous thermal disruption in base-pairing nucleated at an A:T site may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA. An unusual salt dependence of the binding affinity observed previously for IHF/DNA complex, and explained in terms of DNA binding coupled with disruption of a network of salt bridges within the protein (Holbrook et al., 2001, JMB, 310, 379), is reflected in the salt dependence of the observed bending rates. These results suggest that salt-dependent protein conformational changes may be playing a role in the DNA bending process.

  7. Lupus autoantibodies to native DNA preferentially bind DNA presented on PolIV.

    PubMed

    Kumar, Sanjeev; Bunting, Karen A; Kalsi, Jatinderpal; Hinks, John A; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2005-03-01

    While immunoglobulin G (IgG) antibodies to double-stranded (ds)DNA are serological markers of systemic lupus erythematosus (SLE), not all antibodies to DNA (anti-DNA) are able to cause tissue damage to a similar extent. It has been proposed that anti-DNA-induced renal damage could be linked to differences in the fine specificity of the antibodies. In an attempt to gain insight into their fine binding properties, we investigated the cross-reactivity of two human lupus monoclonal IgG anti-dsDNA (B3 and RH14) to a recently described Escherichia coli PolIV (a DNA polymerase). These autoantibodies possess distinct pathogenic properties in severe combined immunodeficient (SCID) mice. Although both antibodies cause proteinuria, only RH14 induces early histological features of lupus nephritis. Both RH14 and B3 bound PolIV; however, they exhibited a marked difference in their reactivity to the PolIV-dsDNA complex. Alhough RH14 exhibited significant activity to the complex, the binding of B3 to PolIV complexed with dsDNA was almost abolished. Furthermore, there was a significant difference in the way the lupus sera recognized naked dsDNA and that presented on PolIV. Although 67% of lupus sera bound naked dsDNA, approximately 90% of these sera (93% calf thymus DNA; 90% synthetic oligonucleotide) reacted to the complex when dsDNA was presented on PolIV. Thus, the IgG anti-dsDNA likely to exist in lupus patients may be distinguished into those that recognize dsDNA in the context of PolIV and those which do not. This difference in binding ability may help to distinguish those dsDNA antibodies that are more pathogenic.

  8. Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

    SciTech Connect

    X Qiu; D Rau; V Parsegian; L Fang; C Knobler; W Gelbart

    2011-12-31

    Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.

  9. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  10. CtIP: A DNA damage response protein at the intersection of DNA metabolism.

    PubMed

    Makharashvili, Nodar; Paull, Tanya T

    2015-08-01

    The mammalian CtIP protein and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. Here we review the current literature supporting the role of CtIP in DNA end processing and the importance of CtIP endonuclease activity in DNA repair. We also examine the regulation of CtIP function by post-translational modifications, and its involvement in transcription- and replication-dependent functions through association with other protein complexes. The tumor suppressor function of CtIP likely is dependent on a combination of these roles in many aspects of DNA metabolism.

  11. Enzyme-guided DNA Sewing Architecture

    NASA Astrophysics Data System (ADS)

    Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

    2015-12-01

    With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5‧-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields.

  12. Enzyme-guided DNA Sewing Architecture.

    PubMed

    Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

    2015-12-04

    With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5'-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields.

  13. Enzyme-guided DNA Sewing Architecture

    PubMed Central

    Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

    2015-01-01

    With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5′-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields. PMID:26634810

  14. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  15. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2006-08-01

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

  16. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi,Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2011-07-05

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

  17. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    PubMed

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  18. Effective delivery of DNA into tumor cells and tissues by electroporation of polymer-DNA complex.

    PubMed

    Kang, Jeong-Hun; Toita, Riki; Niidome, Takuro; Katayama, Yoshiki

    2008-07-01

    Electroporation is a useful means for non-viral gene delivery. Here, we investigated the use of electroporation to deliver polymer-DNA complexes into living cells using a protein kinase C (PKC)alpha-responsive polymer. The polymer was complexed with a luciferase-encoding DNA and electroporated into B16 melanoma cells. Gene expression from polymer-DNA complexes was 3- to 5-fold higher than from naked DNA. Moreover, after introduction of the polymer-DNA complex into tissues, luciferase levels were >2-fold higher in B16 melanoma tumors than in normal skin tissue. These results suggest that the combination of our polymer and electroporation is useful for the effective delivery of DNA into tumors. PMID:18375054

  19. Introduction of heteroplasmic mitochondrial DNA (mtDNA) from a patient with NARP into two human rho degrees cell lines is associated either with selection and maintenance of NARP mutant mtDNA or failure to maintain mtDNA.

    PubMed

    Vergani, L; Rossi, R; Brierley, C H; Hanna, M; Holt, I J

    1999-09-01

    Mitochondria from a patient heteroplasmic at nucleo-tide position 8993 of mitochondrial DNA (mtDNA) were introduced into two human tumour cell lines lacking mtDNA. The donor mitochondria contained between 85 and 95% 8993G:C mtDNA. All detectable mtDNA in the mitochondrially transformed cells contained the pathological 8993G:C mutation 3 months after transformation. These results suggest that 8993G:C mtDNA had a selective advantage over 8993T:A mtDNA in both lung carcinoma and osteo-sarcoma cell backgrounds. In contrast, two other presumed pathological mtDNA variants were lost in favour of 'wild-type' mtDNA molecules in the same lung carcinoma cell background. Taken together, these findings suggest that the transmission bias of mtDNA variants is dependent upon a combination of nuclear background and mtDNA genotype. A second phenomenon observed was a marked decrease in the growth rate of many putative transformed cell lines after 6 weeks of culturing in selective medium, and in these cell lines mtDNA was not readily detectable by Southern blotting. Restriction endonuclease analysis and sequencing of amplified mtDNA demonstrated that the slow growing cells contained little or no mtDNA. It is concluded that these cells represented transient mitochondrial transformants.

