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Sample records for enhanced unscheduled dna

  1. Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes

    SciTech Connect

    Yarosh, D.B.; Kibitel, J.T.; Green, L.A.; Spinowitz, A. )

    1991-07-01

    Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population.

  2. Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres.

    PubMed

    Renier, A; Lévy, F; Pillière, F; Jaurand, M C

    1990-08-01

    Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

  3. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    PubMed

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

  4. Induction of pyrimidine dimers and unscheduled DNA synthesis in cultured mouse epithelial cells exposed to 254-nm- and u. v. -B radiation

    SciTech Connect

    Yotti, L.P.; Ley, R.D.

    1983-01-01

    The induction and fate of pyrimidine dimers and unscheduled DNA synthesis were measured in u.v.-irradiated primary, newborn SENCAR mouse epithelial cells. Unscheduled DNA synthesis was induced in a dose responsive manner by two u.v. sources, a germicidal lamp (254 nm) and an FS40 sunlamp (280--400 nm). Using the endonuclease-sensitive site assay to detect pyrimidine dimer production and excision, we examined the response of the newborn mouse cells to both u.v. sources. We were unable to detect the removal of pyrimidine dimers with either of the two sources of u.v. The speculation is made that primary, newborn mouse epidermal cells excise u.v.-induced pyrimidine dimers to an extent below the level of detection of the endonuclease-sensitive site assay but to an extent sufficient to induce unscheduled DNA synthesis.

  5. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.

    PubMed

    Hoffman, Elizabeth A; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz; Feng, Wenyi

    2015-03-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

  6. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription

    PubMed Central

    Hoffman, Elizabeth A.; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz

    2015-01-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites. PMID:25609572

  7. Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA

    SciTech Connect

    Sega, G.A.; Generoso, E.E.; Brimer, P.A. )

    1990-01-01

    A study of meiotic and postmeiotic germ-cell-stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid-spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg. By using 14C-labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.

  8. Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

    PubMed

    Generoso, W M; Sega, G A; Lockhart, A M; Hughes, L A; Cain, K T; Cacheiro, N L; Shelby, M D

    1996-12-20

    The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

  9. Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

    PubMed

    Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

    2016-01-01

    Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

  10. Induction of unscheduled DNA synthesis in primary cultures of rat pancreatic cells following in vivo and in vitro treatment with genotoxic agents

    SciTech Connect

    Steinmetz, K.L.; Mirsalis, J.C.

    1984-01-01

    The pancreas is a key target tissue in human and animal carcinogenesis, yet few short-term test systems measure genotoxicity in pancreatic cells. A method has been developed for the measurement of DNA repair as unscheduled DNA synthesis (UDS) in primary cultures of rat pancreatic cells (PRP) following in vitro or in vivo exposures to chemicals. PRP were isolated from female Sprague-Dawley (SD) or male Fischer-344 rats by mincing the pancreas in a collagenase solution followed by digestion in dispase. For vitro exposures, PRP were incubated with /sup 3/H-thymidine (/sup 3/H-TdR) in the presence of genotoxic agents for 18-22 hr. For in vivo exposures, male Fischer-344 rats were treated with genotoxic agents 2 hr prior to sacrifice of the animals, and cells were incubated with /sup 3/H-TdR. Cells isolated from female SD rats and treated in vitro with methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the pancreatic carcinogen azaserine all showed UDS. Cells treated in vitro with the hepatocarcinogens 2-acetylaminofluorene (2-AAF) and dimethylnitrosamine (DMN) and with the multisite carcinogen benzo(a) pyrene (B(a)P) yielded no UDS. In vivo treatment with MMS at 100 mg/kg and with azaserine at 100 mg/kg yielded varying degrees of UDS.

  11. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds

    SciTech Connect

    Mirsalis, J.C.; Tyson, C.K.; Steinmetz, K.L.; Loh, E.K.; Hamilton, C.M.; Bakke, J.P. ); Spalding, J.W. )

    1989-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with {sup 3}H-thymidine following in vivo treatment by gavage. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induced UDS in the liver; however, the significant S-phase response induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarinogenicity.

  12. Use of in vivo/in vitro unscheduled DNA synthesis for identification of organ-specific carcinogens.

    PubMed

    Furihata, C; Matsushima, T

    1987-01-01

    There are still only a few in vivo short-term assay methods for predicting potential organ-specific carcinogens and mutagens in mammals, although such methods are required for evaluating the in vivo effects of in vitro mutagens. In the in vivo/in vitro UDS assay methods described here, chemicals are given to experimental animals and induction of UDS in target organs is determined by in vitro organ culture or primary cell culture in the presence of [3H]dThd. Incorporation of [3H]dThd into DNA is measured with a liquid scintillation counter or by autoradiography. These methods have now been applied to the glandular stomach, forestomach, colon, liver, kidney, pancreas, tracheal epithelium, nasal epithelium, and spermatocytes. With minor modifications, they may also be applied to other organs. The present review shows that induction of UDS in various organs correlated well with the induction of cancer in these organs. The present authors have used the present methods to identify some potential organ-specific mutagens and carcinogens in mammals. The present authors found that three dicarbonyl compounds, glyoxal, methylglyoxal, and diacetyl, induced apparent UDS and TDS in the glandular stomach, and other groups found that 2-NT, MA6BT, and CNEt6BT induced UDS in the liver. These in vivo/in vitro UDS assays are better than in vitro UDS assay for identification of potential organ-specific mutagens and carcinogens in mammals and are especially useful for identifying potential mutagens and carcinogens that are specific for certain organs, such as the stomach, liver, and kidney. They are also useful for examining the potential mutagenicities and carcinogenicities of carcinogen analogs. However, these methods are not suitable for general in vivo screening because they are not yet available for all organs. A further advantage of the methods is that they can be used to examine larger numbers of animals at one time than other methods for detecting DNA damage, such as alkaline

  13. Correlation between mixed-function oxidase enzyme induction and aflatoxin B/sub 1/-induced unscheduled DNA synthesis in the chick embryo, in vivo

    SciTech Connect

    Hamilton, J.W.; Bloom, S.E.

    1984-01-01

    The unscheduled DNA synthesis (UDS) technique has been adapted for use in the chick embryo, in vivo, to determine the relationship between induction of the mixed-function oxidase (MFO) enzyme system and genetic damage from an indirect-acting mutagen-carcinogen. Embryos were injected at 6 days of incubation (DI) with either phenobarbital (PB), a specific inducer of P-450-associated enzyme activities, or 3,4,3',4'-tetrachlorobiphenyl (TCB), a specific inducer of P/sub 1/-450-associated enzyme activities. Aflatoxin B/sub 1/ (AFB1) was injected 24 hr later (7 DI), followed by a 5-hr continuous /sup 3/H-thymidine exposure. The livers were removed, prepared for autoradiography, and hepatocytes were scored for an increase in grains/nucleus, indicative of UDS. Aflatoxin B/sub 1/ caused a dose-related increase in UDS in all control and induction groups. Phenobarbital-induced embryos had an increased UDS response while TCB-induced embryos had a decreased UDS response, relative to noninduced embryos, for each dosage of AFB1. This suggests that the genotoxicity of an indirect-acting mutagen-carcinogen can be either increased or decreased, in vivo, depending on the inducer used. The chick embryo provides an excellent system for studying the effect of MFO induction on the genotoxicity of promutagen-carcinogens in a developing system.

  14. Use of in vivo/in vitro unscheduled DNA synthesis for identification of organ-specific carcinogens

    SciTech Connect

    Furihata, C.; Matsushima, T.

    1987-01-01

    There are still only a few in vivo short-term assay methods for predicting potential organ-specific carcinogens and mutagens in mammals, although such methods are required for evaluating the in vivo effects of in vitro mutagens. In the in vivo/in vitro UDS assay methods described here, chemicals are given to experimental animals and induction of UDS in target organs is determined by in vitro organ culture or primary cell culture in the presence of (/sup 3/H)dThd. Incorporation of (/sup 3/H)dThd into DNA is measured with a liquid scintillation counter or by autoradiography. These methods have now been applied to the glandular stomach, forestomach, colon, liver, kidney, pancreas, tracheal epithelium, nasal epithelium, and spermatocytes. With minor modifications, they may also be applied to other organs. The present review shows that induction of UDS in various organs correlated well with the induction of cancer in these organs. The present authors have used the present methods to identify some potential organ-specific mutagens and carcinogens in mammals. The present authors found that three dicarbonyl compounds, glyoxal, methylglyoxal, and diacetyl, induced apparent UDS and TDS in the glandular stomach, and other groups found that 2-NT, MA6BT, and CNEt6BT induced UDS in the liver. These in vivo/in vitro UDS assays are better than in vitro UDS assay for identification of potential organ-specific mutagens and carcinogens in mammals and are especially useful for identifying potential mutagens and carcinogens that are specific for certain organs, such as the stomach, liver, and kidney. They are also useful for examining the potential mutagenicities and carcinogenicities of carcinogen analogs. However, these methods are not suitable for general in vivo screening because they are not yet available for all organs. 113 references.

  15. 5 CFR 550.182 - Unscheduled duty.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Unscheduled duty. 550.182 Section 550.182 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY ADMINISTRATION (GENERAL) Premium Pay Law Enforcement Availability Pay § 550.182 Unscheduled duty. (a) Unscheduled Duty Hours....

  16. 5 CFR 550.182 - Unscheduled duty.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 5 Administrative Personnel 1 2012-01-01 2012-01-01 false Unscheduled duty. 550.182 Section 550.182 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY ADMINISTRATION (GENERAL) Premium Pay Law Enforcement Availability Pay § 550.182 Unscheduled duty. (a) Unscheduled Duty Hours....

  17. 5 CFR 550.182 - Unscheduled duty.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false Unscheduled duty. 550.182 Section 550.182 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY ADMINISTRATION (GENERAL) Premium Pay Law Enforcement Availability Pay § 550.182 Unscheduled duty. (a) Unscheduled Duty Hours....

  18. 5 CFR 550.182 - Unscheduled duty.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 1 2011-01-01 2011-01-01 false Unscheduled duty. 550.182 Section 550.182 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY ADMINISTRATION (GENERAL) Premium Pay Law Enforcement Availability Pay § 550.182 Unscheduled duty. (a) Unscheduled Duty Hours....

  19. 5 CFR 550.182 - Unscheduled duty.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Unscheduled duty. 550.182 Section 550.182 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY ADMINISTRATION (GENERAL) Premium Pay Law Enforcement Availability Pay § 550.182 Unscheduled duty. (a) Unscheduled Duty Hours....

  20. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in primary melanocytes.

    PubMed

    Thompson, Benjamin C; Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2014-07-01

    Cutaneous melanoma is a significant cause of morbidity and mortality. Nicotinamide is a safe, widely available vitamin that reduces the immune suppressive effects of UV, enhances DNA repair in keratinocytes and has shown promise in the chemoprevention of non-melanoma skin cancer. Here, we report the effect of nicotinamide on DNA damage and repair in primary human melanocytes. Nicotinamide significantly enhanced the repair of oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine) and cyclobutane pyrimidine dimers induced by UV exposure. It also enhanced the repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine induced by the culture conditions in unirradiated melanocytes. A significant increase in the percentage of melanocytes undergoing unscheduled but not scheduled DNA synthesis was observed, confirming that nicotinamide enhances DNA repair in human melanocytes. In summary, nicotinamide, by enhancing DNA repair in melanocytes, is a potential agent for the chemoprevention of cutaneous melanoma.

  1. Suomi-NPP VIIRS unscheduled lunar observations

    NASA Astrophysics Data System (ADS)

    Wang, Zhipeng; Xiong, Xiaoxiong; Fulbright, Jon P.

    2016-10-01

    Lunar observations by the Suomi-NPP instrument VIIRS are scheduled on a nearly monthly basis at a phase angle of approximately -51 degrees. The lunar images acquired during scheduled observations have been used for radiometric calibration stability monitoring of the reflective solar bands, band-to-band registration characterization, modulation transfer function derivation and electric crosstalk examination. A satellite roll maneuver is usually necessary for the Moon to be viewed by VIIRS detectors, which results in the loss of approximately 20-minute science data during the period. Without any scheduling, the Moon has also been regularly observed when it intrudes the field of view of the instrument's space view port. Since the launch of Suomi-NPP in late 2011, nearly 200 unscheduled lunar observations have been made with complete lunar images captured by at least two spectral bands. These observations are made at a larger phase angle from -45 to -90 degrees and libration angle range than the scheduled lunar observation. In this paper, the strategies and methodologies of lunar calibration developed for scheduled lunar observations are applied to these unscheduled lunar observations, with necessary adaptation to account for the differences in data format. The result from the unscheduled lunar observations are provided, with the focus of it comparison with the results from scheduled lunar observations as well as solar diffuser (SD) calibration. Overall, the long-term trends of these results agree with each other and the trends from the un-scheduled lunar calibration show more fluctuation. For radiometric calibration, the difference between the lunar calibration and SD calibration strongly depends on phase angles and libration angles. If the VIIRS measurement is accurate, this indicates that the lunar irradiance reference for the radiometric calibration, modeled by the USGS robotic lunar observatory (ROLO), carries systematic error that changes with these photometric

  2. 40 CFR 86.429-78 - Maintenance, unscheduled; test vehicles.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 18 2011-07-01 2011-07-01 false Maintenance, unscheduled; test... Emission Regulations for 1978 and Later New Motorcycles, General Provisions § 86.429-78 Maintenance... advance approval of the Administrator. (1) In the case of unscheduled maintenance, such approval will...

  3. Molecular mechanisms for enhanced DNA vaccine immunogenicity

    PubMed Central

    Li, Lei; Petrovsky, Nikolai

    2016-01-01

    Summary In the two decades since their initial discovery, DNA vaccines technologies have come a long way. Unfortunately, when applied to human subjects inadequate immunogenicity is still the biggest challenge for practical DNA vaccine use. Many different strategies have been tested in preclinical models to address this problem, including novel plasmid vectors and codon optimization to enhance antigen expression, new gene transfection systems or electroporation to increase delivery efficiency, protein or live virus vector boosting regimens to maximise immune stimulation, and formulation of DNA vaccines with traditional or molecular adjuvants. Better understanding of the mechanisms of action of DNA vaccines has also enabled better use of the intrinsic host response to DNA to improve vaccine immunogenicity. This review summarizes recent advances in DNA vaccine technologies and related intracellular events and how these might impact on future directions of DNA vaccine development. PMID:26707950

  4. Molecular mechanisms for enhanced DNA vaccine immunogenicity.

    PubMed

    Li, Lei; Petrovsky, Nikolai

    2016-01-01

    In the two decades since their initial discovery, DNA vaccines technologies have come a long way. Unfortunately, when applied to human subjects inadequate immunogenicity is still the biggest challenge for practical DNA vaccine use. Many different strategies have been tested in preclinical models to address this problem, including novel plasmid vectors and codon optimization to enhance antigen expression, new gene transfection systems or electroporation to increase delivery efficiency, protein or live virus vector boosting regimens to maximise immune stimulation, and formulation of DNA vaccines with traditional or molecular adjuvants. Better understanding of the mechanisms of action of DNA vaccines has also enabled better use of the intrinsic host response to DNA to improve vaccine immunogenicity. This review summarizes recent advances in DNA vaccine technologies and related intracellular events and how these might impact on future directions of DNA vaccine development.

  5. Enhancing the DNA Patent Database

    SciTech Connect

    Walters, LeRoy B.

    2008-02-18

    Final Report on Award No. DE-FG0201ER63171 Principal Investigator: LeRoy B. Walters February 18, 2008 This project successfully completed its goal of surveying and reporting on the DNA patenting and licensing policies at 30 major U.S. academic institutions. The report of survey results was published in the January 2006 issue of Nature Biotechnology under the title “The Licensing of DNA Patents by US Academic Institutions: An Empirical Survey.” Lori Pressman was the lead author on this feature article. A PDF reprint of the article will be submitted to our Program Officer under separate cover. The project team has continued to update the DNA Patent Database on a weekly basis since the conclusion of the project. The database can be accessed at dnapatents.georgetown.edu. This database provides a valuable research tool for academic researchers, policymakers, and citizens. A report entitled Reaping the Benefits of Genomic and Proteomic Research: Intellectual Property Rights, Innovation, and Public Health was published in 2006 by the Committee on Intellectual Property Rights in Genomic and Protein Research and Innovation, Board on Science, Technology, and Economic Policy at the National Academies. The report was edited by Stephen A. Merrill and Anne-Marie Mazza. This report employed and then adapted the methodology developed by our research project and quoted our findings at several points. (The full report can be viewed online at the following URL: http://www.nap.edu/openbook.php?record_id=11487&page=R1). My colleagues and I are grateful for the research support of the ELSI program at the U.S. Department of Energy.