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

    PubMed

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

    2015-09-18

    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.

  1. Methylsorb: a simple method for quantifying DNA methylation using DNA-gold affinity interactions.

    PubMed

    Sina, Abu Ali Ibn; Carrascosa, Laura G; Palanisamy, Ramkumar; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-10-21

    The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

  2. DNA Aptamer Technology for Personalized Medicine

    PubMed Central

    Xing, Hang; Hwang, Kevin; Li, Ji; Torabi, Seyed-Fakhreddin; Lu, Yi

    2014-01-01

    This review highlights recent progress in developing DNA aptamers for personalized medicine, with more focus on in vivo studies for potential clinical applications. Examples include design of aptamers in combination with DNA nanostructures, nanomaterials, or microfluidic devices as diagnostic probes or therapeutic agents for cancers and other diseases. The use of aptamers as targeting agents in drug delivery is also covered. The advantages and future directions of such DNA aptamer-based technology for the continued development of personalized medicine are discussed. PMID:24791224

  3. DNA from plant mitochondria.

    PubMed

    Suyama, Y; Bonner, W D

    1966-03-01

    DNA WAS ISOLATED FROM A MITOCHONDRIAL FRACTION OF EACH OF THE FOLLOWING PLANT MATERIALS: Mung bean (Phaseolus aureus) etiolated hypocotyl; turnip (Brassica rapa) root; sweet potato (Ipomoea batatas) root; and onion (Allium cepa) bulb. It was found that all of these mitochondrial fractions contained DNA, the densities of which were identical (rho=1.706 g.cm(-3)). An additional DNA (rho=1.695) band found in the mitochondrial fraction of Brassica rapa, was identical to DNA separately isolated from the chloroplast-rich fraction. The origin of the second DNA from Allium mitochondrial fraction was not identified.Contrary to the identity of the mitochondrial DNA, DNA from nuclear fractions differed not only with each other but from the corresponding mitochondrial DNA.DNA from Phaseolus and Brassica mitochondria showed the hyperchromicity characteristic of double stranded, native DNA upon heating; Tm's in 0.0195 Na(+) were the same; 72.0 degrees . The amount of DNA within the mitochondrion of Phaseolus was estimated to be 5.0 x 10(-10) mug; this estimate was made by isolating the mitochondrial DNA concomitantly with the known amount of added (15)N(2)H B. subtilis DNA (rho=1.740). Approximately the same amount of DNA was present in the mitochondrion of Brassica or Ipomoea.

  4. Sliding of proteins non-specifically bound to DNA: Brownian dynamics studies with coarse-grained protein and DNA models.

    PubMed

    Ando, Tadashi; Skolnick, Jeffrey

    2014-12-01

    DNA binding proteins efficiently search for their cognitive sites on long genomic DNA by combining 3D diffusion and 1D diffusion (sliding) along the DNA. Recent experimental results and theoretical analyses revealed that the proteins show a rotation-coupled sliding along DNA helical pitch. Here, we performed Brownian dynamics simulations using newly developed coarse-grained protein and DNA models for evaluating how hydrodynamic interactions between the protein and DNA molecules, binding affinity of the protein to DNA, and DNA fluctuations affect the one dimensional diffusion of the protein on the DNA. Our results indicate that intermolecular hydrodynamic interactions reduce 1D diffusivity by 30%. On the other hand, structural fluctuations of DNA give rise to steric collisions between the CG-proteins and DNA, resulting in faster 1D sliding of the protein. Proteins with low binding affinities consistent with experimental estimates of non-specific DNA binding show hopping along the CG-DNA. This hopping significantly increases sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome.

  5. Functionalizing Designer DNA Crystals

    NASA Astrophysics Data System (ADS)

    Chandrasekaran, Arun Richard

    Three-dimensional crystals have been self-assembled from a DNA tensegrity triangle via sticky end interaction. The tensegrity triangle is a rigid DNA motif containing three double helical edges connected pair-wise by three four-arm junctions. The symmetric triangle contains 3 unique strands combined in a 3:3:1 ratio: 3 crossover, 3 helical and 1 central. The length of the sticky end reported previously was two nucleotides (nt) (GA:TC) and the motif with 2-helical turns of DNA per edge diffracted to 4.9 A at beam line NSLS-X25 and to 4 A at beam line ID19 at APS. The purpose of these self-assembled DNA crystals is that they can be used as a framework for hosting external guests for use in crystallographic structure solving or the periodic positioning of molecules for nanoelectronics. This thesis describes strategies to improve the resolution and to incorporate guests into the 3D lattice. The first chapter describes the effect of varying sticky end lengths and the influence of 5'-phosphate addition on crystal formation and resolution. X-ray diffraction data from beam line NSLS-X25 revealed that the crystal resolution for 1-nt (G:C) sticky end was 3.4 A. Motifs with every possible combination of 1-nt and 2-nt sticky-ended phosphorylated strands were crystallized and X-ray data were collected. The position of the 5'-phosphate on either the crossover (strand 1), helical (strand 2), or central strand (3) had an impact on the resolution of the self-assembled crystals with the 1-nt 1P-2-3 system diffracting to 2.62 A at APS and 3.1 A at NSLS-X25. The second chapter describes the sequence-specific recognition of DNA motifs with triplex-forming oligonucleotides (TFOs). This study examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The TFO 5'-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine.oligopyrimidine binding site. As triplex formation involving cytidine

  6. Unravelling the structural plasticity of stretched DNA under torsional constraint

    NASA Astrophysics Data System (ADS)

    King, Graeme A.; Peterman, Erwin J. G.; Wuite, Gijs J. L.

    2016-06-01

    Regions of the genome are often held under torsional constraint. Nevertheless, the influence of such constraint on DNA-protein interactions during genome metabolism is still poorly understood. Here using a combined optical tweezers and fluorescence microscope, we quantify and explain how torsional constraint influences the structural stability of DNA under applied tension. We provide direct evidence that concomitant basepair melting and helical unwinding can occur in torsionally constrained DNA at forces >~50 pN. This striking result indicates that local changes in linking number can be absorbed by the rest of the DNA duplex. We also present compelling new evidence that an overwound DNA structure (likely P-DNA) is created (alongside underwound structures) at forces >~110 pN. These findings substantiate previous theoretical predictions and highlight a remarkable structural plasticity of torsionally constrained DNA. Such plasticity may be required in vivo to absorb local changes in linking number in DNA held under torsional constraint.

  7. LCAT DNA shearing.

    PubMed

    Okabe, Yuka; Lee, Abraham P

    2014-04-01

    We present a novel method to fragment DNA by using lateral cavity acoustic transducers (LCATs). DNA solution is placed within a microfluidic device containing LCATs. The LCATs cause microstreaming, which fragments DNA within the solution without any need for purification or downstream processing. The LCAT-based DNA fragmentation method offers an easy-to-use, low-cost, low-energy way to fragment DNA that is amenable to integration on microfluidic platforms to further automate DNA processing. Furthermore, the LCAT microdevice requires less than 10 µL of sample, and no external equipment is needed besides a piezoelectric transducer. PMID:23850863

  8. Assembly of highly aligned DNA strands onto Si chips.

    PubMed

    Zhang, Jianming; Ma, Yufeng; Stachura, Sylwia; He, Huixin

    2005-04-26

    This paper reports a robust and efficient approach to assemble highly aligned DNA strands onto Si chips. The method combines advantages from molecular combing and microcontact printing to realize controlling both the density and direction of DNA strands on the Si chip. In addition, it also can be utilized to prepare stretched DNA structures on solid surfaces. Compared to approaches that use molecular combing directly on silanated surfaces, the stretched single-chain DNA structures are straighter. Furthermore, by exploiting the hydrophobic property of the intrinsic poly(dimethylsiloxane) stamp, this study also describes a simple way to produce straight bundled DNA arrays on Si and other substrates.

  9. Concentrating Genomic Length DNA in a Microfabricated Array

    NASA Astrophysics Data System (ADS)

    Chen, Yu; Abrams, Ezra S.; Boles, T. Christian; Pedersen, Jonas N.; Flyvbjerg, Henrik; Austin, Robert H.; Sturm, James C.

    2015-05-01

    We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to 40 μ m /s , if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting the DNA molecules to minimal coil size using polyethylene glycol (PEG) derived depletion forces. We map out the sweet spot, where concentration occurs, as a function of PEG concentration and flow speed using a combination of theoretical analysis and experiment. Purification of DNA from enzymatic reactions for next-generation DNA-sequencing libraries will be an important application of this development.

  10. [Uracil-DNA glycosylases].

    PubMed

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  11. Electrokinetic acceleration of DNA hybridization in microsystems.

    PubMed

    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. PMID:25863384

  12. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N.; Mariella, Jr., Raymond P.; Christian, Allen T.; Young, Jennifer A.; Clague, David S.

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  13. Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

    PubMed Central

    Takahashi, Hirokazu; Yamazaki, Hiroyuki; Akanuma, Satoshi; Kanahara, Hiroko; Saito, Toshiyuki; Chimuro, Tomoyuki; Kobayashi, Takayoshi; Ohtani, Toshio; Yamamoto, Kimiko; Sugiyama, Shigeru; Kobori, Toshiro

    2014-01-01

    We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents. PMID:24505243

  14. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  15. DNA tagged microparticles

    DOEpatents

    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.

  16. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

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

  17. Strategies and hurdles using DNA vaccines to fish.

    PubMed

    Hølvold, Linn B; Myhr, Anne I; Dalmo, Roy A

    2014-01-01

    DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen - and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.

  18. Strategies and hurdles using DNA vaccines to fish

    PubMed Central

    2014-01-01

    DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen – and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish. PMID:24552235

  19. DNA nanoarchitectonics: assembled DNA at interfaces.

    PubMed

    Howorka, Stefan

    2013-06-18

    DNA is a powerful biomaterial for creating rationally designed and functionally enhanced nanostructures. DNA nanoarchitectures positioned at substrate interfaces can offer unique advantages leading to improved surface properties relevant to biosensing, nanotechnology, materials science, and cell biology. This Perspective highlights the benefits and challenges of using assembled DNA as a nanoscale building block for interfacial layers and surveys their applications in three areas: homogeneous dense surface coatings, bottom-up nanopatterning, and 3D nanoparticle lattices. Possible future research developments are discussed at the end of the Perspective.

  20. A DNA 3′-phosphatase functions in active DNA demethylation in Arabidopsis

    PubMed Central

    Martínez-Macías, María Isabel; Qian, Weiqiang; Miki, Daisuke; Pontes, Olga; Liu, Yunhua; Tang, Kai; Liu, Renyi; Morales-Ruiz, Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

    2012-01-01

    SUMMARY DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway. PMID:22325353

  1. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  2. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma

    PubMed Central

    Cottini, Francesca; Hideshima, Teru; Suzuki, Rikio; Tai, Yu-Tzu; Bianchini, Giampaolo; Richardson, Paul G.; Anderson, Kenneth C.; Tonon, Giovanni

    2015-01-01

    Ongoing DNA damage is a common feature of epithelial cancers. Here we show that tumor cells derived from multiple myeloma (MM), a disease of clonal plasma cells, demonstrate DNA replicative stress leading to DNA damage. We identified a poor prognosis subset of MM with extensive chromosomal instability and replicative stress which rely on ATR to compensate for DNA replicative stress; conversely, silencing of ATR or treatment with a specific ATR inhibitor triggers MM cell apoptosis. We show that oncogenes such as MYC induce DNA damage in MM cells not only by increased replicative stress, but also via increased oxidative stress, and that ROS-inducer piperlongumine triggers further DNA damage and apoptosis. Importantly, ATR inhibition combined with piperlongumine triggers synergistic MM cytotoxicity. This synthetic lethal approach, enhancing oxidative stress while concomitantly blocking replicative stress response, provides a novel combination targeted therapy to address an unmet medical need in this subset of MM. PMID:26080835

  3. DNA Sequencing apparatus

    DOEpatents

    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.