  6. Polymer multilayer tattooing for enhanced DNA vaccination

    NASA Astrophysics Data System (ADS)

    Demuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

    2013-04-01

    DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

  7. 36 CFR 1238.26 - What are the restrictions on use for permanent and unscheduled microform records?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... permanent and unscheduled microform records? (a) Agencies must not use the silver gelatin master microform or duplicate silver gelatin microform of permanent or unscheduled records created in accordance...

  8. 36 CFR 1238.26 - What are the restrictions on use for permanent and unscheduled microform records?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... permanent and unscheduled microform records? (a) Agencies must not use the silver gelatin master microform or duplicate silver gelatin microform of permanent or unscheduled records created in accordance...

  9. 36 CFR 1238.26 - What are the restrictions on use for permanent and unscheduled microform records?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... permanent and unscheduled microform records? (a) Agencies must not use the silver gelatin master microform or duplicate silver gelatin microform of permanent or unscheduled records created in accordance...

  10. 36 CFR 1238.26 - What are the restrictions on use for permanent and unscheduled microform records?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... permanent and unscheduled microform records? (a) Agencies must not use the silver gelatin master microform or duplicate silver gelatin microform of permanent or unscheduled records created in accordance...

  11. 36 CFR 1238.26 - What are the restrictions on use for permanent and unscheduled microform records?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... permanent and unscheduled microform records? (a) Agencies must not use the silver gelatin master microform or duplicate silver gelatin microform of permanent or unscheduled records created in accordance...

  12. Enhanced tumorigenicity by mitochondrial DNA mild mutations.

    PubMed

    Cruz-Bermúdez, Alberto; Vallejo, Carmen G; Vicente-Blanco, Ramiro J; Gallardo, María Esther; Fernández-Moreno, Miguel Ángel; Quintanilla, Miguel; Garesse, Rafael

    2015-05-30

    To understand how mitochondria are involved in malignant transformation we have generated a collection of transmitochondrial cybrid cell lines on the same nuclear background (143B) but with mutant mitochondrial DNA (mtDNA) variants with different degrees of pathogenicity. These include the severe mutation in the tRNALys gene, m.8363G>A, and the three milder yet prevalent Leber's hereditary optic neuropathy (LHON) mutations in the MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C) mitochondrial genes. We found that 143B ρ0 cells devoid of mtDNA and cybrids harboring wild type mtDNA or that causing severe mitochondrial dysfunction do not produce tumors when injected in nude mice. By contrast cybrids containing mild mutant mtDNAs exhibit different tumorigenic capacities, depending on OXPHOS dysfunction.The differences in tumorigenicity correlate with an enhanced resistance to apoptosis and high levels of NOX expression. However, the final capacity of the different cybrid cell lines to generate tumors is most likely a consequence of a complex array of pro-oncogenic and anti-oncogenic factors associated with mitochondrial dysfunction.Our results demonstrate the essential role of mtDNA in tumorigenesis and explain the numerous and varied mtDNA mutations found in human tumors, most of which give rise to mild mitochondrial dysfunction.

  13. Propulsion element requirements using electrical power system unscheduled power

    NASA Technical Reports Server (NTRS)

    Zimmermann, Frank; Hodge, Kathy

    1989-01-01

    The suitability of using the electrical energy from the Space Station's Electrical Power System (EPS) during the periods of peak solar insolation which is currently not specifically allocated (unscheduled power) to produce propulsion propellants, gaseous hydrogen, and oxygen by electrolyzing water is investigated. Reboost propellant requirements are emphasized, but the results are more generally relevant because the balance of recurring propellant requirements are an order of magnitude smaller and the nonrecurring requirements are not significant on an average basis.

  14. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.

    PubMed

    Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2013-05-01

    Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

  15. Arsenic Exposure Induces Unscheduled Mitotic S Phase Entry Coupled with Cell Death in Mouse Cortical Astrocytes

    PubMed Central

    Htike, Nang T. T.; Maekawa, Fumihiko; Soutome, Haruka; Sano, Kazuhiro; Maejima, Sho; Aung, Kyaw H.; Tokuda, Masaaki; Tsukahara, Shinji

    2016-01-01

    There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 μM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings

  16. Unscheduled return visits to a Dutch inner-city emergency department

    PubMed Central

    2014-01-01

    Background Unscheduled return visits to the emergency department (ED) may reflect shortcomings in care. This study characterized ED return visits with respect to incidence, risk factors, reasons and post-ED disposition. We hypothesized that risk factors for unscheduled return and reasons for returning would differ from previous studies, due to differences in health care systems. Methods All unscheduled return visits occurring within 1 week and related to the initial ED visit were selected. Multivariable logistic regression was conducted to determine independent factors associated with unscheduled return, using patient information available at the initial visit. Reasons for returning unscheduled were categorized into illness-, patient- or physician-related. Post-ED disposition was compared between patients with unscheduled return visits and the patients who did not return. Results Five percent (n = 2,492) of total ED visits (n = 49,341) were unscheduled return visits. Patients with an urgent triage level, patients presenting during the night shift, with a wound or local infection, abdominal pain or urinary problems were more likely to return unscheduled. Reasons to revisit unscheduled were mostly illness-related (49%) or patient-related (41%). Admission rates for returning patients (16%) were the same as for the patients who did not return (17%). Conclusions Apart from abdominal complaints, risk factors for unscheduled return differ from previous studies. Short-term follow-up at the outpatient clinic or general practitioner for patients with urgent triage levels and suffering from wounds or local infections, abdominal pain or urinary problem might prevent unscheduled return. PMID:25045407

  17. A Conceptual Model for Episodes of Acute, Unscheduled Care.

    PubMed

    Pines, Jesse M; Lotrecchiano, Gaetano R; Zocchi, Mark S; Lazar, Danielle; Leedekerken, Jacob B; Margolis, Gregg S; Carr, Brendan G

    2016-10-01

    We engaged in a 1-year process to develop a conceptual model representing an episode of acute, unscheduled care. Acute, unscheduled care includes acute illnesses (eg, nausea and vomiting), injuries, or exacerbations of chronic conditions (eg, worsening dyspnea in congestive heart failure) and is delivered in emergency departments, urgent care centers, and physicians' offices, as well as through telemedicine. We began with a literature search to define an acute episode of care and to identify existing conceptual models used in health care. In accordance with this information, we then drafted a preliminary conceptual model and collected stakeholder feedback, using online focus groups and concept mapping. Two technical expert panels reviewed the draft model, examined the stakeholder feedback, and discussed ways the model could be improved. After integrating the experts' comments, we solicited public comment on the model and made final revisions. The final conceptual model includes social and individual determinants of health that influence the incidence of acute illness and injury, factors that affect care-seeking decisions, specific delivery settings where acute care is provided, and outcomes and costs associated with the acute care system. We end with recommendations for how researchers, policymakers, payers, patients, and providers can use the model to identify and prioritize ways to improve acute care delivery.

  18. Unscheduled Telephone Calls to Measure Percent Syllables Stuttered during Clinical Trials

    ERIC Educational Resources Information Center

    Karimi, Hamid; O'Brian, Sue; Onslow, Mark; Jones, Mark; Menzies, Ross; Packman, Ann

    2013-01-01

    Purpose: Researchers have used unscheduled telephone calls for many years during clinical trials to measure adult stuttering severity before and after treatment. Because variability is a hallmark of stuttering severity with adults, it is questionable whether an unscheduled telephone call is truly representative of their everyday speech. Method:…

  19. 15 CFR Supplement No. 1 to Part 715 - Definition of an Unscheduled Discrete Organic Chemical

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Organic Chemical No. Supplement No. 1 to Part 715 Commerce and Foreign Trade Regulations Relating to... WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 1 Supplement No. 1 to Part 715—Definition of an Unscheduled Discrete Organic Chemical...

  20. 15 CFR Supplement No. 1 to Part 715 - Definition of an Unscheduled Discrete Organic Chemical

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Organic Chemical No. Supplement No. 1 to Part 715 Commerce and Foreign Trade Regulations Relating to... WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 1 Supplement No. 1 to Part 715—Definition of an Unscheduled Discrete Organic Chemical...

  1. 15 CFR Supplement No. 1 to Part 715 - Definition of an Unscheduled Discrete Organic Chemical

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Organic Chemical No. Supplement No. 1 to Part 715 Commerce and Foreign Trade Regulations Relating to... WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 1 Supplement No. 1 to Part 715—Definition of an Unscheduled Discrete Organic Chemical...

  2. 15 CFR Supplement No. 1 to Part 715 - Definition of an Unscheduled Discrete Organic Chemical

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Organic Chemical No. Supplement No. 1 to Part 715 Commerce and Foreign Trade Regulations Relating to... WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 1 Supplement No. 1 to Part 715—Definition of an Unscheduled Discrete Organic Chemical...

  3. 15 CFR Supplement No. 1 to Part 715 - Definition of an Unscheduled Discrete Organic Chemical

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Organic Chemical No. Supplement No. 1 to Part 715 Commerce and Foreign Trade Regulations Relating to... WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 1 Supplement No. 1 to Part 715—Definition of an Unscheduled Discrete Organic Chemical...

  4. 15 CFR 715.1 - Annual declaration requirements for production by synthesis of unscheduled discrete organic...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... production by synthesis of unscheduled discrete organic chemicals (UDOCs). 715.1 Section 715.1 Commerce and... DISCRETE ORGANIC CHEMICALS (UDOCs) § 715.1 Annual declaration requirements for production by synthesis of unscheduled discrete organic chemicals (UDOCs). (a) Declaration of production by synthesis of UDOCs...

  5. Therapeutic Options for Unscheduled Bleeding Associated with Long-Acting Reversible Contraception.

    PubMed

    Friedlander, EmmaKate; Kaneshiro, Bliss

    2015-12-01

    Long-acting reversible contraception (LARC) is the most effective form of reversible contraception. Although most women are satisfied with LARC methods, unscheduled bleeding and spotting are common reasons for method dissatisfaction and discontinuation. This systematic analysis of the current literature delineates treatment options for unscheduled bleeding related to LARC use. Although consistent results are lacking, all devices seem to have the best response to nonsteroidal antiinflammatory drugs for 5 to 7 days or the antifibrinolytic agent tranexamic acid. Additional studies are necessary to identify improved treatment interventions for unscheduled bleeding with LARC use.

  6. Active DNA demethylation at enhancers during the vertebrate phylotypic period.

    PubMed

    Bogdanović, Ozren; Smits, Arne H; de la Calle Mustienes, Elisa; Tena, Juan J; Ford, Ethan; Williams, Ruth; Senanayake, Upeka; Schultz, Matthew D; Hontelez, Saartje; van Kruijsbergen, Ila; Rayon, Teresa; Gnerlich, Felix; Carell, Thomas; Veenstra, Gert Jan C; Manzanares, Miguel; Sauka-Spengler, Tatjana; Ecker, Joseph R; Vermeulen, Michiel; Gómez-Skarmeta, José Luis; Lister, Ryan

    2016-04-01

    The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

  7. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    SciTech Connect

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  8. 76 FR 16691 - Western Electric Coordinating Council Qualified Transfer Path Unscheduled Flow Relief Regional...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-25

    ... Federal Power Act, the Commission approves regional Reliability Standard of the Western Electricity... Electricity Coordinating Council (WECC) IRO-006-WECC-1 (Qualified Transfer Path Unscheduled Flow Relief) and...: Balancing Authorities and Reliability Coordinator in the Western Electricity Coordinating Council....

  9. Enhancing the Immunogenicity of a Tetravalent Dengue DNA Vaccine

    DTIC Science & Technology

    2016-08-01

    AWARD NUMBER: W81XWH-15-2-0029 TITLE: Enhancing the Immunogenicity of a Tetravalent Dengue DNA Vaccine PRINCIPAL INVESTIGATOR: Maya...TITLE AND SUBTITLE Enhancing the Immunogenicity of a Tetravalent Dengue DNA 5a. CONTRACT NUMBER Vaccine 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...the top infectious diseases that afflict US Military personnel deployed overseas. Developing a successful vaccine to prevent dengue fever in DoD

  10. Direct surface-enhanced Raman scattering analysis of DNA duplexes.

    PubMed

    Guerrini, Luca; Krpetić, Željka; van Lierop, Danny; Alvarez-Puebla, Ramon A; Graham, Duncan

    2015-01-19

    The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface-enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label-free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5-methylated cytosine and N6-methylated Adenine) in duplexes.

  11. Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy.

    PubMed

    Barhoumi, Aoune; Halas, Naomi J

    2011-12-15

    Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.

  12. Enhanced structural stability of DNA origami nanostructures by graphene encapsulation

    NASA Astrophysics Data System (ADS)

    Matković, Aleksandar; Vasić, Borislav; Pešić, Jelena; Prinz, Julia; Bald, Ilko; Milosavljević, Aleksandar R.; Gajić, Radoš

    2016-02-01

    We demonstrate that a single-layer graphene replicates the shape of DNA origami nanostructures very well. It can be employed as a protective layer for the enhancement of structural stability of DNA origami nanostructures. Using the AFM based manipulation, we show that the normal force required to damage graphene encapsulated DNA origami nanostructures is over an order of magnitude greater than for the unprotected ones. In addition, we show that graphene encapsulation offers protection to the DNA origami nanostructures against prolonged exposure to deionized water, and multiple immersions. Through these results we demonstrate that graphene encapsulated DNA origami nanostructures are strong enough to sustain various solution phase processing, lithography and transfer steps, thus extending the limits of DNA-mediated bottom-up fabrication.

  13. Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA

    SciTech Connect

    Leadon, S.A.; Hanawalt, P.C.

    1984-11-01

    Excision repair of bulky adducts in alpha DNA of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have found that u.v. irradiation of these cells results in the enhanced removal of both aflatoxin B1 (AFB1) and acetylaminofluorene (AAF) adducts from the alpha DNA sequences without affecting repair in the bulk of the DNA. The degree of enhanced removal of AFB1 is dependent upon the u.v. dose and the time interval between irradiation and AFB1 treatment. The u.v. enhancement is not inhibited by cycloheximide. Exposure of the cells to dimethylsulfate or gamma-rays does not affect AFB1 adduct repair. The formation and removal of N-acetoxy-2-acetylaminofluorene (NA-AAF) adducts from alpha and bulk DNA was studied in detail. A higher initial level of the acetylated C8 adduct of guanine was found in alpha DNA than in bulk DNA. Although both the acetylated and deacetylated C8 adducts were removed from the two DNA species, the level of repair was significantly greater in the bulk DNA. Irradiation of cells with u.v. prior to treatment with NA-AAF enhanced the removal of both adducts from alpha DNA with little or no effect on repair in bulk DNA. We conclude that the presence of u.v. photoproducts or some intermediate in their processing alters the chromatin structure of alpha DNA thereby rendering bulky adducts accessible to repair enzymes. In addition, the differential formation and repair of AAF adducts in alpha DNA compared with that in the bulk of the genome supports the hypothesis of an altered chromatin structure for alpha domains.

  14. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

    NASA Technical Reports Server (NTRS)

    Nordheim, A.; Rich, A.

    1983-01-01

    Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

  15. Effect of aging and dietary restriction on DNA repair

    SciTech Connect

    Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

    1989-03-01

    DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

  16. Loneliness and pregnancy in an urban Latino community: associations with maternal age and unscheduled hospital utilization.

    PubMed

    Geller, Jeffrey S

    2004-01-01

    The objective is to compare loneliness in a pregnant population to a non-pregnant control group, and to evaluate loneliness and unscheduled hospital visits during pregnancy. A prospective cohort study in a Latino urban community including 53 consecutive pregnant women in their first trimester, and 61 non-pregnant women as a control. The UCLA Loneliness Scale version 3, and demographic information was collected. A chart review after delivery determined total number of unscheduled pregnancy related hospital visits. Appropriate data analysis using t-test and regression analysis was used. Forty-eight women continued to delivery. There was no difference in mean loneliness scores between pregnant (41) and non-pregnant groups (43), or that of normal populations (41). There was a significant association between UCLA loneliness scores and total pregnancy related unscheduled hospital visits p = 0.042, beta = 0.06, r= 0.29. There was a significant association between increasing age and increasing loneliness during pregnancy p = 0.007, beta = 0.21, r= 0.36, not seen in the non-pregnant group p = 0.98. Loneliness, when controlling for age, yielded a stronger association with unscheduled hospital visits p = 0.018, beta = 0.076, and r = 0.40. The findings were that increased loneliness is associated with increased unscheduled pregnancy related hospital utilization during pregnancy. Older pregnant women had higher loneliness scores. Loneliness was more significant than age in predicting higher unscheduled hospital visits. The combination of increased loneliness and younger age predicted the highest number of unscheduled hospital visits.