  4. Racemic DNA crystallography.

    PubMed

    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.

  5. Mutagenic DNA repair in enterobacteria.

    PubMed Central

    Sedgwick, S G; Ho, C; Woodgate, R

    1991-01-01

    Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD' fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD' antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium umu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention. Images PMID:1885540

  6. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.

  7. DNA barcoding for plants.

    PubMed

    de Vere, Natasha; Rich, Tim C G; Trinder, Sarah A; Long, Charlotte

    2015-01-01

    DNA barcoding uses specific regions of DNA in order to identify species. Initiatives are taking place around the world to generate DNA barcodes for all groups of living organisms and to make these data publically available in order to help understand, conserve, and utilize the world's biodiversity. For land plants the core DNA barcode markers are two sections of coding regions within the chloroplast, part of the genes, rbcL and matK. In order to create high quality databases, each plant that is DNA barcoded needs to have a herbarium voucher that accompanies the rbcL and matK DNA sequences. The quality of the DNA sequences, the primers used, and trace files should also be accessible to users of the data. Multiple individuals should be DNA barcoded for each species in order to check for errors and allow for intraspecific variation. The world's herbaria provide a rich resource of already preserved and identified material and these can be used for DNA barcoding as well as by collecting fresh samples from the wild. These protocols describe the whole DNA barcoding process, from the collection of plant material from the wild or from the herbarium, how to extract and amplify the DNA, and how to check the quality of the data after sequencing.

  8. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  9. Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

    PubMed Central

    Kuznetsov, Serguei V.; Sugimura, Sawako; Vivas, Paula; Crothers, Donald M.; Ansari, Anjum

    2006-01-01

    Regulation of gene expression involves formation of specific protein–DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from ≈2 ms−1 at ≈37°C to ≈40 ms−1 at ≈10°C and correspond to an activation energy of ≈14 ± 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF. PMID:17124171

  10. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  11. Oxidation of DNA: damage to nucleobases.

    PubMed

    Kanvah, Sriram; Joseph, Joshy; Schuster, Gary B; Barnett, Robert N; Cleveland, Charles L; Landman, Uzi

    2010-02-16

    All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and

  12. A DNA barcode for land plants.

    PubMed

    2009-08-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

  13. Improving DNA vaccine performance through vector design.

    PubMed

    Williams, James A

    2014-01-01

    DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

  14. A DNA barcode for land plants

    PubMed Central

    Hollingsworth, Peter M.; Forrest, Laura L.; Spouge, John L.; Hajibabaei, Mehrdad; Ratnasingham, Sujeevan; van der Bank, Michelle; Chase, Mark W.; Cowan, Robyn S.; Erickson, David L.; Fazekas, Aron J.; Graham, Sean W.; James, Karen E.; Kim, Ki-Joong; Kress, W. John; Schneider, Harald; van AlphenStahl, Jonathan; Barrett, Spencer C.H.; van den Berg, Cassio; Bogarin, Diego; Burgess, Kevin S.; Cameron, Kenneth M.; Carine, Mark; Chacón, Juliana; Clark, Alexandra; Clarkson, James J.; Conrad, Ferozah; Devey, Dion S.; Ford, Caroline S.; Hedderson, Terry A.J.; Hollingsworth, Michelle L.; Husband, Brian C.; Kelly, Laura J.; Kesanakurti, Prasad R.; Kim, Jung Sung; Kim, Young-Dong; Lahaye, Renaud; Lee, Hae-Lim; Long, David G.; Madriñán, Santiago; Maurin, Olivier; Meusnier, Isabelle; Newmaster, Steven G.; Park, Chong-Wook; Percy, Diana M.; Petersen, Gitte; Richardson, James E.; Salazar, Gerardo A.; Savolainen, Vincent; Seberg, Ole; Wilkinson, Michael J.; Yi, Dong-Keun; Little, Damon P.

    2009-01-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

  15. Multiplexed Sequence Encoding: A Framework for DNA Communication.

    PubMed

    Zakeri, Bijan; Carr, Peter A; Lu, Timothy K

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  16. Multiplexed Sequence Encoding: A Framework for DNA Communication

    PubMed Central

    Zakeri, Bijan; Carr, Peter A.; Lu, Timothy K.

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication—data encoding, data transfer & data extraction—and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system—Multiplexed Sequence Encoding (MuSE)—that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  17. Targeting DNA Replication Stress for Cancer Therapy.

    PubMed

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-01-01

    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. PMID:27548226

  18. DNA-nanostructure-assembly by sequential spotting

    PubMed Central

    2011-01-01

    Background The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures. Conclusions The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element. PMID:22099392

  19. DNA hydration studied by neutron fiber diffraction

    SciTech Connect

    Fuller, W.; Forsyth, V.T.; Mahendrasingam, A.; Langan, P.; Pigram, W.J.

    1994-12-31

    The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institute Laue-Langevin, Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformations] transitions in the DNA double-helix.