  17. New approaches to biochemical radioprotection: antioxidants and DNA repair enhancement

    NASA Astrophysics Data System (ADS)

    Riklis, E.; Emerit, I.; Setlow, R. B.

    Chemical repair may be provided by radioprotective compounds present during exposure to ionizing radiation. Considering DNA as the most sensitive target it is feasible to biochemically improve protection by enhancing DNA repair mechanisms. Protection of DNA by reducing the amount of damage (by radical scavenging and chemical repair) followed by enhanced repair of DNA will provide much improved protection and recovery. Furthermore, in cases of prolonged exposure, such as is possible in prolonged space missions, or of unexpected variations in the intensity of radiation, as is possible when encountering solar flares, it is important to provide long-acting protection, and this may be provided by antioxidants and well functioning DNA repair systems. It has also become important to provide protection from the potentially damaging action of long-lived clastogenic factors which have been found in plasma of exposed persons from Hiroshima & Nagasaki, radiation accidents, radiotherapy patients and recently in ``liquidators'' - persons involved in salvage operations at the Chernobyl reactor. The clastogenic factor, which causes chromatid breaks in non-exposed plasma, might account for late effects and is posing a potential carcinogenic hazard /1/. The enzyme superoxide dismutase (SOD) has been shown to eliminate the breakage factor from cultured plasma of exposed persons /2/. Several compounds have been shown to enhance DNA repair: WR-2721 /3/, nicotinamide /4/, glutathione monoester (Riklis et al., unpublished) and others. The right combination of such compounds may prove effective in providing protection from a wide range of radiation exposures over a long period of time.

  18. B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles

    NASA Technical Reports Server (NTRS)

    Gruskin, E. A.; Rich, A.

    1993-01-01

    During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

  19. AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA.

    PubMed Central

    Guo, W; Tang, W J; Bu, X; Bermudez, V; Martin, M; Folk, W R

    1996-01-01

    An early step in the initiation of polyomavirus DNA replication is viral large-T-antigen-mediated unwinding of the origin. We report that components of the AP1 transcription factor, Fos and Jun, interact with T antigen in vitro to enhance unwinding of the viral origin. This provides a biochemical basis for the capacity of AP1 to activate viral DNA replication in vivo. PMID:8763994

  20. 15 CFR Supplement No. 2 to Part 715 - Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Organic Chemicals (UDOCs) and UDOC Production No. Supplement No. 2 to Part 715 Commerce and Foreign Trade... COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 2 Supplement No. 2 to Part 715—Examples of Unscheduled Discrete...

  1. 15 CFR Supplement No. 2 to Part 715 - Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Organic Chemicals (UDOCs) and UDOC Production No. Supplement No. 2 to Part 715 Commerce and Foreign Trade... COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 2 Supplement No. 2 to Part 715—Examples of Unscheduled Discrete...

  2. 15 CFR Supplement No. 2 to Part 715 - Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Organic Chemicals (UDOCs) and UDOC Production No. Supplement No. 2 to Part 715 Commerce and Foreign Trade... COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 2 Supplement No. 2 to Part 715—Examples of Unscheduled Discrete...

  3. 15 CFR Supplement No. 2 to Part 715 - Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Organic Chemicals (UDOCs) and UDOC Production No. Supplement No. 2 to Part 715 Commerce and Foreign Trade... COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING UNSCHEDULED DISCRETE ORGANIC CHEMICALS (UDOCs) Pt. 715, Supp. 2 Supplement No. 2 to Part 715—Examples of Unscheduled Discrete...

  4. Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone

    SciTech Connect

    Rinaldy, A.; Bellew, T.; Egli, E.; Lloyd, R.S. )

    1990-09-01

    Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells to the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure.

  5. Suppression and enhancement of transcriptional noise by DNA looping

    NASA Astrophysics Data System (ADS)

    Vilar, Jose M. G.; Saiz, Leonor

    2014-06-01

    DNA looping has been observed to enhance and suppress transcriptional noise but it is uncertain which of these two opposite effects is to be expected for given conditions. Here, we derive analytical expressions for the main quantifiers of transcriptional noise in terms of the molecular parameters and elucidate the role of DNA looping. Our results rationalize paradoxical experimental observations and provide the first quantitative explanation of landmark individual-cell measurements at the single molecule level on the classical lac operon genetic system [Choi, L. Cai, K. Frieda, and X. S. Xie, Science 322, 442 (2008), 10.1126/science.1161427].

  6. Fluorescence enhancement of DNA-silver nanoclusters from guanine proximity

    SciTech Connect

    Yeh, Hsin-chih; Sharma, Jaswinder; Yoo, Hyojong; Martinez, Jennifer S

    2010-01-01

    Oligonucleotide-templated, silver nanoclusters (DNA/Ag NCs) are a versatile set of fluorophores and have already been used for live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. Compared to commonly used organic dyes, these fluorescent nanoclusters have much better photostability and are often a few times brighter. Owing to their small size, simple preparation, and biocompatibility (i.e. made of nontoxic metals), DNA/Ag NCs should find more applications in biological imaging and chemical detection in the years to come. While clearly promising as new fluorophores, DNA/Ag NCs possess a unique and poorly understood dynamic process not shared by organic dyes or photoluminescent nanocrystals - the conversion among different NC species due to silver oxidation/reduction or NC regrouping. While this environmental sensitivity can be viewed as a drawback, in the appropriate context, it can be used as a sensor or reporter. Often reversible, conversions among different NC species have been found to depend upon a number of factors, including time, temperature, oxygen and salt content. In this communication, we report significant fluorescence enhancement of DNA/Ag NCs via interactions with guanine-rich DNA sequences. Moreover, we demonstrated this property can be used for sensitive detection of specific target DNA from a human oncogene (i.e. Braf gene).

  7. Enhanced photoacoustic signal from DNA assembled gold nanoparticle networks

    NASA Astrophysics Data System (ADS)

    Buchkremer, A.; Beckmann, M. F.; Linn, M.; Ruff, J.; Rosencrantz, R. R.; von Plessen, G.; Schmitz, G.; Simon, U.

    2014-12-01

    We report an experimental finding of photoacoustic signal enhancement from finite sized DNA-gold nanoparticle networks. We synthesized DNA-functionalized hollow and solid gold nanospheres (AuNS) to form finite sized networks, which were characterized by means of optical extinction spectroscopy, dynamic light scattering, and scanning electron microscopy in transmission mode. It is shown that the signal amplification scales with network size for networks comprising either hollow or solid AuNS as well as networks consisting of both types of nanoparticles. The laser intensities applied in our multispectral setup (λ = 650 nm, 850 nm, 905 nm) were low enough to maintain the structural integrity of the networks. This reflects that the binding and recognition properties of the temperature-sensitive cross-linking DNA-molecules are retained.

  8. DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

    PubMed Central

    Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B.

    2013-01-01

    Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. PMID:24101469

  9. 15 CFR 715.1 - Annual declaration requirements for production by synthesis of unscheduled discrete organic...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... production by synthesis of unscheduled discrete organic chemicals (UDOCs). 715.1 Section 715.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING...

  10. 15 CFR 715.1 - Annual declaration requirements for production by synthesis of unscheduled discrete organic...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... production by synthesis of unscheduled discrete organic chemicals (UDOCs). 715.1 Section 715.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING...

  11. 15 CFR 715.1 - Annual declaration requirements for production by synthesis of unscheduled discrete organic...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... production by synthesis of unscheduled discrete organic chemicals (UDOCs). 715.1 Section 715.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING...

  12. Histone H2A significantly enhances in vitro DNA transfection.

    PubMed Central

    Balicki, D.; Beutler, E.

    1997-01-01

    BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery. PMID:9407553

  13. DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

    PubMed

    Kou, Bei-Bei; Zhang, Li; Xie, Hua; Wang, Ding; Yuan, Ya-Li; Chai, Ya-Qin; Yuan, Ruo

    2016-09-07

    Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases.

  14. Enhancing intersystem crossing in phenotiazinium dyes by intercalation into DNA.

    PubMed

    Nogueira, Juan J; Oppel, Markus; González, Leticia

    2015-03-27

    Phenothiazinium dyes are used as photosensitizers in photodynamic therapy. Their mode of action is related to the generation of triplet excited states by intersystem crossing. Therefore, rationalizing the factors that influence intersystem crossing is crucial to improve the efficacy of photodynamic therapy. Here we employ quantum mechanics/molecular mechanics calculations to investigate the effect of aqueous and nucleic acid environments on the intersystem crossing mechanism in methylene blue. We find that the mechanism by which the triplet states are generated depends strongly on the environment. While intersystem crossing in water is mediated exclusively by vibronic spin-orbit coupling, it is enhanced in DNA due to a second pathway driven by electronic spin-orbit coupling. Competing charge-transfer processes, which are also possible in the presence of DNA, can therefore be suppressed by a suitable structural functionalization, thereby increasing the efficacy of photodynamic therapy.

  15. Surface-enhanced Raman scattering spectroscopy of topotecan-DNA complexes: Binding to DNA induces topotecan dimerization

    NASA Astrophysics Data System (ADS)

    Mochalov, K. E.; Strel'Tsov, S. A.; Ermishov, M. A.; Grokhovskii, S. L.; Zhuze, A. L.; Ustinova, O. A.; Sukhanova, A. V.; Nabiev, I. R.; Oleinikov, V. A.

    2002-09-01

    The interaction of topotecan (TPT), antitumor inhibitor of human DNA topoisomerase I, with calf thymus DNA was studied by surface-enhanced Raman scattering (SERS) spectroscopy. The SERS spectra of TPT are found to depend on its concentration in solution, which is associated with the dimerization of TPT. The spectral signatures of dimerization are identified. It is shown that binding to DNA induces the formation of TPT dimers. The formation of DNA-TPT-TPT-DNA complexes is considered as one of the possible mechanisms of human DNA topoisomerase I inhibition.

  16. Resonance Enhanced Multi-photon Spectroscopy of DNA

    NASA Astrophysics Data System (ADS)

    Ligare, Marshall Robert

    For over 50 years DNA has been studied to better understand its connection to life and evolution. These past experiments have led to our understanding of its structure and function in the biological environment but the interaction of DNA with UV radiation at the molecular level is still not very well understood. Unique mechanisms in nucleobase chromaphores protect us from adverse chemical reactions after UV absorption. Studying these processes can help develop theories for prebiotic chemistry and the possibility of alternative forms of DNA. Using resonance enhanced multi-photon spectroscopic techniques in the gas phase allow for the structure and dynamics of individual nucleobases to be studied in detail. Experiments studying different levels of structure/complexity with relation to their biological function are presented. Resonant IR multiphoton dissociation spectroscopy in conjunction with molecular mechanics and DFT calculations are used to determine gas phase structures of anionic nucleotide clusters. A comparison of the identified structures with known biological function shows how the hydrogen bonding of the nucleotides and their clusters free of solvent create favorable structures for quick incorporation into enzymes such as DNA polymerase. Resonance enhanced multi-photon ionization (REMPI) spectroscopy techniques such as resonant two photon ionization (R2PI) and IR-UV double resonance are used to further elucidate the structure and excited state dynamics of the bare nucleobases thymine and uracil. Both exhibit long lived excited electronic states that have been implicated in DNA photolesions which can ultimately lead to melanoma and carcinoma. Our experimental data in comparison with many quantum chemical calculations suggest a new picture for the dynamics of thymine and uracil in the gas phase. A high probability of UV absorption from a vibrationally hot ground state to the excited electronic state shows that the stability of thymine and uracil comes from

  17. Pain-related unscheduled contact with healthcare services after outpatient surgery.

    PubMed

    Brix, L D; Bjørnholdt, K T; Thillemann, T M; Nikolajsen, L

    2017-04-10

    This prospective, observational study explored the need for pain-related unscheduled contact with healthcare services after outpatient surgery. We hypothesised that 10% of outpatients would have pain-related unscheduled contact with healthcare services, and that the incidence would differ depending on the type of surgical procedure. In total, 905 patients who had undergone one of five common outpatient surgical procedures (knee or shoulder arthroscopy, surgical correction of hallux valgus, laparoscopic cholecystectomy or laparoscopic gynaecological procedures) completed an electronic questionnaire one week and eight weeks after surgery. Data from 732 patients (81%) were available for analysis. Within the first eight weeks after surgery, 150 patients (20.5%) had made unscheduled contact with healthcare professionals, in 247 cases due to pain that was most frequent in the first postoperative week. Risk factors were female sex, unemployment and laparoscopic cholecystectomy. The most frequent healthcare contact was with the general practitioner (46.4%), and the most frequent outcome was further information and guidance (41.2%). We have demonstrated that a minority of patients still needed to make contact with health services after outpatient surgery, most often due to inadequate pain management. This finding should be considered when planning postoperative monitoring and care, and developing postoperative patient education.

  18. Inhibitors of Histone Deacetylases Enhance Neurotoxicity of DNA Damage

    PubMed Central

    Vashishta, A.

    2014-01-01

    The nonselective inhibitors of class I/II histone deacetylases (HDACs) including trichostatin A and the clinically used suberoylanilide hydroxamic acid (SAHA, vorinostat) are neuroprotective in several models of neuronal injury. Here, we report that in cultured cortical neurons from newborn rats and in the cerebral cortex of whole neonate rats, these HDAC inhibitors exacerbated cytotoxicity of the DNA double-strand break (DSB)-inducing anticancer drug etoposide by enhancing apoptosis. Similar neurotoxic interactions were also observed in neurons that were treated with other DNA damaging drugs including cisplatin and camptothecin. In addition, in rat neonates, SAHA increased cortical neuron apoptosis that was induced by a single injection of the NMDA receptor antagonist dizocilpine (MK801). In etoposide-treated neurons, the nonselective HDAC inhibition resulted in more DSBs. It also potentiated etoposide-induced accumulation and phosphorylation of the pro-apoptotic transcription factor p53. Moreover, nonselective HDAC inhibition exacerbated neuronal apoptosis that was induced by the overexpressed p53. Importantly, such effects cannot be fully explained by inhibition of HDAC1, which is known to play a role in DSB repair and regulation of p53. The specific HDAC1 inhibitor MS275 only moderately enhanced etoposide-induced neuronal death. Although in etoposide-treated neurons MS275 increased DSBs, it did not affect activation of p53. Our findings suggest that besides HDAC1, there are other class I/II HDACs that participate in neuronal DNA damage response attenuating neurotoxic consequences of genotoxic insults to the developing brain. PMID:25063076

  19. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex

    PubMed Central

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  20. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

    PubMed

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

    2016-10-04

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

  1. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment

    PubMed Central

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Cate, Rosemarie ten; Rodermond, Hans M.; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A.P.

    2016-01-01

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation. The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found. In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome. PMID:27602767

  2. Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

    PubMed Central

    Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

    2015-01-01

    A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

  3. Enhanced malignant transformation is accompanied by increased survival recovery after ionizing radiation in Chinese hamster embryo fibroblasts

    SciTech Connect

    Boothman, D.A.

    1994-04-01

    Transformed Chinese hamster embryo fibroblasts (CHEF), which gradually increase in tumor-forming ability in nude mice, were isolated from normal diploid CHEF/18 cells. Transformed CHEF cells (i.e., T30-4 > 21-2M3 > 21-2 > normal CHEF/18) showed gradual increases in potentially lethal damage (PLD) survival recovery. {beta}-Lapachone and camptothecin, modulators of topoisomerase I (Topo I) activity, not only prevented survival recovery in normal as well as in tumor cells, but enhanced unscheduled DNA synthesis. These seemingly conflicting results are due to the fact that Topo I activity can be modulated by inhibitors to convert single-stranded DNA lesions into double-stranded breaks. Increases in unscheduled DNA synthesis may result from a continual supply of free ends, on which DNA repair processes may act. Altering Topo I activity with modulators appears to increase X-ray lethality via a DNA lesion modification suicide pathway. Cells down-regulate Topo I immediately after ionizing radiation to prevent Topo I-mediated lesion modification and to enhance survival recovery. 16 refs., 3 figs., 1 tab.