  20. Targeting DNA Replication Stress for Cancer Therapy

    PubMed Central

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-01-01

    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. PMID:27548226

  1. Targeting DNA Replication Stress for Cancer Therapy.

    PubMed

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-08-19

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

  2. Visualizing the Formation and Collapse of DNA Toroids

    PubMed Central

    van den Broek, Bram; Noom, Maarten C.; van Mameren, Joost; Battle, Christopher; MacKintosh, Fred C.; Wuite, Gijs J.L.

    2010-01-01

    Abstract In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau. PMID:20441754

  3. DNA nanotechnology from the test tube to the cell.

    PubMed

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  4. DNA nanotechnology from the test tube to the cell

    NASA Astrophysics Data System (ADS)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  5. Charting the Structure and Energetics of Packaged DNA in Bacteriophages

    NASA Astrophysics Data System (ADS)

    Qiu, Xiangyun; Rau, Donald C.; Parsegian, V. Adrian; Fang, Li Tai; Knobler, Charles M.; Gelbart, William M.

    2009-03-01

    Many bacterial viruses resort to pressure in order to infect bacteria, e.g., lambda phage stores its dsDNA genome at surprisingly high pressure and then uses this pressure to drive delivery of the genome. We report on a biophysical interrogation of the DNA configuration and pressure in lambda phage by combining structural and thermodynamic measurements with theoretical modeling. Changes in DNA organization in the capsid are monitored using solution small angle x-ray scattering (SAXS). We vary the DNA-DNA repulsion and DNA bending contributions to the capsid pressure by changing salt concentrations and packaged length, and augment SAXS data with osmotic stress measurements to elicit the evolving structure and energetics of the packaged DNA.

  6. Chemical method for introducing haptens on to DNA probes

    SciTech Connect

    Keller, G.H.; Cumming, C.U.; Huang, D.P.; Manak, M.M.; Ting, R.

    1988-05-01

    The authors developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. They have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 x 10/sup -17/ mol (1 x 10/sup 6/ copies) of HBV DNA in total DNA from human serum.

  7. Geant4-DNA: overview and recent developments

    NASA Astrophysics Data System (ADS)

    Štěpán, Václav

    Space travel and high altitude flights are inherently associated with prolonged exposure to cosmic and solar radiation. Understanding and simulation of radiation action on cellular and subcellular level contributes to precise assessment of the associated health risks and remains a challenge of today’s radiobiology research. The Geant4-DNA project (http://geant4-dna.org) aims at developing an experimentally validated simulation platform for modelling of the damage induced by ionizing radiation at DNA level. The platform is based on the Geant4 Monte Carlo simulation toolkit. This project extends specific functionalities of Geant4 in following areas: The step-by-step single scattering modelling of elementary physical interactions of electrons, protons, alpha particles and light ions with liquid water and DNA bases, for the so-called “physical” stage. The modelling of the “physico-chemical and chemical” stages corresponding to the production, the diffusion, the chemical reactions occurring between chemical species produced by water radiolysis, and to the radical attack on the biological targets. Physical and chemical stage simulations are combined with biological target models on several scales, from DNA double helix, through nucleosome, to chromatin segments and cell geometries. In addition, data mining clustering algorithms have been developed and optimised for the purpose of DNA damage scoring in simulated tracks. Experimental measurements on pBR322 plasmid DNA are being carried out in order to validate the Geant4-DNA models. The plasmid DNA has been irradiated in dry conditions by protons with energies from 100 keV to 30 MeV and in aqueous conditions, with and without scavengers, by 30 MeV protons, 290 MeV/u carbon and 500 MeV/u iron ions. Agarose gel electrophoresis combined with enzymatic treatment has been used to measure the resulting DNA damage. An overview of the developments undertaken by the Geant4-DNA collaboration including a description of

  8. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  9. A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

    PubMed Central

    Godonoga, Maia; Lin, Ting-Yu; Oshima, Azusa; Sumitomo, Koji; Tang, Marco S. L.; Cheung, Yee-Wai; Kinghorn, Andrew B.; Dirkzwager, Roderick M.; Zhou, Cunshan; Kuzuya, Akinori; Tanner, Julian A.; Heddle, Jonathan G.

    2016-01-01

    DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology. PMID:26891622

  10. Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone.

    PubMed

    Awasthi, Sita; Lubinski, John M; Shaw, Carolyn E; Barrett, Shana M; Cai, Michael; Wang, Fushan; Betts, Michael; Kingsley, Susan; Distefano, Daniel J; Balliet, John W; Flynn, Jessica A; Casimiro, Danilo R; Bryan, Janine T; Friedman, Harvey M

    2011-10-01

    Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.

  11. Strandedness and Complementarity of DNA from Long-Term RNA-Dependent DNA Polymerase Reactions of Soehner-Dmochowski Murine Sarcoma Virus

    PubMed Central

    East, James L.; Knesek, John E.; Allen, Patton T.; Dmochowski, Leon

    1973-01-01

    The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA. PMID:4358160

  12. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  13. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.

  14. Electrocatalysis in DNA Sensors.

    PubMed

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  15. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

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

    DOEpatents

    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.

  17. Hysteresis in Pressure-Driven DNA Denaturation

    PubMed Central

    Hernández-Lemus, Enrique; Nicasio-Collazo, Luz Adriana; Castañeda-Priego, Ramón

    2012-01-01

    In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like) charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue. PMID:22496765

  18. Tracking earthworm communities from soil DNA.

    PubMed

    Bienert, Friederike; De Danieli, Sébastien; Miquel, Christian; Coissac, Eric; Poillot, Carole; Brun, Jean-Jacques; Taberlet, Pierre

    2012-04-01

    Earthworms are known for their important role within the functioning of an ecosystem, and their diversity can be used as an indicator of ecosystem health. To date, earthworm diversity has been investigated through conventional extraction methods such as handsorting, soil washing or the application of a mustard solution. Such techniques are time consuming and often difficult to apply. We showed that combining DNA metabarcoding and next-generation sequencing facilitates the identification of earthworm species from soil samples. The first step of our experiments was to create a reference database of mitochondrial DNA (mtDNA) 16S gene for 14 earthworm species found in the French Alps. Using this database, we designed two new primer pairs targeting very short and informative DNA sequences (about 30 and 70 bp) that allow unambiguous species identification. Finally, we analysed extracellular DNA taken from soil samples in two localities (two plots per locality and eight samples per plot). The two short metabarcode regions led to the identification of a total of eight earthworm species. The earthworm communities identified by the DNA-based approach appeared to be well differentiated between the two localities and are consistent with results derived from inventories collected using the handsorting method. The possibility of assessing earthworm communities from hundreds or even thousands of localities through the use of extracellular soil DNA will undoubtedly stimulate further ecological research on these organisms. Using the same DNA extracts, our study also illustrates the potential of environmental DNA as a tool to assess the diversity of other soil-dwelling animal taxa.

  19. Binary electrokinetic separation of target DNA from background DNA primers.

    SciTech Connect

    James, Conrad D.; Derzon, Mark Steven

    2005-10-01

    This report contains the summary of LDRD project 91312, titled ''Binary Electrokinetic Separation of Target DNA from Background DNA Primers''. This work is the first product of a collaboration with Columbia University and the Northeast BioDefense Center of Excellence. In conjunction with Ian Lipkin's lab, we are developing a technique to reduce false positive events, due to the detection of unhybridized reporter molecules, in a sensitive and multiplexed detection scheme for nucleic acids developed by the Lipkin lab. This is the most significant problem in the operation of their capability. As they are developing the tools for rapidly detecting the entire panel of hemorrhagic fevers this technology will immediately serve an important national need. The goal of this work was to attempt to separate nucleic acid from a preprocessed sample. We demonstrated the preconcentration of kilobase-pair length double-stranded DNA targets, and observed little preconcentration of 60 base-pair length single-stranded DNA probes. These objectives were accomplished in microdevice formats that are compatible with larger detection systems for sample pre-processing. Combined with Columbia's expertise, this technology would enable a unique, fast, and potentially compact method for detecting/identifying genetically-modified organisms and multiplexed rapid nucleic acid identification. Another competing approach is the DARPA funded IRIS Pharmaceutical TIGER platform which requires many hours for operation, and an 800k$ piece of equipment that fills a room. The Columbia/SNL system could provide a result in 30 minutes, at the cost of a few thousand dollars for the platform, and would be the size of a shoebox or smaller.

  20. DNA-based machines.

    PubMed

    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.

  1. DNA-based machines.

    PubMed

    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. PMID:24647836

  2. Multiprotein DNA Looping

    NASA Astrophysics Data System (ADS)

    Vilar, Jose M. G.; Saiz, Leonor

    2006-06-01

    DNA looping plays a fundamental role in a wide variety of biological processes, providing the backbone for long range interactions on DNA. Here we develop the first model for DNA looping by an arbitrarily large number of proteins and solve it analytically in the case of identical binding. We uncover a switchlike transition between looped and unlooped phases and identify the key parameters that control this transition. Our results establish the basis for the quantitative understanding of fundamental cellular processes like DNA recombination, gene silencing, and telomere maintenance.

  3. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    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.

  4. DNA polymerase profiling.

    PubMed

    Summerer, Daniel

    2008-01-01

    We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alteration is reported by fluorescence resonance energy transfer between two dyes introduced into the molecular beacon stem. We describe real-time reaction profiling of two model DNA polymerases. We demonstrate kinetic characterization, rapid optimization of reaction conditions, and inhibitor profiling using the presented assay. Furthermore, to supersede purification steps in screening procedures of DNA polymerase mutant libraries, detection of enzymatic activity in bacterial expression lysates is described.

  5. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  6. Combined Cancer Immunotherapy Against Aurora Kinase A.

    PubMed

    Kaštánková, Iva; Poláková, Ingrid; Dušková, Martina; Šmahel, Michal

    2016-05-01

    Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka(220-228) nonapeptide representing an H-2Kb epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity-only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3(-)CD25(+) cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect. PMID:27070447

  7. Human DNA adduct measurements: state of the art.

    PubMed Central

    Poirier, M C; Weston, A

    1996-01-01

    Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either 32P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presented that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points. PMID:8933030

  8. Mechanochemical regulations of RPA's binding to ssDNA.

    PubMed

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J; Yan, Jie

    2015-01-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA. PMID:25787788

  9. Mechanochemical regulations of RPA's binding to ssDNA

    NASA Astrophysics Data System (ADS)

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J.; Yan, Jie

    2015-03-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

  10. Using evolutionary and structural information to predict DNA-binding sites on DNA-binding proteins.

    PubMed

    Kuznetsov, Igor B; Gou, Zhenkun; Li, Run; Hwang, Seungwoo

    2006-07-01

    Proteins that interact with DNA are involved in a number of fundamental biological activities such as DNA replication, transcription, and repair. A reliable identification of DNA-binding sites in DNA-binding proteins is important for functional annotation, site-directed mutagenesis, and modeling protein-DNA interactions. We apply Support Vector Machine (SVM), a supervised pattern recognition method, to predict DNA-binding sites in DNA-binding proteins using the following features: amino acid sequence, profile of evolutionary conservation of sequence positions, and low-resolution structural information. We use a rigorous statistical approach to study the performance of predictors that utilize different combinations of features and how this performance is affected by structural and sequence properties of proteins. Our results indicate that an SVM predictor based on a properly scaled profile of evolutionary conservation in the form of a position specific scoring matrix (PSSM) significantly outperforms a PSSM-based neural network predictor. The highest accuracy is achieved by SVM predictor that combines the profile of evolutionary conservation with low-resolution structural information. Our results also show that knowledge-based predictors of DNA-binding sites perform significantly better on proteins from mainly-alpha structural class and that the performance of these predictors is significantly correlated with certain structural and sequence properties of proteins. These observations suggest that it may be possible to assign a reliability index to the overall accuracy of the prediction of DNA-binding sites in any given protein using its sequence and structural properties. A web-server implementation of the predictors is freely available online at http://lcg.rit.albany.edu/dp-bind/.