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

    PubMed Central

    Liu, Jinhuai; Liu, Jinyun; Yang, Liangbao; Chen, Xing; Zhang, Meiyun; Meng, Fanli; Luo, Tao; Li, Minqiang

    2009-01-01

    Detection of DNA sequences has received broad attention due to its potential applications in a variety of fields. As sensitivity of DNA biosensors is determined by signal variation of hybridization events, the signal enhancement is of great significance for improving the sensitivity in DNA detection, which still remains a great challenge. Nanomaterials, which possess some unique chemical and physical properties caused by nanoscale effects, provide a new opportunity for developing novel nanomaterial-based signal-enhancers for DNA biosensors. In this review, recent progress concerning this field, including some newly-developed signal enhancement approaches using quantum-dots, carbon nanotubes and their composites reported by our group and other researchers are comprehensively summarized. Reports on signal enhancement of DNA biosensors by non-nanomaterials, such as enzymes and polymer reagents, are also reviewed for comparison. Furthermore, the prospects for developing DNA biosensors using nanomaterials as signal-enhancers in future are also indicated. PMID:22399999

  5. Short thio-multi-walled carbon nanotubes and Au nanoparticles enhanced electrochemical DNA biosensor for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Guo, Feng; Zhang, Jimei; Dai, Zhao; Zheng, Guo

    2010-07-01

    A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a thio group (MWNTs-SH) and gold nanoparticles (GNPs) for covalent DNA immobilization and enhanced hybridization detection is described. The key step for developing this novel DNA biosensor is to cut the pristine MWNT into short and generate lots of active sites simultaneously. With this approach, the target DNA could be quantified in a linear range from 8.5×10-10 to 1.5×10-5 mol/L, with a detection limit of 1.67×10-11 mol/L by 3σ.

  6. Extracellular Genomic DNA Mediates Enhancement of Xylella fastidiosa Biofilm Formation in Vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) produces extracellular DNA in PD3 liquid medium. This extracellular DNA could enhance biofilm formation, a factor in successful establishment of Xf in planta. The relative amounts of extracellular DNA were positively correlated with planktonic growth and biofilm formation in ...

  7. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

    SciTech Connect

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

    2013-06-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

  8. TopBP1-mediated DNA processing during mitosis.

    PubMed

    Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

    2016-01-01

    Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

  9. A fluorescence enhancement assay for cellular DNA damage. [X Radiation

    SciTech Connect

    Kanter, P.M.; Schwartz, H.S.

    1982-07-01

    A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based upon the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA, and is sufficiently sensitive to detect damage induced with 100 rads of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, beta. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents in unlabeled murine leukemia cells.

  10. Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

    PubMed Central

    Cravens, Shannen L.; Schonhoft, Joseph D.; Rowland, Meng M.; Rodriguez, Alyssa A.; Anderson, Breeana G.; Stivers, James T.

    2015-01-01

    Intracellular space is at a premium due to the high concentrations of biomolecules and is expected to have a fundamental effect on how large macromolecules move in the cell. Here, we report that crowded solutions promote intramolecular DNA translocation by two human DNA repair glycosylases. The crowding effect increases both the efficiency and average distance of DNA chain translocation by hindering escape of the enzymes to bulk solution. The increased contact time with the DNA chain provides for redundant damage patrolling within individual DNA chains at the expense of slowing the overall rate of damaged base removal from a population of molecules. The significant biological implication is that a crowded cellular environment could influence the mechanism of damage recognition as much as any property of the enzyme or DNA. PMID:25845592

  11. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    SciTech Connect

    Nishiyama, Y.; Rapp, F.

    1981-04-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants.

  12. Protocol investigating the clinical outcomes and cost-effectiveness of cognitive–behavioural therapy delivered remotely for unscheduled care users with health anxiety: randomised controlled trial

    PubMed Central

    Patel, Shireen; Malins, Sam; Guo, Boliang; James, Marilyn; Kai, Joe; Kaylor-Hughes, Catherine; Rowley, Emma; Simpson, Jayne; Smart, David; Stubley, Michelle; Tyrer, Helen

    2016-01-01

    Background Health anxiety and medically unexplained symptoms cost the National Health Service (NHS) an estimated £3 billion per year in unnecessary costs with little evidence of patient benefit. Effective treatment is rarely taken up due to issues such as stigma or previous negative experiences with mental health services. An approach to overcome this might be to offer remotely delivered psychological therapy, which can be just as effective as face-to-face therapy and may be more accessible and suitable. Aims To investigate the clinical outcomes and cost-effectiveness of remotely delivered cognitive–behavioural therapy (CBT) to people with high health anxiety repeatedly accessing unscheduled care (trial registration: NCT02298036). Method A multicentre randomised controlled trial (RCT) will be undertaken in primary and secondary care providers of unscheduled care across the East Midlands. One hundred and forty-four eligible participants will be equally randomised to receive either remote CBT (6–12 sessions) or treatment as usual (TAU). Two doctoral research studies will investigate the barriers and facilitators to delivering the intervention and the factors contributing to the optimisation of therapeutic outcome. Results This trial will be the first to test the clinical outcomes and cost-effectiveness of remotely delivered CBT for the treatment of high health anxiety. Conclusions The findings will enable an understanding as to how this intervention might fit into a wider care pathway to enhance patient experience of care. Declaration of interest None. Copyright and usage © The Royal College of Psychiatrists 2016. This is an open access article distributed under the terms of the Creative Commons Non-commercial, No Derivatives (CC BY-NC-ND) licence. PMID:27703758

  13. An enhanced MITOMAP with a global mtDNA mutational phylogeny

    PubMed Central

    Ruiz-Pesini, Eduardo; Lott, Marie T.; Procaccio, Vincent; Poole, Jason C.; Brandon, Marty C.; Mishmar, Dan; Yi, Christina; Kreuziger, James; Baldi, Pierre; Wallace, Douglas C.

    2007-01-01

    The MITOMAP () data system for the human mitochondrial genome has been greatly enhanced by the addition of a navigable mutational mitochondrial DNA (mtDNA) phylogenetic tree of ∼3000 mtDNA coding region sequences plus expanded pathogenic mutation tables and a nuclear-mtDNA pseudogene (NUMT) data base. The phylogeny reconstructs the entire mutational history of the human mtDNA, thus defining the mtDNA haplogroups and differentiating ancient from recent mtDNA mutations. Pathogenic mutations are classified by both genotype and phenotype, and the NUMT sequences permits detection of spurious inclusion of pseudogene variants during mutation analysis. These additions position MITOMAP for the implementation of our automated mtDNA sequence analysis system, Mitomaster. PMID:17178747

  14. 15 CFR Supplement No. 2 to Part 715 - Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... end product: (i) Acetophenone (CAS #98-86-2); (ii) 6-Chloro-2-methyl aniline (CAS #87-63-8); (iii) 2... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Examples of Unscheduled Discrete Organic Chemicals (UDOCs) and UDOC Production No. Supplement No. 2 to Part 715 Commerce and Foreign...

  15. 14 CFR 135.267 - Flight time limitations and rest requirements: Unscheduled one- and two-pilot crews.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... requirements: Unscheduled one- and two-pilot crews. 135.267 Section 135.267 Aeronautics and Space FEDERAL... two-pilot crews. (a) No certificate holder may assign any flight crewmember, and no flight crewmember may accept an assignment, for flight time as a member of a one- or two-pilot crew if that...

  16. 14 CFR 135.269 - Flight time limitations and rest requirements: Unscheduled three- and four-pilot crews.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... requirements: Unscheduled three- and four-pilot crews. 135.269 Section 135.269 Aeronautics and Space FEDERAL... four-pilot crews. (a) No certificate holder may assign any flight crewmember, and no flight crewmember may accept an assignment, for flight time as a member of a three- or four-pilot crew if...

  17. Ultrasound enhancement of in vitro transfection of plasmid DNA by a cationized gelatin.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2002-05-01

    In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.

  18. Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13.

    PubMed

    Lee, Na-Ra; Kwon, Hyun-Mi; Park, Kkothanahreum; Oh, Sangtaek; Jeong, Yong-Joo; Kim, Dong-Eun

    2010-11-01

    SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5'-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5'-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5'-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5'-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5'-overhang account for the observed enhanced processivity of DNA unwinding.

  19. Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

    PubMed

    Sharma, Himanshu; Wood, Jennifer B; Lin, Sophia; Corn, Robert M; Khine, Michelle

    2014-09-23

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

  20. DNA Diagnostics: Nanotechnology-enhanced Electrochemical Detection of Nucleic Acids

    PubMed Central

    Wei, Fang; Lillehoj, Peter B.; Ho, Chih-Ming

    2010-01-01

    The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been employed for DNA detection, electrochemical sensors show great promise since they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection for point-of-care (POC) clinical diagnostics. PMID:20075759

  1. Enhancement of therapeutic drug and DNA delivery into cells by electroporation* Enhancement of therapeutic drug and DNA delivery into cells by electroporation

    NASA Astrophysics Data System (ADS)

    Rabussay, Dietmar; Dev, Nagendu B.; Fewell, Jason; Smith, Louis C.; Widera, Georg; Zhang, Lei

    2003-02-01

    The effectiveness of potentially powerful therapeutics, including DNA, is often limited by their inability to permeate the cell membrane efficiently. Electroporation (EP) also referred to as `electropermeabilization' of the outer cell membrane renders this barrier temporarily permeable by inducing `pores' across the lipid bilayer. For in vivo EP, the drug or DNA is delivered into the interstitial space of the target tissue by conventional means, followed by local EP. EP pulses of micro- to millisecond duration and field strengths of 100-1500 V cm-1 generally enhance the delivery of certain chemotherapeutic drugs by three to four orders of magnitude and intracellular delivery of DNA several hundred-fold. We have used EP in clinical studies for human cancer therapy and in animals for gene therapy and DNA vaccination. Late stage squamous cell carcinomas of the head and neck were treated with intratumoural injection of bleomycin and subsequent EP. Of the 69 tumours treated, 25% disappeared completely and another 32% were reduced in volume by more than half. Residence time of bleomycin in electroporated tumours was significantly greater than in non-electroporated lesions. Histological findings and gene expression patterns after bleomycin-EP treatment indicated rapid apoptosis of the majority of tumour cells. In animals, we demonstrated the usefulness of EP for enhanced DNA delivery by achieving normalization of blood clotting times in haemophilic dogs, and by substantially increasing transgene expression in smooth muscle cells of arterial walls using a novel porous balloon EP catheter. Finally, we have found in animal experiments that the immune response to DNA vaccines can be dramatically enhanced and accelerated by EP and co-injection of micron-sized particles. We conclude that EP represents an effective, economical and safe approach to enhance the intracellular delivery, and thus potency, of important drugs and genes for therapeutic purposes. The safety and pharmaco

  2. Nucleosome linker proteins HMGB1 and histone H1 differentially enhance DNA ligation reactions.

    PubMed

    Yamanaka, Shiho; Katayama, Eisaku; Yoshioka, Ken-ichi; Nagaki, Sumiko; Yoshida, Michiteru; Teraoka, Hirobumi

    2002-03-22

    We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both intra-molecular and inter-molecular ligations. In this paper, we found that histone H1 differentially enhances ligation reaction of DNA double-strand breaks (DSB). Histone H1 stimulated exclusively inter-molecular ligation reaction of DSB with DNA ligase IIIbeta and IV, whereas HMGB1 enhanced mainly intra-molecular ligation reaction. Electron microscopy of direct DNA-protein interaction without chemical cross-linking visualized that HMGB1 bends and loops linear DNA to form compact DNA structure and that histone H1 is capable of assembling DNA in tandem arrangement with occasional branches. These results suggest that differences in the enhancement of DNA ligation reaction are due to those in alteration of DNA configuration induced by these two linker proteins. HMGB1 and histone H1 may function in non-homologous end-joining of DSB repair and V(D)J recombination in different manners.

  3. Three enhancements to the inference of statistical protein-DNA potentials.

    PubMed

    AlQuraishi, Mohammed; McAdams, Harley H

    2013-03-01

    The energetics of protein-DNA interactions are often modeled using so-called statistical potentials, that is, energy models derived from the atomic structures of protein-DNA complexes. Many statistical protein-DNA potentials based on differing theoretical assumptions have been investigated, but little attention has been paid to the types of data and the parameter estimation process used in deriving the statistical potentials. We describe three enhancements to statistical potential inference that significantly improve the accuracy of predicted protein-DNA interactions: (i) incorporation of binding energy data of protein-DNA complexes, in conjunction with their X-ray crystal structures, (ii) use of spatially-aware parameter fitting, and (iii) use of ensemble-based parameter fitting. We apply these enhancements to three widely-used statistical potentials and use the resulting enhanced potentials in a structure-based prediction of the DNA binding sites of proteins. These enhancements are directly applicable to all statistical potentials used in protein-DNA modeling, and we show that they can improve the accuracy of predicted DNA binding sites by up to 21%.

  4. Label-Free Ag+ Detection by Enhancing DNA Sensitized Tb3+ Luminescence

    PubMed Central

    Kleinke, Kimberly; Saran, Runjhun; Liu, Juewen

    2016-01-01

    In this work, the effect of Ag+ on DNA sensitized Tb3+ luminescence was studied initially using the Ag+-specific RNA-cleaving DNAzyme, Ag10c. While we expected to observe luminescence quenching by Ag+, a significant enhancement was produced. Based on this observation, simple DNA oligonucleotide homopolymers were used with systematically varied sequence and length. We discovered that both poly-G and poly-T DNA have a significant emission enhancement by Ag+, while the absolute intensity is stronger with the poly-G DNA, indicating that a G-quadruplex DNA is not required for this enhancement. Using the optimized length of the G7 DNA (an oligo constituted with seven guanines), Ag+ was measured with a detection limit of 57.6 nM. The signaling kinetics, G7 DNA conformation, and the binding affinity of Tb3+ to the DNA in the presence or absence of Ag+ are also studied to reveal the mechanism of emission enhancement. This observation is useful not only for label-free detection of Ag+, but also interesting for the rational design of new biosensors using Tb3+ luminescence. PMID:27571082

  5. Cleavage enhancement of specific chemical bonds in DNA-Cisplatin complexes induced by X-rays

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Yao, Xiaobin; Luo, Xinglan; Fu, Xianzhi

    2014-04-01

    The chemical bond transformation of cisplatin-DNA complexes can be probed efficiently by XPS which provides a concomitant X-ray irradiation source as well. The presence to Pt could considerably increase formation of the SE induced by X-ray and that the further interaction of these LEE with DNA leads to the enhancement of bond cleavages.

  6. Amphiphilic Block Copolymers Enhance Cellular Uptake and Nuclear Entry of Polyplex-Delivered DNA

    PubMed Central

    Yang, Zhihui; Sahay, Gaurav; Sriadibhatla, Srikanth; Kabanov, Alexander V.

    2008-01-01

    This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IκB and subsequent NFκB nuclear localization as well as upregulate numerous NFκB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes (“polyplexes”) in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFκB–binding sites, but have no effect on import of pDNA without these sites. All together, our studies suggest that Pluronics rapidly activate NFκB, which binds cytosolic pDNA that possesses promoters containing NFκB binding sites and consequently increases nuclear import of pDNA through NFκB nuclear translocation. PMID:18729495

  7. Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells

    SciTech Connect

    Protic, M.; Hirschfeld, S.; Tsang, A.P.; Wagner, M.; Dixon, K.; Levine, A.S. )

    1989-10-01

    Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, the authors have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. They have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.

  8. CDK1 Enhances Mitochondrial Bioenergetics for Radiation-Induced DNA Repair

    PubMed Central

    Qin, Lili; Fan, Ming; Candas, Demet; Jiang, Guochun; Papadopoulos, Stelios; Tian, Lin; Woloschak, Gayle; Grdina, David J.; Li, Jian Jian

    2015-01-01

    SUMMARY Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy consuming process; however, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of cell cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is significantly reduced in cells harboring CDK1 phosphorylation deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair are also severely compromised in cells harboring mitochondrial-targeted kinase deficient CDK1. These results demonstrate a mechanism governing the communication between mitochondria and nucleus, by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress. PMID:26670043

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

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  10. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents.

    PubMed

    Lezin, George; Kuehn, Michael R; Brunelli, Luca

    2011-08-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.

  11. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

    PubMed Central

    Lezin, George; Kuehn, Michael R.; Brunelli, Luca

    2011-01-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

  12. Current-induced enhancement of DNA bubble creation

    NASA Astrophysics Data System (ADS)

    Gu, Lei; Fu, Hua-Hua

    2016-05-01

    Current-induced heating of short double-stranded DNA chains is studied within a two-probe transport setup by using the Langevin approach. The electrons are modeled by a tight-binding Hamiltonian. The DNA atomic motion is described by the Peyrard-Bishop-Dauxois atomic potential, coupled with electrons through the Holstein interaction. The solvent environment is accounted for as a classical heat bath. Voltage biases of 0.1˜ 0.5 {{V}} can effectively break the base pairs and lead to the melting transition, which can be detected from the resulting significant reduction of the conductance. When the bias increases, the opening of base pairs near the leads with higher chemical potential is suppressed and bubble (localized separation of the double strand) formation becomes asymmetric. Our results suggest that the voltage bias can excite the base pairs, hence increases the chemical activity of DNA.

  13. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    SciTech Connect

    Gupta, P.K.; Sirover, M.A.

    1984-10-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. 62 references, 3 figures, 2 tables.

  14. Enhancement of anion-exchange chromatography of DNA using compaction agents

    NASA Technical Reports Server (NTRS)

    Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.

  15. Gentiana asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticles-induced DNA damage.

    PubMed

    Hudecová, Alexandra; Kusznierewicz, Barbara; Hašplová, Katarína; Huk, Anna; Magdolenová, Zuzana; Miadoková, Eva; Gálová, Eliška; Dušinská, Mária

    2012-09-01

    Exposure to high levels of different environmental pollutants is known to be associated with induction of DNA damage in humans. Thus DNA repair is of great importance in preventing mutations and contributes crucially to the prevention of cancer. In our study we have focused on quantitative analysis of Gentiana asclepiadea aqueous or methanolic extracts obtained from flower and haulm, their antioxidant potency in ABTS post-column derivatisation, and their potential ability to enhance DNA repair in human lymphocytes after hydrogen peroxide (H(2)O(2)) treatment (250 μM, 5 min). We also studied DNA repair in human kidney HEK 293 cells after exposure to 20 nm silver nanoparticles (AgNPs) (100 μg/ml, 30 min) in the presence and absence of the plant extract. We have found that mangiferin along with unidentified polar compounds are the most pronounced antioxidants in the studied extracts. Extract from haulm exhibited slightly stronger antioxidant properties compared to flower extracts. However, all four extracts showed significant ability to enhance DNA repair in both cell types after H(2)O(2) and AgNP treatments.

  16. Extracellular Xylella fastidiosa genomic DNA enhances biofilm formation in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) is a Gram negative, xylem-limited bacterium that causes Pierce’s Disease (PD) of grapevine, as well as other diseases of economically important crops and landscape plants. Many bacteria produce large amounts of extracellular DNA, which may function as a matrix component in b...

  17. DNA detection using plasmonic enhanced near-infrared photoluminescence of gallium arsenide.

    PubMed

    Tang, Longhua; Chun, Ik Su; Wang, Zidong; Li, Jinghong; Li, Xiuling; Lu, Yi

    2013-10-15

    Efficient near-infrared detection of specific DNA with single nucleotide polymorphism selectivity is important for diagnostics and biomedical research. Herein, we report the use of gallium arsenide (GaAs) as a sensing platform for probing DNA immobilization and targeting DNA hybridization, resulting in ∼8-fold enhanced GaAs photoluminescence (PL) at ∼875 nm. The new signal amplification strategy, further coupled with the plasmonic effect of Au nanoparticles, is capable of detecting DNA molecules with a detection limit of 0.8 pM and selectivity against single base mismatches. Such an ultrasensitive near-infrared sensor can find a wide range of biochemical and biomedical applications.

  18. Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion

    PubMed Central

    McLean, Meghan M; Chang, Yong; Dhar, Gautam; Heiss, John K; Johnson, Reid C

    2013-01-01

    Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI: http://dx.doi.org/10.7554/eLife.01211.001 PMID:24151546

  19. NonO enhances the association of many DNA-binding proteins to their targets.

    PubMed

    Yang, Y S; Yang, M C; Tucker, P W; Capra, J D

    1997-06-15

    NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

  20. "Cap-and-Catch" Purification for Enhancing the Quality of Libraries of DNA Conjugates.

    PubMed

    Franzini, Raphael M; Biendl, Stefan; Mikutis, Gediminas; Samain, Florent; Scheuermann, Jörg; Neri, Dario

    2015-07-13

    The potential of DNA-encoded combinatorial libraries (DECLs) as tools for hit discovery crucially relies on the availability of methods for their synthesis at acceptable purity and quality. Incomplete reactions in the presence of DNA can noticeably affect the purity of DECLs and methods to selectively remove unreacted oligonucleotide-based starting products would likely enhance the quality of DECL screening results. We describe an approach to selectively remove unreacted oligonucleotide starting products from reaction mixtures and demonstrate its applicability in the context of acylation of amino-modified DNA. Following an amide bond forming reaction, we treat unreacted amino-modified DNAs with biotinylating reagents and isolate the corresponding biotinylated oligonucleotides from the reaction mixture by affinity capture on streptavidin-coated sepharose. This approach, which yields the desired DNA-conjugate at enhanced purity, can be applied both to reactions performed in solution and to procedures in which DNA is immobilized on an anion exchange solid support.

  1. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    PubMed

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-03-04

    Food grade Lactococcus lactis (L. lactis) has been widely used as an antigen and DNA delivery vehicle. We had previously reported the use of non-invasive L. lactis for the delivery of newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, we outline the construction of dual recombinant L. lactis expressing Internalin A of Listeria monocytogenes and harbouring pPERDBY (LL InlA+ pPERDBY) to enhance the DNA delivery efficiency of L. lactis. After confirmation and validation of LL InlA+ pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around three fold increase in number of Enhanced Green Fluorescent Protein expressing Caco- cells. Thus, these findings reinforce the prospective application of invasive strain of L. lactis in delivery of DNA/RNA and antigens.

  2. Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer.

    PubMed

    Muvarak, Nidal E; Chowdhury, Khadiza; Xia, Limin; Robert, Carine; Choi, Eun Yong; Cai, Yi; Bellani, Marina; Zou, Ying; Singh, Zeba N; Duong, Vu H; Rutherford, Tyler; Nagaria, Pratik; Bentzen, Søren M; Seidman, Michael M; Baer, Maria R; Lapidus, Rena G; Baylin, Stephen B; Rassool, Feyruz V

    2016-10-10

    Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.

  3. Cleavage enhancement of specific chemical bonds in DNA by cisplatin radiosensitization.

    PubMed

    Xiao, Fangxing; Luo, Xinglan; Fu, Xianzhi; Zheng, Yi

    2013-05-02

    X-ray photoelectron spectroscopy (XPS) is harnessed as an in situ efficient characterization technique for monitoring chemical bond transformation in DNA and cisplatin-DNA complexes under synergic X-ray irradiation. By analyzing the variation of relative peak area of core elements of DNA as a function of irradiation time, we find that the most vulnerable scission sites in DNA are those containing phosphate and glycosidic bonds. Compared to DNA, the effective rate constants of the corresponding phosphodiester and glycosidic bond cleavages for cisplatin-DNA complexes are 1.8 and 1.9 folds larger. These damages and their enhancements are similar to those induced by low energy electrons (LEE). Consistently, the magnitude of the secondary electron distribution produced by the X-rays on the cisplatin-DNA complexes is considerably increased compared to that of pristine DNA. The data suggest that DNA radiosensization by cisplatin results not only from the sensitization of DNA to the action of LEE, but also from an increase the production of LEE at the site of binding of the cisplatin. The results provide new insights into the mechanisms of cisplatin-induced sensitization of DNA under X-ray irradiation, which could be helpful in the design of new cisplatin-based antitumor drugs.

  4. The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair

    PubMed Central

    Costantini, Silvia; Pegoraro, Silvia; Ros, Gloria; Penzo, Carlotta; Triolo, Gianluca; Demarchi, Francesca; Sgarra, Riccardo; Vindigni, Alessandro; Manfioletti, Guidalberto

    2016-01-01

    The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens. PMID:27723831

  5. The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair.

    PubMed

    Pellarin, Ilenia; Arnoldo, Laura; Costantini, Silvia; Pegoraro, Silvia; Ros, Gloria; Penzo, Carlotta; Triolo, Gianluca; Demarchi, Francesca; Sgarra, Riccardo; Vindigni, Alessandro; Manfioletti, Guidalberto

    2016-01-01

    The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

  6. Enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic 'touch DNA' evidence.

    PubMed

    Farash, Katherine; Hanson, Erin K; Ballantyne, Jack

    2015-03-09

    DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

  7. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

    PubMed

    Agirre, Xabier; Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C; Beekman, Renée; Rodríguez-Madoz, Juan R; San José-Enériz, Edurne; Fang, Fang; Gutiérrez, Norma C; García-Verdugo, José M; Robson, Michael I; Schirmer, Eric C; Guruceaga, Elisabeth; Martens, Joost H A; Gut, Marta; Calasanz, Maria J; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F San; Melnick, Ari; Stunnenberg, Hendrik G; Gut, Ivo G; Prosper, Felipe; Martín-Subero, José I

    2015-04-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

  8. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers

    PubMed Central

    Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C.; Beekman, Renée; Rodríguez-Madoz, Juan R.; José-Enériz, Edurne San; Fang, Fang; Gutiérrez, Norma C.; García-Verdugo, José M.; Robson, Michael I.; Schirmer, Eric C.; Guruceaga, Elisabeth; Martens, Joost H.A.; Gut, Marta; Calasanz, Maria J.; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F. San; Melnick, Ari; Stunnenberg, Hendrik G.; Gut, Ivo G.

    2015-01-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. PMID:25644835

  9. G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

    NASA Astrophysics Data System (ADS)

    Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

    2015-07-01

    Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially

  10. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    PubMed Central

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2016-01-01

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic. PMID:26544157

  11. Dna-Enhanced Dye-Sensitized Solar Cells

    NASA Astrophysics Data System (ADS)

    Pollum, Marvin; Crespo-Hernandez, Carlos E.

    2013-06-01

    Dye-sensitized solar cells (DSSCs) are a promising alternative to the current silicon solar cell technologies. DSSCs have a lower manufacturing cost, can be made to be flexible, semi-transparent, and in any variety of colors for aesthetic applications. Despite these advantageous properties, the stability and power conversion efficiency of DSSCs are still lacking. Recently, we have shown that the adsorption of DNA onto the semiconductor surface of a typical DSSC improves its overall performance. Structure-function analysis, in conjunction with steady-state and time-resolved spectroscopic studies, are currently being done to understand this phenomenon and to uncover the mechanism by which DNA boosts the overall performance of DSSCs. This new knowledge is expected to facilitate the rational design of DSSCs that exhibit higher power conversion efficiency than those currently available.

  12. An AC electrokinetic method for enhanced detection of DNA nanoparticles.

    PubMed

    Krishnan, Rajaram; Heller, Michael J

    2009-04-01

    In biomedical research and diagnostics it is a challenge to isolate and detect low levels of nanoparticles and nanoscale biomarkers in blood and other biological samples. While highly sensitive epifluorescent microscope systems are available for ultra low level detection, the isolation of the specific entities from large sample volumes is often the bigger limitation. AC electrokinetic techniques like dielectrophoresis (DEP) offer an attractive mechanism for specifically concentrating nanoparticles into microscopic locations. Unfortunately, DEP requires significant sample dilution thus making the technology unsuitable for biological applications. Using a microelectrode array device, special conditions have been found for the separation of hmw-DNA and nanoparticles under high conductance (ionic strength) conditions. At AC frequencies in the 3000-10 000 Hz range, 10 mum microspheres and human T lymphocytes can be isolated into the DEP low field regions, while hmw-DNA and nanoparticles can be concentrated into microscopic high field regions for subsequent detection using an epifluorescent system.

  13. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    DOE PAGES

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; ...

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides themore » first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.« less

  14. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    SciTech Connect

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

  15. Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines

    NASA Astrophysics Data System (ADS)

    Wang, Chun; Ge, Qing; Ting, David; Nguyen, David; Shen, Hui-Rong; Chen, Jianzhu; Eisen, Herman N.; Heller, Jorge; Langer, Robert; Putnam, David

    2004-03-01

    Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

  16. Using silver nanowire antennas to enhance the conversion efficiency of photoresponsive DNA nanomotors.

    PubMed

    Yuan, Quan; Zhang, Yunfei; Chen, Yan; Wang, Ruowen; Du, Chaoling; Yasun, Emir; Tan, Weihong

    2011-06-07

    Plasmonic near-field coupling can induce the enhancement of photoresponsive processes by metal nanoparticles. Advances in nanostructured metal synthesis and theoretical modeling have kept surface plasmons in the spotlight. Previous efforts have resulted in significant intensity enhancement of organic dyes and quantum dots and increased absorption efficiency of optical materials used in solar cells. Here, we report that silver nanostructures can enhance the conversion efficiency of an interesting type of photosensitive DNA nanomotor through coupling with incorporated azobenzene moieties. Spectral overlap between the azobenzene absorption band and plasmonic resonances of silver nanowires increases light absorption of photon-sensitive DNA motor molecules, leading to 85% close-open conversion efficiency. The experimental results are consistent with our theoretical calculations of the electric field distribution. This enhanced conversion of DNA nanomotors holds promise for the development of new types of molecular nanodevices for light manipulative processes and solar energy harvesting.

  17. Using silver nanowire antennas to enhance the conversion efficiency of photoresponsive DNA nanomotors

    PubMed Central

    Yuan, Quan; Zhang, Yunfei; Chen, Yan; Wang, Ruowen; Du, Chaoling; Yasun, Emir; Tan, Weihong

    2011-01-01

    Plasmonic near-field coupling can induce the enhancement of photoresponsive processes by metal nanoparticles. Advances in nanostructured metal synthesis and theoretical modeling have kept surface plasmons in the spotlight. Previous efforts have resulted in significant intensity enhancement of organic dyes and quantum dots and increased absorption efficiency of optical materials used in solar cells. Here, we report that silver nanostructures can enhance the conversion efficiency of an interesting type of photosensitive DNA nanomotor through coupling with incorporated azobenzene moieties. Spectral overlap between the azobenzene absorption band and plasmonic resonances of silver nanowires increases light absorption of photon-sensitive DNA motor molecules, leading to 85% close-open conversion efficiency. The experimental results are consistent with our theoretical calculations of the electric field distribution. This enhanced conversion of DNA nanomotors holds promise for the development of new types of molecular nanodevices for light manipulative processes and solar energy harvesting. PMID:21596999

  18. Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer

    PubMed Central

    Munye, Mustafa M.; Tagalakis, Aristides D.; Barnes, Josephine L.; Brown, Rachel E.; McAnulty, Robin J.; Howe, Steven J.; Hart, Stephen L.

    2016-01-01

    Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials. PMID:26975732

  19. A DNA/HDAC dual-targeting drug CY190602 with significantly enhanced anticancer potency

    PubMed Central

    Liu, Chuan; Ding, Hongyu; Li, Xiaoxi; Pallasch, Christian P; Hong, Liya; Guo, Dianwu; Chen, Yi; Wang, Difei; Wang, Wei; Wang, Yajie; Hemann, Michael T; Jiang, Hai

    2015-01-01

    Genotoxic drugs constitute a major treatment modality for human cancers; however, cancer cells' intrinsic DNA repair capability often increases the threshold of lethality and renders these drugs ineffective. The emerging roles of HDACs in DNA repair provide new opportunities for improving traditional genotoxic drugs. Here, we report the development and characterization of CY190602, a novel bendamustine-derived drug with significantly enhanced anticancer potency. We show that CY190602's enhanced potency can be attributed to its newly gained ability to inhibit HDACs. Using this novel DNA/HDAC dual-targeting drug as a tool, we further explored HDAC's role in DNA repair. We found that HDAC activities are essential for the expression of several genes involved in DNA synthesis and repair, including TYMS, Tip60, CBP, EP300, and MSL1. Importantly, CY190602, the first-in-class example of such DNA/HDAC dual-targeting drugs, exhibited significantly enhanced anticancer activity in vitro and in vivo. These findings provide rationales for incorporating HDAC inhibitory moieties into genotoxic drugs, so as to overcome the repair capacity of cancer cells. Systematic development of similar DNA/HDAC dual-targeting drugs may represent a novel opportunity for improving cancer therapy. PMID:25759362

  20. C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

    SciTech Connect

    Tong Tiezhu; Fan Huiying; Tan Yadi; Xiao Shaobo; Ling Jieyu; Chen Huanchun; Guo Aizhen . E-mail: aizhen@mail.hzau.edu.cn

    2006-09-08

    Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28{sub 4} were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d{sub 3} DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD{sub 5}) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. The IL-4 level for sgC-C3d{sub 3} DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response.