  11. DNA chips with conjugated polyelectrolytes in resonance energy transfer mode.

    PubMed

    Wigenius, Jens A; Magnusson, Karin; Björk, Per; Andersson, Olof; Inganäs, Olle

    2010-03-01

    We show how to use well-defined conjugated polyelectrolytes (CPEs) combined with surface energy patterning to fabricate DNA chips utilizing fluorescence signal amplification. Cholesterol-modified DNA strands in complex with a CPE are adsorbed to a surface energy pattern, formed by printing with soft elastomer stamps. Hybridization of the surface bound DNA strands with a short complementary strand from solution is monitored using both fluorescence microscopy and imaging surface plasmon resonance. The CPEs act as antennas, enhancing resonance energy transfer to the dye-labeled DNA when complementary hybridization of the double strand occurs.

  12. PNA Directed Sequence Addressed Self-Assembly of DNA Nanostructures

    NASA Astrophysics Data System (ADS)

    Nielsen, Peter E.

    2008-10-01

    Peptide nucleic acids (PNA) can be designed to target duplex DNA with very high sequence specificity and efficiency via various binding modes. We have designed three domain PNA clamps, that bind stably to predefined decameric homopurine targets in large dsDNA molecules and via a third PNA domain sequence specifically recognize another PNA oligomer. We describe how such three domain PNAs have utility for assembling dsDNA grid and clover leaf structures, and in combination with SNAP-tag technology of protein dsDNA structures.

  13. Mitochondrial DNA, restoring Beethovens music.

    PubMed

    Merheb, Maxime; Vaiedelich, Stéphane; Maniguet, Thiérry; Hänni, Catherine

    2016-01-01

    Great ancient composers have endured many obstacles and constraints which are very difficult to understand unless we perform the restoration process of ancient music. Species identification in leather used during manufacturing is the key step to start such a restoration process in order to produce a facsimile of a museum piano. Our study reveals the species identification in the leather covering the hammer head in a piano created by Erard in 1802. This is the last existing piano similar to the piano that Beethoven used with its leather preserved in its original state. The leather sample was not present in a homogeneous piece, yet combined with glue. Using a DNA extraction method that avoids PCR inhibitors; we discovered that sheep and cattle are the origin of the combination. To identify the species in the leather, we focused on the amounts of mitochondrial DNA in both leather and glue and results have led us to the conclusion that the leather used to cover the hammer head in this piano was made of cattle hide. PMID:24617463

  14. Mitochondrial DNA, restoring Beethovens music.

    PubMed

    Merheb, Maxime; Vaiedelich, Stéphane; Maniguet, Thiérry; Hänni, Catherine

    2016-01-01

    Great ancient composers have endured many obstacles and constraints which are very difficult to understand unless we perform the restoration process of ancient music. Species identification in leather used during manufacturing is the key step to start such a restoration process in order to produce a facsimile of a museum piano. Our study reveals the species identification in the leather covering the hammer head in a piano created by Erard in 1802. This is the last existing piano similar to the piano that Beethoven used with its leather preserved in its original state. The leather sample was not present in a homogeneous piece, yet combined with glue. Using a DNA extraction method that avoids PCR inhibitors; we discovered that sheep and cattle are the origin of the combination. To identify the species in the leather, we focused on the amounts of mitochondrial DNA in both leather and glue and results have led us to the conclusion that the leather used to cover the hammer head in this piano was made of cattle hide.

  15. DNA-PKcs-Mediated Transcriptional Regulation Drives Prostate Cancer Progression and Metastasis.

    PubMed

    Goodwin, Jonathan F; Kothari, Vishal; Drake, Justin M; Zhao, Shuang; Dylgjeri, Emanuela; Dean, Jeffry L; Schiewer, Matthew J; McNair, Christopher; Jones, Jennifer K; Aytes, Alvaro; Magee, Michael S; Snook, Adam E; Zhu, Ziqi; Den, Robert B; Birbe, Ruth C; Gomella, Leonard G; Graham, Nicholas A; Vashisht, Ajay A; Wohlschlegel, James A; Graeber, Thomas G; Karnes, R Jeffrey; Takhar, Mandeep; Davicioni, Elai; Tomlins, Scott A; Abate-Shen, Cory; Sharifi, Nima; Witte, Owen N; Feng, Felix Y; Knudsen, Karen E

    2015-07-13

    Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies. PMID:26175416

  16. DNA-PKcs mediated transcriptional regulation drives prostate cancer progression and metastasis

    PubMed Central

    Goodwin, Jonathan F.; Kothari, Vishal; Drake, Justin M.; Zhao, Shuang; Dylgjeri, Emanuela; Dean, Jeffry L.; Schiewer, Matthew J.; McNair, Christopher; Jones, Jennifer K.; Aytes, Alvaro; Magee, Michael S.; Snook, Adam E.; Zhu, Ziqi; Den, Robert B.; Birbe, Ruth C.; Gomella, Leonard G.; Graham, Nicholas A.; Vashisht, Ajay A.; Wohlschlegel, James A.; Graeber, Thomas G.; Karnes, R. Jeffrey; Takhar, Mandeep; Davicioni, Elai; Tomlins, Scott A.; Abate-Shen, Cory; Sharifi, Nima; Witte, Owen N.; Feng, Felix Y.; Knudsen, Karen E.

    2015-01-01

    SUMMARY Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease, and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies. PMID:26175416

  17. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  18. DNA barcoding, phylogenetic relationships and speciation of snappers (genus Lutjanus).