  1. A DNA/HDAC dual-targeting drug CY190602 with significantly enhanced anticancer potency.

    PubMed

    Liu, Chuan; Ding, Hongyu; Li, Xiaoxi; Pallasch, Christian P; Hong, Liya; Guo, Dianwu; Chen, Yi; Wang, Difei; Wang, Wei; Wang, Yajie; Hemann, Michael T; Jiang, Hai

    2015-03-09

    Genotoxic drugs constitute a major treatment modality for human cancers; however, cancer cells' intrinsic DNA repair capability often increases the threshold of lethality and renders these drugs ineffective. The emerging roles of HDACs in DNA repair provide new opportunities for improving traditional genotoxic drugs. Here, we report the development and characterization of CY190602, a novel bendamustine-derived drug with significantly enhanced anticancer potency. We show that CY190602's enhanced potency can be attributed to its newly gained ability to inhibit HDACs. Using this novel DNA/HDAC dual-targeting drug as a tool, we further explored HDAC's role in DNA repair. We found that HDAC activities are essential for the expression of several genes involved in DNA synthesis and repair, including TYMS, Tip60, CBP, EP300, and MSL1. Importantly, CY190602, the first-in-class example of such DNA/HDAC dual-targeting drugs, exhibited significantly enhanced anticancer activity in vitro and in vivo. These findings provide rationales for incorporating HDAC inhibitory moieties into genotoxic drugs, so as to overcome the repair capacity of cancer cells. Systematic development of similar DNA/HDAC dual-targeting drugs may represent a novel opportunity for improving cancer therapy.

  2. DNA vaccination by electroporation and boosting with recombinant proteins enhances the efficacy of DNA vaccines for Schistosomiasis japonica.

    PubMed

    Dai, Yang; Zhu, Yinchang; Harn, Donald A; Wang, Xiaoting; Tang, Jianxia; Zhao, Song; Lu, Fei; Guan, Xiaohong

    2009-12-01

    Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Control programs combining chemotherapy and snail killing have not been able to block transmission of infection in lakes and marsh regions. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we wanted to determine if the efficacies of DNA vaccines encoding the 23-kDa tetraspanin membrane protein (SjC23), triose phosphate isomerase (SjCTPI), and sixfold-repeated genes of the complementarity determining region 3 (CDR3) in the H chain of NP30 could be enhanced by boosting via electroporation in vivo and/or with cocktail protein vaccines. Mice vaccinated with cocktail DNA vaccines showed a significant worm reduction of 32.88% (P < 0.01) and egg reduction of 36.20% (P < 0.01). Vaccine efficacy was enhanced when animals were boosted with cocktail protein vaccines; adult worm and liver egg burdens were reduced 45.35% and 48.54%, respectively. Nearly identical results were obtained in mice boosted by electroporation in vivo, with adult worm and egg burdens reduced by 45.00% and 50.88%, respectively. The addition of a protein vaccine boost to this regimen further elevated efficacy to approximately 60% for adult worm burden and greater than 60% for liver egg reduction. The levels of interleukin-2, gamma interferon, and the ratios of immunoglobulin G2a (IgG2a)/IgG1 clearly showed that cocktail DNA vaccines induced CD4(+) Th1-type responses. Boosting via either electroporation or with recombinant proteins significantly increased associated immune responses over those seen in mice vaccinated solely with DNA vaccines. Thus, schistosome DNA vaccine efficacy was significantly enhanced via boosting by electroporation in vivo and/or cocktail protein vaccines.

  3. Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function.

    PubMed

    Ege, T; Reisbig, R R; Rogne, S

    1984-11-01

    A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk-) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 X 10(6) D) was used as a carrier, the presence of either 20 mM NH4Cl, 1 microM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-Pi) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 X 10(6) D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH4Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-Pi coprecipitate, whereas NH4Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.

  4. Ultrasound enhances in vivo tumor expression of plasmid DNA by PEG-introduced cationized dextran.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2005-11-28

    This study is an investigation to experimentally confirm whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of plasmid DNA in tumor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow to polyionically complex with a plasmid DNA. The cationized dextran prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have an active ester and methoxy groups at each terminal, to obtain cationized dextran with different percentages of PEG introduced. Various cationized dextrans with or without PEG introduction were mixed with a plasmid DNA of LacZ to form cationized dextran-plasmid DNA complexes. Electrophoretical examination revealed that the plasmid DNA was complexed both with the cationized dextran and PEG-introduced cationized dextran, irrespective of the PEG introduction percentage, although the higher N/P ratio was needed for plasmid DNA complexation with the latter. By complexation with the cationized dextran, the zeta potential of plasmid DNA was changed to be positive. The charge of PEG-introduced cationized dextran-plasmid DNA complexes became close to 0 mV as their percentage of PEG introduced increased, although the molecular size was about 250 nm, irrespective of the PEG introduction. When cationized dextran-plasmid DNA complexes with or without PEG introduction were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass and the subsequent US irradiation to the tumor mass percutaneously, the PEG-introduced cationized dextran-plasmid DNA complex plus US irradiation enhanced the tumor level of gene expression to a significantly high extent compared with the cationized dextran-plasmid DNA complex and free plasmid DNA with or without US irradiation. The enhanced level depended on the time period and timing of US irradiation. Fluorescent microscopic studies revealed that the localization of plasmid DNA and the gene expression were observed in

  5. Two Adhesive Sites Can Enhance the Knotting Probability of DNA

    PubMed Central

    2015-01-01

    Self-entanglement, or knotting, is entropically favored in long polymers. Relatively short polymers such as proteins can knot as well, but in this case the entanglement is mainly driven by fine-tuned, sequence-specific interactions. The relation between the sequence of a long polymer and its topological state is here investigated by means of a coarse-grained model of DNA. We demonstrate that the introduction of two adhesive regions along the sequence of a self-avoiding chain substantially increases the probability of forming a knot. PMID:26136125

  6. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  7. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

    PubMed Central

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

    2013-01-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

  8. Arc-repressor dimerization on DNA: folding rate enhancement by colocalization.

    PubMed

    Marcovitz, Amir; Levy, Yaakov

    2009-05-20

    Multimeric proteins are ubiquitous in many cellular processes that require high levels of regulation. Eukaryotic gene expression is often regulated by a mechanism of combinatorial control that involves the binding of dimeric transcription factors to DNA together with the coordinated activity of additional proteins. In this study, we investigated the dimerization of the Arc-repressor on DNA with the aim of achieving microscopic insight into the possible advantages of interacting with DNA as a complex rather than as a monomeric single-domain protein. We used a computational coarse-grained model in which the protein dynamics was governed by native interactions and protein-DNA interactions were dictated by electrostatic forces. Inspired by previous experimental work that showed an enhanced refolding rate for the Arc-repressor in the presence of DNA and other polyanions, we focused on the mechanism and kinetics of the assembly of Arc monomers in the presence of single- (ssDNA) and double-stranded DNA (dsDNA) molecules in a low-salt concentration environment. The electrostatic interactions that attract the protein to the dsDNA were shown to be fundamental in colocalizing the unfolded Arc chains and in accelerating refolding. Arc monomers bind the dsDNA efficiently and nonspecifically, and search for each other via one-dimensional diffusion. The fastest folding of Arc is observed for DNA of 30 bp. Longer DNA is significantly less efficient in accelerating the Arc refolding rate, since the two subunits search distinct regions of the one-dimensional DNA and are therefore much less colocalized. The probability that the two unfolded chains will meet on 200 bp DNA is similar to that in the bulk. The colocalization of Arc subunits on ssDNA results in much faster folding compared to that obtained on dsDNA of the same length. Differences in the rate of Arc refolding, cooperativity, and the structure of its transition state ensemble introduced by ssDNA and dsDNA molecules

  9. Intercalation between antitumor anthracyclines and DNA as probed by resonance and surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Smulevich, G.; Mantini, A. R.; Casu, M.; Marzocchi, M. P.

    1991-05-01

    The antiturnor anthracyclincs, idarubicin (IDA ), adrianiycin (ADM), epirubicin (EPI), carminomycin (CAR) and 1 1-deoxycarminornycin (DCM), whose siructural formula includes a substituted hydroxyanthraquirionc chrornophore and a sugar residue, form intercalation complexes with DNA. The stacking interaction between the chromophore and the base-pairs of DNA gives rise to noticeable ciTects on resonance Raman (RR) and surface-enhanced resonance Raman (SERRS) scattering as well as on the absorption (ABS), its second derivative (D2) and fluorescence emission (FEM) spectra.

  10. Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

    PubMed Central

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2008-01-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

  11. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    SciTech Connect

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  12. Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas

    NASA Astrophysics Data System (ADS)

    Acuna, G. P.; Möller, F. M.; Holzmeister, P.; Beater, S.; Lalkens, B.; Tinnefeld, P.

    2012-10-01

    We introduce self-assembled nanoantennas to enhance the fluorescence intensity in a plasmonic hotspot of zeptoliter volume. The nanoantennas are prepared by attaching one or two gold nanoparticles (NPs) to DNA origami structures, which also incorporated docking sites for a single fluorescent dye next to one NP or in the gap between two NPs. We measured the dependence of the fluorescence enhancement on NP size and number and compare it to numerical simulations. A maximum of 117-fold fluorescence enhancement was obtained for a dye molecule positioned in the 23-nanometer gap between 100-nanometer gold NPs. Direct visualization of the binding and unbinding of short DNA strands, as well as the conformational dynamics of a DNA Holliday junction in the hotspot of the nanoantenna, show the compatibility with single-molecule assays.

  13. Enhanced electrophoretic DNA separation in photonic crystal fiber.

    PubMed

    Sun, Yi; Nguyen, Nam-Trung; Kwok, Yien Chian

    2009-07-01

    Joule heating generated by the electrical current in capillary electrophoresis leads to a temperature gradient along the separation channel and consequently affects the separation quality. We describe a method of reducing the Joule heating effect by incorporating photonic crystal fiber into a micro capillary electrophoresis chip. The photonic crystal fiber consists of a bundle of extremely narrow hollow channels, which ideally work as separation columns. Electrophoretic separation of DNA fragments was simultaneously but independently carried out in 54 narrow capillaries with a diameter of 3.7 microm each. The capillary bundle offers more efficient heat dissipation owing to the high surface-to-volume ratio. Under the same electrical field strength, notable improvement in resolution was obtained in the capillary bundle chip.

  14. The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

    PubMed

    Shaw, Kirsty J; Thain, Lauren; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-10-12

    DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

  15. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    PubMed

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  16. Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in chitosan/DNA complexes.

    PubMed

    Chen, Liulin; Zhu, Junming; Li, Yuhong; Lu, Jie; Gao, Li; Xu, Huibi; Fan, Mingwen; Yang, Xiangliang

    2013-01-01

    The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.

  17. Effect of dactyloscopic powders on DNA profiling from enhanced fingerprints: results from an experimental study.

    PubMed

    Tozzo, Pamela; Giuliodori, Alice; Rodriguez, Daniele; Caenazzo, Luciana

    2014-03-01

    We conducted a study on the effect of fingerprint enhancement methods on subsequent short tandem repeat profiling. First, we performed a study typing blood traces deposited on 5 different surfaces, treated with 8 types of dactyloscopic powders. Three different DNA extraction methods were used. Subsequently, we analyzed latent fingerprints on the same 5 surfaces enhanced with the 8 different powders used in the first part of the study. This study has demonstrated that DNA profiling can be performed on fingerprints left on different substrates, and the substrate will affect the amount of DNA that can be recovered for DNA typing. In the first phase of the study, a profile was obtained in 92% of the 120 samples analyzed; in the second part, in 55% of the 80 samples analyzed, we obtained a profile complete in 32.5% of the cases. From the results obtained, it seems that the powders used in latent fingerprints enhancement, rather than having a direct inhibitory effect on extraction and amplification of DNA, may cause partial degradation of DNA, reducing the efficiency of amplification reaction. It should not be forgotten that these results were obtained under laboratory conditions, and in real caseworks, there may still be different problems involved.

  18. ARRB1 enhances the chemosensitivity of lung cancer through the mediation of DNA damage response

    PubMed Central

    Shen, Hongchang; Wang, Liguang; Zhang, Jiangang; Dong, Wei; Zhang, Tiehong; Ni, Yang; Cao, Hongxin; Wang, Kai; Li, Yun; Wang, Yibing; Du, Jiajun

    2017-01-01

    ARRB1 (also known as β-arrestin-1) serves as a multifunctional adaptor contributing to the regulation of signaling pathways. ARRB1 may be involved in DNA damage accumulation; however the underlying mechanism involved is unclear. In the present study, non-small cell lung cancer (NSCLC) cell lines (H520 and SK-MES-1) were transfected with ARRB1 plasmids or small interfering ribonucleic acid (siRNA) and received treatment with DNA-damaging agents (cisplatin and etoposide). A mouse xenograft model was used to assess the impact of ARRB1 on the efficacy of cisplatin in vivo. A total of 30 surgically resected NSCLC patients were recruited for the present study and qRT-PCR was performed to determine the mRNA levels in cancer tissues compared with para-carcinoma tissues. Our data showed that DNA damage was abrogated in the ARRB1-knockdown cells and enhanced in the ARRB1-overexpressing cells. ATR and Chk1 were more activated in the ARRB1-overexpressing cells compared to the ARRB1-knockdown cells, followed by increased H2AX phosphorylation. DNA damage and apoptosis were increased in the ARRB1-overexpressing cells treated with cisplatin. These data provided strong evidence that ARRB1 contributes to the response of NSCLC to DNA-damaging agents and is essential for DNA damage response (DDR). ARRB1 may enhance the efficacy of DNA-damaging agents in NSCLC. PMID:28035404

  19. Advances in SCA and RF-DNA Fingerprinting Through Enhanced Linear Regression Attacks and Application of Random Forest Classifiers

    DTIC Science & Technology

    2014-09-18

    ADVANCES IN SCA AND RF-DNA FINGERPRINTING THROUGH ENHANCED LINEAR REGRESSION ATTACKS AND APPLICATION OF RANDOM FOREST CLASSIFIERS DISSERTATION Hiren...SCA AND RF-DNA FINGERPRINTING THROUGH ENHANCED LINEAR REGRESSION ATTACKS AND APPLICATION OF RANDOM FOREST CLASSIFIERS DISSERTATION Presented to the...APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED AFIT-ENG-DS-14-S-03 ADVANCES IN SCA AND RF-DNA FINGERPRINTING THROUGH ENHANCED LINEAR REGRESSION ATTACKS

  20. Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

    PubMed Central

    Stuiver, M H; van der Vliet, P C

    1990-01-01

    The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI. Images PMID:2293667

  1. Weak operator binding enhances simulated Lac repressor-mediated DNA looping.

    PubMed

    Colasanti, Andrew V; Grosner, Michael A; Perez, Pamela J; Clauvelin, Nicolas; Lu, Xiang-Jun; Olson, Wilma K

    2013-12-01

    The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding.

  2. Unscheduled load flow effect due to large variation in the distributed generation in a subtransmission network

    NASA Astrophysics Data System (ADS)

    Islam, Mujahidul

    from the vast network. A path tracing methodology is developed to identify the power lines that are vulnerable to an unscheduled flow effect in the sub-transmission network. It is much harder to aggregate power system network sensitivity information or data from measuring load flow physically than to simulate in software. System dynamics is one of the key factors to determine an appropriate dynamic control mechanism at an optimum network location. Once a model of deterministic but variable power generator is used, the simulation can be meaningful in justifying this claim. The method used to model the variable generator is named the two-components phase distortion model. The model was validated from the high resolution data collected from three pilot photovoltaic sites in Florida - two in the city of St. Petersburg and one in the city of Tampa. The high resolution data was correlated with weather radar closest to the sites during the design stage of the model. Technically the deterministic model cannot replicate a stochastic model which is more realistically applicable for solar isolation and involves a Markov chain. The author justified the proposition based on the fact that for analysis of the response functions of different systems, the excitation function should be common for comparison. Moreover, there could be many possible simulation scenarios but fewer worst cases. Almost all commercial systems are protected against potential faults and contingencies to a certain extent. Hence, the proposed model for worst case studies was designed within a reasonable limit. The simulation includes steady state and transient mode using multiple software modules including MatlabRTM, PSCADRTM and Paladin Design BaseRTM. It is shown that by identifying vulnerable or sensitive branches in the network, the control mechanisms can be coordinated reliably. In the long run this can save money by preventing unscheduled power flow in the network and eventually stabilizing the energy market.

  3. DNA-guided assembly of three-dimensional nanostructures for surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Li-An; Lin, Yu-Ting; Chen, Yih-Fan

    2015-03-01

    Surface enhancement Raman spectroscopy (SERS) has drawn much attention in recent years because its ability to greatly enhance Raman signals to allow for the detection of molecules at low concentration. When using metallic nanoparticles as SERS substrates, many studies have shown that the size of the interparticle gap significantly affects the enhancement of the Raman signals. Given that the optimal interparticle gap is as small as a few nanometers, fabricating sensitive, uniform, and reproducible SERS substrates remains challenging. Here we report a three-dimensional SERS substrate created through the assembly of core-shell nanoparticles using DNA. By using DNA of appropriate sequence and length, DNA-functionalized nanoparticles were assembled into ordered and highly packed nanostructures. The interparticle distance was precisely controlled by adjusting the design of the DNA and the thickness of the silver shell coated on the gold nanoparticles. Compared with randomly aggregated nanoparticles, the interparticle distance in the synthesized nanostructures can be more uniform and better controlled. In addition, the DNA-guided assembly process allows us to create precise nanostructures without using complex and expensive fabrication methods. The study demonstrates that the synthesized nanostructures can be used as effective SERS substrates to successfully measure the Raman signals of malachite green, a toxic compound that is sometimes illegally used on fish, as well as Fluorescein isothiocyanate (FITC) at low concentrations.

  4. Enhanced nucleotide excision repair capacity in lung cancer cells by preconditioning with DNA-damaging agents.

    PubMed

    Choi, Ji Ye; Park, Jeong-Min; Yi, Joo Mi; Leem, Sun-Hee; Kang, Tae-Hong

    2015-09-08

    The capacity of tumor cells for nucleotide excision repair (NER) is a major determinant of the efficacy of and resistance to DNA-damaging chemotherapeutics, such as cisplatin. Here, we demonstrate that using lesion-specific monoclonal antibodies, NER capacity is enhanced in human lung cancer cells after preconditioning with DNA-damaging agents. Preconditioning of cells with a nonlethal dose of UV radiation facilitated the kinetics of subsequent cisplatin repair and vice versa. Dual-incision assay confirmed that the enhanced NER capacity was sustained for 2 days. Checkpoint activation by ATR kinase and expression of NER factors were not altered significantly by the preconditioning, whereas association of XPA, the rate-limiting factor in NER, with chromatin was accelerated. In preconditioned cells, SIRT1 expression was increased, and this resulted in a decrease in acetylated XPA. Inhibition of SIRT1 abrogated the preconditioning-induced predominant XPA binding to DNA lesions. Taking these data together, we conclude that upregulated NER capacity in preconditioned lung cancer cells is caused partly by an increased level of SIRT1, which modulates XPA sensitivity to DNA damage. This study provides some insights into the molecular mechanism of chemoresistance through acquisition of enhanced DNA repair capacity in cancer cells.

  5. Shrink-Induced Silica Multiscale Structures for Enhanced Fluorescence from DNA Microarrays

    PubMed Central

    2015-01-01

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30–45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays. PMID:25191785

  6. Using surface-enhanced Raman spectroscopy to probe for genetic markers on single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Moody, Benjamin; Leotaud, John; McCarty, Gregory S.

    2010-03-01

    Methods capable of quickly and inexpensively collecting genetic information are of increasing importance. We report a method of using surface-enhanced Raman spectroscopy to probe single-stranded DNA for genetic markers. This unique approach is used to analyze unmodified genes of moderate length for genetic markers by hybridizing native test oligonucleotides into a surface-enhanced Raman complex, vastly increasing detection sensitivity as compared to traditional Raman spectroscopy. The Raman complex is formed by sandwiching the test DNA between 40-nm gold nanoparticles and a photolithographically defined gold surface. With this design, we are able to collect characteristic Raman spectra about the test DNA and to detect genetic markers such as single-nucleotide polymorphisms (SNPs) and polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant trends regarding Raman intensity.

  7. Assessing the Risk of Secondary Transfer Via Fingerprint Brush Contamination Using Enhanced Sensitivity DNA Analysis Methods.

    PubMed

    Bolivar, Paula-Andrea; Tracey, Martin; McCord, Bruce

    2016-01-01

    Experiments were performed to determine the extent of cross-contamination of DNA resulting from secondary transfer due to fingerprint brushes used on multiple items of evidence. Analysis of both standard and low copy number (LCN) STR was performed. Two different procedures were used to enhance sensitivity, post-PCR cleanup and increased cycle number. Under standard STR typing procedures, some additional alleles were produced that were not present in the controls or blanks; however, there was insufficient data to include the contaminant donor as a contributor. Inclusion of the contaminant donor did occur for one sample using post-PCR cleanup. Detection of the contaminant donor occurred for every replicate of the 31 cycle amplifications; however, using LCN interpretation recommendations for consensus profiles, only one sample would include the contaminant donor. Our results indicate that detection of secondary transfer of DNA can occur through fingerprint brush contamination and is enhanced using LCN-DNA methods.

  8. Ultrasensitive detection of potassium ions based on target induced DNA conformational switch enhanced fluorescence polarization.

    PubMed

    Hu, Kun; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Wu, Qiang; Zhang, Guohai; Jiang, Jing

    2012-06-21

    We have developed a simple, highly sensitive and selective fluorescence polarization assay for the detection of potassium ions based on target induced DNA conformational switch from hairpin to G-quadruplex enhanced fluorescence polarization. The assay was applied in the detection of low nM concentrations of potassium ions and was highly selective over other cations.

  9. Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos.

    PubMed

    Bohrer, Rodrigo Camponogara; Duggavathi, Raj; Bordignon, Vilceu

    2014-01-01

    Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos.

  10. Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination.

    PubMed

    Henley, Robert Y; Vazquez-Pagan, Ana G; Johnson, Michael; Kanavarioti, Anastassia; Wanunu, Meni

    2015-01-01

    Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.

  11. DNA sequence detection using surface-enhanced resonance Raman spectroscopy in a homogeneous multiplexed assay.

    PubMed

    MacAskill, Alexandra; Crawford, David; Graham, Duncan; Faulds, Karen

    2009-10-01

    Detection of specific DNA sequences is central to modern molecular biology and also to molecular diagnostics where identification of a particular disease is based on nucleic acid identification. Many methods exist, and fluorescence spectroscopy dominates the detection technologies employed with different assay formats. This study demonstrates the use of surface-enhanced resonance Raman scattering (SERRS) to detect specific DNA sequences when coupled with modified SERRS-active probes that have been designed to modify the affinity of double- and single-stranded DNA for the surface of silver nanoparticles resulting in discernible differences in the SERRS which can be correlated to the specific DNA hybridization event. The principle of the assay lies on the lack of affinity of double-stranded DNA for silver nanoparticle surfaces; therefore, hybridization of the probe to the target results in a reduction in the SERRS signal. Use of locked nucleic acid (LNA) residues in the DNA probes resulted in greater discrimination between exact match and mismatches when used in comparison to unmodified labeled DNA probes. Polymerase chain reaction (PCR) products were detected using this methodology, and ultimately a multiplex detection of sequences relating to a hospital-acquired infection, namely, methicillin-resistant Staphylococcus aureus (MRSA), demonstrated the versatility and applicability of this approach to real-life situations.

  12. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.

    PubMed Central

    Tang, W J; Berger, S L; Triezenberg, S J; Folk, W R

    1987-01-01

    The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein. Images PMID:3037332

  13. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    PubMed

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

  14. Enhanced memory persistence is blocked by a DNA methyltransferase inhibitor in the snail Lymnaea stagnalis.

    PubMed

    Lukowiak, Ken; Heckler, Benjamin; Bennett, Thomas E; Schriner, Ellen K; Wyrick, Kathryn; Jewett, Cynthia; Todd, Ryan P; Sorg, Barbara A

    2014-08-15

    Lymnaea stagnalis provides an excellent model system for studying memory because these snails have a well-described set of neurons, a single one of which controls expression of long-term memory of operantly conditioned respiratory behavior. We have shown that several different manipulations, including pre-training exposure to serotonin (5-HT) or methamphetamine, submersion of snails after training to prevent memory interference, and exposure to effluent from predatory crayfish (CE), enhance memory persistence. Changes in DNA methylation underlie formation of strong memories in mammals and 5-HT-enhanced long-term facilitation in Aplysia. Here we determined the impact of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-AZA; 87 μmol l(-1)), on enhanced memory persistence by all four manipulations. We found that 5-HT (100 μmol l(-1)) enhanced memory persistence, which was blocked by 5-AZA pretreatment. Snails pre-exposed to 3.3 μmol l(-1) Meth 4 h prior to training demonstrated memory 72 h later, which was not present in controls. This memory-enhancing effect was blocked by pre-treatment with 87 μmol l(-1) 5-AZA. Similarly, submersion to prevent interference learning as well as training in CE produced memory that was not present in controls, and these effects were blocked by pre-treatment with 87 μmol l(-1) 5-AZA. In contrast, 5-AZA injection did not alter expression of normal (non-enhanced) memory, suggesting that these four stimuli enhance memory persistence by increasing DNA methyltransferase activity, which, in turn, increases expression of memory-enhancing genes and/or inhibits memory suppressor genes. These studies lay important groundwork for delineating gene methylation changes that are common to persistent memory produced by different stimuli.

  15. Microwave-accelerated metal-enhanced fluorescence: an ultra-fast and sensitive DNA sensing platform.

    PubMed

    Aslan, Kadir; Malyn, Stuart N; Bector, Geetika; Geddes, Chris D

    2007-11-01

    In this paper, we investigated the effects of low-power microwave heating on the components of the recently described new approach to surface DNA hybridization assays, based on the Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) platform technology. Thiolated oligonucleotides have been linked to surface-bound silver nanostructures which partially coat a glass slide. The addition of a complementary fluorescein-labeled oligonucleotide results in metal-enhanced fluorescein emission as the probe is brought into close proximity to the silver upon hybridization. In addition, the combined use with low-power microwave heating, which is thought to locally heat around the silvered surface, affords for both the assay kinetics and optical amplification to also be localized to the surface. In our model DNA target assay reported here, we can detect 23-mer targets in less than 20 s, up to a 600-fold decrease in the assay run time as compared to control samples hybridized to completion at room temperature. Importantly, the use of MAMEF also reduces the extent of unwanted non-specific DNA absorption, further increasing specific DNA target detection limits. It was also found that low-power microwave heating did not denature DNA and the bulk temperature increase near to silver nanoparticles was only ca. 1 degrees C.

  16. Feasibility of Single Molecule DNA Sequencing using Surface-Enhanced Raman Scattering

    SciTech Connect

    Talley, C E; Reboredo, F; Chan, J; Lane, S M

    2006-02-03

    We have used a combined theoretical and experimental approach in order to assess the feasibility of using surface-enhanced Raman scattering (SERS) for DNA sequencing at the single molecule level. We have developed a numerical tool capable of calculating the E-field and resulting SERS enhancement factors for metallic structures of arbitrary size and shape. Measurements of the additional SERS enhancement by combining SERS with coherent antistokes Raman scattering (CARS) show that only modest increases in the signal are achievable due to thermal damage at higher laser powers. Finally, measurements of the SERS enhancement from nanoparticles coated with an insulating layer show that the SERS enhancement is decreased by as much as two orders of magnitude when the molecule is not in contact with the metal surface.

  17. Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding.

    PubMed Central

    Khoroshilova, N; Beinert, H; Kiley, P J

    1995-01-01

    In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein. Images Fig. 4 PMID:7708673

  18. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    PubMed

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi

    2014-08-11

    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  19. DNA-directed gold nanodimers with tailored ensemble surface-enhanced Raman scattering properties.

    PubMed

    Lan, Xiang; Chen, Zhong; Lu, Xuxing; Dai, Gaole; Ni, Weihai; Wang, Qiangbin

    2013-11-13

    Gold nanodimers (GNDs) are assembled with high uniformity as ideal surface-enhanced Raman scattering (SERS) substrates through DNA-directed self-assembly of gold nanoparticles. The interparticle distance within GNDs is precisely tailored on the order of a few nanometers with changing the molecule length of DNA bridge. The ensemble SERS activity of monodispersed GNDs is then rationally engineered by modifying the structural parameters of GNDs including the particle size and interparticle distance. Theoretical studies on the level of single GND evidence the particle size- and interparticle-distance-dependent SERS effects, consistent with the ensemble averaged measurements.

  20. DNA Sensing using Nano-crystalline Surface Enhanced Al2O3 Nanopore Sensors

    PubMed Central

    Venkatesan, B. M.; Shah, A.B.; Zuo, J.M.; Bashir, R.

    2013-01-01

    A new solid-state, Al2O3 nanopore sensor with enhanced surface properties for the real-time detection and analysis of individual DNA molecules is reported. Nanopore formation using electron beam based decomposition transformed the local nanostructure and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a hetero-phase crystalline network, deficient in O (O to Al ratio of ~0.6). Direct metallization of the pore region was observed during irradiation, thereby permitting the potential fabrication of nano-scale metallic contacts in the pore region with potential application to nanopore-based DNA sequencing. Dose dependent phase transformations to purely γ and/or α-phase nanocrystallites were also observed during pore formation allowing for surface charge engineering at the nanopore/fluid interface. DNA transport studies revealed an order of magnitude reduction in translocation velocities relative to alternate solid-state architectures, accredited to high surface charge density and the nucleation of charged nanocrystalline domains. The unique surface properties of Al2O3 nanopore sensors makes them ideal for the detection and analysis of ssDNA, dsDNA, RNA secondary structures and small proteins. These nano-scale sensors may also serve as a useful tool in studying the mechanisms driving biological processes including DNA-protein interactions and enzyme activity at the single molecule level. PMID:23335871

  1. Enhanced electrochemical detection of DNA hybridization with carbon nanotube modified paste electrode.

    PubMed

    Nie, Libo; Guo, Huishi; He, Quanguo; Chen, Jianrong; Miao, Yuqing

    2007-02-01

    A novel electrochemical genesensor using twice hybridization enhancement of gold nanoparticles based on carbon paste modified electrode is described. The carbon nanotube modified carbon paste electrode (CNTPE) and mesoporous molecular sieve SBA-15 modified carbon paste electrode (MSCPE) were investigated. The assay relies on the immobilization of streptavidin-biotin labeled target oligonucleotides onto the electrode surface and its hybridization to the gold nanoparticle-labeled DNA probe. After twice hybridization enhanced connection of gold nanoparticles to the hybridized system, the differential pulse voltammetry (DPV) signal of total gold nanoparticles was monitored. It was found that the adsorption of oligonucleotide and hybridized DPV signal on CNTPE were both enhanced in comparison with that of pure carbon paste electrode (CPE). But this trend was reverse on MSCPE. The DPV detection of twice hybridized gold nanoparticles indicated that the sensitivity of the genesensor enhanced about one order of magnitude compared with one-layer hybridization. One-base mismatched DNA and complementary DNA could be distinguished clearly. However, no distinct advantage of MSCPE over CPE was found.

  2. Hybrid Structures for Surface-Enhanced Raman Scattering: DNA Origami/Gold Nanoparticle Dimer/Graphene.

    PubMed

    Prinz, Julia; Matković, Aleksandar; Pešić, Jelena; Gajić, Radoš; Bald, Ilko

    2016-10-01

    A combination of three innovative materials within one hybrid structure to explore the synergistic interaction of their individual properties is presented. The unique electronic, mechanical, and thermal properties of graphene are combined with the plasmonic properties of gold nanoparticle (AuNP) dimers, which are assembled using DNA origami nanostructures. This novel hybrid structure is characterized by means of correlated atomic force microscopy and surface-enhanced Raman scattering (SERS). It is demonstrated that strong interactions between graphene and AuNPs result in superior SERS performance of the hybrid structure compared to their individual components. This is particularly evident in efficient fluorescence quenching, reduced background, and a decrease of the photobleaching rate up to one order of magnitude. The versatility of DNA origami structures to serve as interface for complex and precise arrangements of nanoparticles and other functional entities provides the basis to further exploit the potential of the here presented DNA origami-AuNP dimer-graphene hybrid structures.

  3. Novel Oxidatively Activated Agents Modify DNA and are Enhanced by Ercc1 Silencing

    PubMed Central

    Jones, Amy R.; Bell-Horwath, Tiffany R.; Li, Guorui; Rollmann, Stephanie M.; Merino, Edward J.

    2012-01-01

    Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq2, which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and is found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed the agents add to 2′-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2′-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2′-deoxyadenosine and 2′-deoxycytidine, but not thymidine or 2′-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq2 compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between two and four fold above controls. Benzetheno-nucleobase addition to double stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi based approach, Ercc1 was silenced and survival monitored after injection of an agent. As expected, bulky cross-linking DNA modifying agents, cisplatin and

  4. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  5. Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.