    PubMed

    Wang, ZhongDuo; Guo, YuSong; Tan, Wei; Li, Lu; Tang, EnPu; Liu, ChuWu; Liu, Yun

    2010-08-01

    The phylogenetic relationships of 13 snapper species from the South China Sea have been established using the combined DNA sequences of three full-length mitochondrial genes (COI, COII and CYTB) and two partial nuclear genes (RAG1, RAG2). The 13 species (genus Lutjanus) were selected after DNA barcoding 72 individuals, representing 20 species. Our study suggests that although DNA barcoding aims to develop species identification systems, it may also be useful in the construction of phylogenies by aiding the selection of taxa. Combined mitochondrial and nuclear gene data has an advantage over an individual dataset because of its higher resolving power.

  19. Hierarchical-Multiplex DNA Patterns Mediated by Polymer Brush Nanocone Arrays That Possess Potential Application for Specific DNA Sensing.

    PubMed

    Liu, Wendong; Liu, Xueyao; Ge, Peng; Fang, Liping; Xiang, Siyuan; Zhao, Xiaohuan; Shen, Huaizhong; Yang, Bai

    2015-11-11

    This paper provides a facile and cost-efficient method to prepare single-strand DNA (ssDNA) nanocone arrays and hierarchical DNA patterns that were mediated by poly(2-hydroxyethyl methacrylate) (PHEMA) brush. The PHEMA brush nanocone arrays with different morphology and period were fabricated via colloidal lithography. The hierarchical structure was prepared through the combination of colloidal lithography and traditional photolithography. The DNA patterns were easily achieved via grafting the amino group modified ssDNA onto the side chain of polymer brush, and the anchored DNA maintained their reactivity. The as-prepared ssDNA nanocone arrays can be applied for target DNA sensing with the detection limit reaching 1.65 nM. Besides, with the help of introducing microfluidic ideology, the hierarchical-multiplex DNA patterns on the same substrate could be easily achieved with each kind of pattern possessing one kind of ssDNA, which are promising surfaces for the preparation of rapid, visible, and multiplex DNA sensors.

  20. Differential Nuclear and Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and Ancient DNA Studies

    PubMed Central

    Higgins, Denice; Rohrlach, Adam B.; Kaidonis, John; Townsend, Grant; Austin, Jeremy J.

    2015-01-01

    Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard

  1. DNA extraction from liquid blood using QIAamp.

    PubMed

    Scherczinger, C A; Bourke, M T; Ladd, C; Lee, H C

    1997-09-01

    The implementation of convicted felon DNA databases by increasing numbers of forensic science laboratories has engendered the need for a quick, efficient, and cost-effective method for the isolation of DNA from liquid blood samples. Because of the large numbers of samples involved, the ideal method would combine high throughput capability with maximal yield, high quality, and minimal time. We have found that the QIAGEN QIAamp Blood Kit/Tissue Kit satisfy all of these requirements. This simple, low cost spin column procedure yields purified DNA of approximately 20-30 kb that can be used directly in PCR or other enzymatic reactions without further purification. We compared the QIAamp isolation procedure to the standard SDS-Proteinase K/organic extraction/microcon purification procedure currently used by many forensic laboratories. The QIAamp procedure consistently gave a two- to four-fold increased yield relative to the organic extraction procedure. The DNA obtained was of high molecular weight, exhibited little degradation, and was suitable for RFLP and PCR analyses. We have found QIAGEN's QIAamp DNA isolation procedure to be ideally suited for preparation of samples for DNA databasing.

  2. A new approach for DNA detection by SERRS.

    PubMed

    Faulds, Karen; Fruk, Ljiljana; Robson, David C; Thompson, David G; Enright, Alexis; Smith, W Ewen; Graham, Duncan

    2006-01-01

    A new approach for the detection of DNA using surface enhance resonance Raman scattering (SERRS) is reported. The majority of existing techniques use fluorescence spectroscopy with advanced probe design to provide information on the identity of specific DNA sequences down to single base resolution. A new approach to the labelling of DNA is discussed which uses Michael addition to couple thiolated DNA to dye labels specifically designed to attach to silver surfaces. When combined with existing strategies for sensitive detection of DNA using commercially available labels, a new class of biomolecular probe known as a SERRS Beacon was produced. The detection techniques of fluorescence and surface enhanced resonance Raman scattering (SERRS) are combined to give a sensitive and selective system for use in the development and creation of novel assays for specifically defined targets. It demonstrates improved potential for multiplexing analysis. PMID:16833121

  3. A new approach for DNA detection by SERRS.

    PubMed

    Faulds, Karen; Fruk, Ljiljana; Robson, David C; Thompson, David G; Enright, Alexis; Smith, W Ewen; Graham, Duncan

    2006-01-01

    A new approach for the detection of DNA using surface enhance resonance Raman scattering (SERRS) is reported. The majority of existing techniques use fluorescence spectroscopy with advanced probe design to provide information on the identity of specific DNA sequences down to single base resolution. A new approach to the labelling of DNA is discussed which uses Michael addition to couple thiolated DNA to dye labels specifically designed to attach to silver surfaces. When combined with existing strategies for sensitive detection of DNA using commercially available labels, a new class of biomolecular probe known as a SERRS Beacon was produced. The detection techniques of fluorescence and surface enhanced resonance Raman scattering (SERRS) are combined to give a sensitive and selective system for use in the development and creation of novel assays for specifically defined targets. It demonstrates improved potential for multiplexing analysis.

  4. Routine DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  5. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  6. Translesion DNA synthesis

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Woodgate, Roger

    2014-01-01

    All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell’s replicase. Under these situations, cells are forced to choose between recombination-mediated “damage avoidance” pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. PMID:26442823

  7. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530