    PubMed

    Fox, A; Gittos, M; Harbison, S A; Fleming, R; Wivell, R

    2014-05-01

    Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling.

  6. Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment

    SciTech Connect

    Protic, M.; Roilides, E.; Levine, A.S.; Dixon, K.

    1988-07-01

    To determine whether DNA excision repair is enhanced in mammalian cells in response to DNA damage, as it is in bacteria as part of the SOS response, we used an expression vector-host cell reactivation assay to measure cellular DNA repair capacity. When UV-damaged chloramphenicol acetyltransferase (CAT) vector DNA was introduced into monkey cells (CV-1), the level of CAT activity was inversely related to the UV fluence due to inhibition of CAT gene expression by UV photoproducts. When CV-1 cells were treated with either UV radiation or mitomycin C, 24-48 h before transfection, CAT expression from the UV-irradiated plasmid was increased. This increase also occurred in a line of normal human cells, but not in repair-deficient human xeroderma pigmentosum cells. We confirmed that this increase in CAT expression was due to repair, and not to production of damage-free templates by recombination; the frequency of generation of supF+ recombinants after transfection with UV-irradiated pZ189 vectors carrying different point mutations in the supF gene did not significantly increase in carcinogen-treated CV-1 cells. From these results we conclude that carcinogen treatment enhances the excision-repair capacity of normal mammalian cells.

  7. DNA origami based assembly of gold nanoparticle dimers for surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Thacker, Vivek V.; Herrmann, Lars O.; Sigle, Daniel O.; Zhang, Tao; Liedl, Tim; Baumberg, Jeremy J.; Keyser, Ulrich F.

    2014-03-01

    Plasmonic sensors are extremely promising candidates for label-free single-molecule analysis but require exquisite control over the physical arrangement of metallic nanostructures. Here we employ self-assembly based on the DNA origami technique for accurate positioning of individual gold nanoparticles. Our innovative design leads to strong plasmonic coupling between two 40 nm gold nanoparticles reproducibly held with gaps of 3.3±1 nm. This is confirmed through far field scattering measurements on individual dimers which reveal a significant red shift in the plasmonic resonance peaks, consistent with the high dielectric environment due to the surrounding DNA. We use surface-enhanced Raman scattering (SERS) to demonstrate local field enhancements of several orders of magnitude through detection of a small number of dye molecules as well as short single-stranded DNA oligonucleotides. This demonstrates that DNA origami is a powerful tool for the high-yield creation of SERS-active nanoparticle assemblies with reliable sub-5 nm gap sizes.

  8. Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage.

    PubMed

    Xie, Chengzhi; Edwards, Holly; Caldwell, J Timothy; Wang, Guan; Taub, Jeffrey W; Ge, Yubin

    2015-02-01

    Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan-Bcl-2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double-strand breaks (DSBs) preceded a decrease of Mcl-1 levels, nuclear translocation of Bcl-2, Bcl-xL, and Mcl-1, and apoptosis. These results indicate that obatoclax enhances cytarabine-induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA-damaging agents in AML and possibly a broader range of malignancies.

  9. DNA methylation of oestrogen-regulated enhancers defines endocrine sensitivity in breast cancer

    PubMed Central

    Stone, Andrew; Zotenko, Elena; Locke, Warwick J.; Korbie, Darren; Millar, Ewan K. A.; Pidsley, Ruth; Stirzaker, Clare; Graham, Peter; Trau, Matt; Musgrove, Elizabeth A.; Nicholson, Robert I.; Gee, Julia M. W.; Clark, Susan J.

    2015-01-01

    Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at oestrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine-responsive ESR1-positive cancer. Cumulatively, these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy. PMID:26169690

  10. Enhanced purification of plasmid DNA isoforms by exploiting ionic strength effects during ultrafiltration.

    PubMed

    Li, Ying; Currie, David; Zydney, Andrew L

    2016-04-01

    The solution structure of plasmid DNA is known to be a strong function of solution conditions due to intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. The objective of this work was to determine whether it was possible to enhance the use of ultrafiltration for separation of different plasmid isoforms by proper selection of the solution ionic strength and ion type. Experiments were performed with a 3.0 kbp plasmid using composite regenerated cellulose ultrafiltration membranes. The transmission of the linear isoform was nearly independent of solution ionic strength, but increased significantly with increasing filtrate flux due to the elongation of the highly flexible plasmid in the converging flow field into the membrane pores. In contrast, the transmission of the open-circular and supercoiled plasmids both increased with increasing NaCl or MgCl2 concentration due to the change in plasmid size and conformational flexibility. The effect of ionic strength was greatest for the supercoiled plasmid, providing opportunities for enhanced purification of this therapeutically active isoform. This behavior was confirmed using experiments performed with binary mixtures of the different isoforms. These results clearly demonstrate the potential for enhancing the performance of membrane systems for plasmid DNA separations by proper selection of the ionic conditions.

  11. Enhancing DNA delivery into the skin with a motorized microneedle device.

    PubMed

    Yan, Guang; Arelly, Naresh; Farhan, Nashid; Lobo, Shabbir; Li, Henan

    2014-02-14

    The purpose of this study was to evaluate a motorized microneedle device in delivery of DNA into skin for gene expression. A plasmid DNA encoding both luciferase (Luc) and enhanced green fluorescent protein (EGFP) was delivered into rat skin by puncturing the skin with the microneedle device. Puncturing rat skin with a pre-applied DNA solution on the skin showed much higher luciferase gene expression than that with the procedure of puncturing the skin first then applied the DNA solution. The microneedle puncturing method was more efficient than intradermal injection method in generating high gene expression in the skin. There was no significant difference in the skin gene expression when rat skin was punctured with the microneedle device of different microneedle lengths (0.25 mm, 0.5mm or 0.75 mm). On the other hand, there was a significant difference in the skin gene expression between the short (10s) and the long puncturing durations (30 or 60s), with longer puncturing duration showed higher gene expression. Puncturing the skin with longer needles (0.75 mm) caused some skin damage, while puncturing the skin with shorter microneedle length (0.25 mm) caused only minimal skin damage. The EGFP gene expression was observed predominately in the epidermis layer of the skin from the puncturing method in delivery of DNA into the skin. In summary, the motorized microneedle device could have great potential in skin gene delivery.

  12. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans

    PubMed Central

    2013-01-01

    Background Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called “universal” primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. Results Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first–time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. Conclusions These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory. PMID:24020880

  13. Myeloperoxidase Enhances Etoposide and Mitoxantrone-Mediated DNA Damage: A Target for Myeloprotection in Cancer Chemotherapy

    PubMed Central

    Atwal, Mandeep; Lishman, Emma L.; Austin, Caroline A.

    2017-01-01

    Myeloperoxidase is expressed exclusively in granulocytes and immature myeloid cells and transforms the topoisomerase II (TOP2) poisons etoposide and mitoxantrone to chemical forms that have altered DNA damaging properties. TOP2 poisons are valuable and widely used anticancer drugs, but they are associated with the occurrence of secondary acute myeloid leukemias. These factors have led to the hypothesis that myeloperoxidase inhibition could protect hematopoietic cells from TOP2 poison-mediated genotoxic damage and, therefore, reduce the rate of therapy-related leukemia. We show here that myeloperoxidase activity leads to elevated accumulation of etoposide- and mitoxantrone-induced TOP2A and TOP2B-DNA covalent complexes in cells, which are converted to DNA double-strand breaks. For both drugs, the effect of myeloperoxidase activity was greater for TOP2B than for TOP2A. This is a significant finding because TOP2B has been linked to genetic damage associated with leukemic transformation, including etoposide-induced chromosomal breaks at the MLL and RUNX1 loci. Glutathione depletion, mimicking in vivo conditions experienced during chemotherapy treatment, elicited further MPO-dependent increase in TOP2A and especially TOP2B-DNA complexes and DNA double-strand break formation. Together these results support targeting myeloperoxidase activity to reduce genetic damage leading to therapy-related leukemia, a possibility that is enhanced by the recent development of novel specific myeloperoxidase inhibitors for use in inflammatory diseases involving neutrophil infiltration. PMID:27974636

  14. Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

    NASA Astrophysics Data System (ADS)

    Swasey, Steven M.; Gwinn, Elisabeth G.

    2016-04-01

    The thermal and chemical fragility of DNA nanomaterials assembled by Watson-Crick (WC) pairing constrain the settings in which these materials can be used and how they can be functionalized. Here we investigate use of the silver cation, Ag+, as an agent for more robust, metal-mediated self-assembly, focusing on the simplest duplex building blocks that would be required for more elaborate Ag+-DNA nanostructures. Our studies of Ag+-induced assembly of non-complementary DNA oligomers employ strands of 2-24 bases, with varied base compositions, and use electrospray ionization mass spectrometry to determine product compositions. High yields of duplex products containing narrowly distributed numbers of Ag+ can be achieved by optimizing solution conditions. These Ag+-mediated duplexes are stable to at least 60 mM Mg2+, higher than is necessary for WC nanotechnology schemes such as tile assemblies and DNA origami, indicating that sequential stages of Ag+-mediated and WC-mediated assembly may be feasible. Circular dichroism spectroscopy suggests simple helical structures for Ag+-mediated duplexes with lengths to at least 20 base pairs, and further indicates that the structure of cytosine-rich duplexes is preserved at high urea concentrations. We therefore propose an approach towards dynamic DNA nanomaterials with enhanced thermal and chemical stability through designs that combine sturdy silver-mediated ‘frames’ with WC paired ‘pictures’.

  15. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2

    PubMed Central

    Kaur, Parminder; Wu, Dong; Lin, Jiangguo; Countryman, Preston; Bradford, Kira C.; Erie, Dorothy A.; Riehn, Robert; Opresko, Patricia L.; Wang, Hong

    2016-01-01

    Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways. PMID:26856421

  16. Functional analysis of the individual enhancer core sequences of polyomavirus: Cell-specific uncoupling of DNA replication from transcription

    SciTech Connect

    Campbell, B.A.; Villarreal, L.P.

    1988-05-01

    Polyomavirus (Py) enhancer core elements were compared for their ability to activate Py early transcription and DNA replication in mouse 3T6 cells, lymphoid cell lines, and undifferentiated embryonal carcinoma cells. By examining the pattern of genetic change in a number of cell-specific Py variants, the authors identified subenhancer sequences that may be functionally important for virus replication. Four such distinct enhancer consensus sequences were synthesized and designated as the A core (homologous with adenovirus 5 E1A enhancer), B core (homologous to the simian virus 40 A enhancer core), C core (containing an inverted repeat within the Py B enhancer), and BPV core (homologous to the bovine papillomavirus enhancer). When used to replace the complete Py B enhancer, single copies of all but the BPV element were able to fully activate Py DNA replication after transfection, but this activation was usually cell type specific. In the PCC4 embryonal carcinoma cells, only the A-core sequence was able to activate transcription and DNA replication. The BPV core sequence containing the Py F441 point change was unable to activate DNA replication in the F9 embryonal carcinoma or any other cell line. No single insertion element was dominant nor did these elements display the wild-type enhancer pattern of cell-specific activation of DNA replication. In addition, differential effects were often observed on the activation of transcription versus DNA replication.

  17. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

    PubMed

    Ehrlich, Kenneth C; Paterson, Heather L; Lacey, Michelle; Ehrlich, Melanie

    2016-12-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

  18. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression

    PubMed Central

    Ehrlich, Kenneth C.; Paterson, Heather L.; Lacey, Michelle; Ehrlich, Melanie

    2016-01-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1’s super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation. PMID:28018137

  19. Effect of DNA Superhelicity and Bound Proteins on Mechanistic Aspects of the Hin-Mediated and Fis-Enhanced Inversion

    PubMed Central

    Huang, Jing; Zhang, Qing; Schlick, Tamar

    2003-01-01

    Using a recently developed inhomogeneous, macroscopic model for long DNA bound to proteins, we examine topological and geometric aspects of DNA/protein structures and dynamics on various stages of the Hin inversion pathway. This biological reaction involves exchange of DNA in a synaptic complex that brings together several DNA sites bound to Hin dimers as well as Fis enhancers. Brownian dynamics simulations in the millisecond timescale allow us to follow and analyze the DNA/protein dynamics trajectories and to examine the effects of DNA superhelicity and protein binding on various reaction steps. Analysis of the generated kinetic pathways helps explain mechanistic aspects regarding the process by which two or three protein-bound DNA sites come to close spatial proximity and show that how topological selectivity (two trapped supercoils), enhancer binding, and properties of supercoiled DNA play critical roles in regulating the inversion reaction. Specifically, a critical amount of DNA superhelicity (e.g., |σ| > 0.02) leads to an optimal interplay for the first reaction step—two-site juxtaposition—between large-scale random rearrangements of Hin-bound DNA and local slithering within branches of plectonemes. The three-site juxtaposition, the second step, is significantly accelerated by the presence of an enhancer protein that, due to severe local bending, also alters juxtaposition mechanisms, especially for superhelical density magnitude greater than around 0.04. PMID:12885629

  20. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    SciTech Connect

    Chowdhury, E.H.

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  1. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    PubMed

    Speit, Günter; Schütz, Petra; Bausinger, Julia

    2016-06-01

    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  2. Metal enhanced fluorescence improved protein and DNA detection by zigzag Ag nanorod arrays.

    PubMed

    Ji, Xiaofan; Xiao, Chenyu; Lau, Wai-Fung; Li, Jianping; Fu, Junxue

    2016-08-15

    As metal nano-arrays show great potential on metal enhanced fluorescence (MEF) than random nanostructures, MEF of Ag zigzag nanorod (ZNR) arrays made by oblique angle deposition has been studied for biomolecule-protein interaction and DNA hybridization. By changing the folding number and the deposition substrate temperature, a 14-fold enhancement factor (EF) is obtained for biotin-neutravidin detection. The optimal folding number is decided as Z=7, owing to the high scattering intensity of Ag ZNRs. The substrate temperature T=25°C and 0°C slightly alters the morphology of Ag ZNRs but has no big difference in EF. Further, Ag ZNRs deposited on a layer of Ag film have been introduced to the DNA hybridization and a significant signal enhancement has been observed through the fluorescence microscope. Through a detailed quantitative EF analysis, which excludes the enhancing effect from the increased surface area of ZNRs and only considers the contribution of MEF, an EF of 28 is achieved for the hybridization of two single-stranded oligonucleotides with 33 bases. Furthermore, a limit of detection is determined as 0.01pM. We believe that the Ag ZNR arrays can serve as a universal and sensitive bio-detection platform.

  3. Multiplexed hybridization detection of quantum dot-conjugated DNA sequences using surface plasmon enhanced fluorescence microscopy and spectrometry.

    PubMed

    Robelek, Rudolf; Niu, Lifang; Schmid, Evelyne L; Knoll, Wolfgang

    2004-10-15

    In this study, the general suitability of quantum dot (QD)-DNA conjugates for the surface plasmon enhanced fluorescence spectroscopy technique is demonstrated. Furthermore, the QD-DNA system is transferred to the platform of surface plasmon enhanced fluorescence microscopy. Using this technique together with a microarray format, in which the sensor-bound single-stranded catcher probes are organized in individual surface spots, results in a simultaneous qualitative analysis of QD-conjugated analyte DNA strands as multicolor images. A clear decomposition of different QD(x)()-DNA(y)() mixtures can be achieved for sequential, as well as mixture injections. Besides this, the study describes the successful approach of measuring spectrally resolved surface plasmon enhanced fluorescence signals derived from catcher probe hybridized QD-DNA conjugates.

  4. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    PubMed

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-05

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

  5. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

    PubMed

    Nagaki, S; Yamamoto, M; Yumoto, Y; Shirakawa, H; Yoshida, M; Teraoka, H

    1998-05-08

    DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

  6. Surface-enhanced Raman spectroscopy (SERS) for sub-micromolar detection of DNA/RNA mononucleotides.

    PubMed

    Bell, Steven E J; Sirimuthu, Narayana M S

    2006-12-13

    Surface-enhanced Raman (SER) spectra of all the DNA/RNA mononucleotides have been obtained with high sensitivity using citrate-reduced silver colloids aggregated with MgSO4, rather than the more usual halide ions, which were found to prevent enhancement of these compounds. The SERS spectra of adenine, guanine, thymine, cytosine, and uracil were recorded along with their corresponding nucleosides and 5'-deoxynucleotides. For the cytosine series, all three spectra had similar relative band intensities but the spectra of adenine were different from those of adenosine and dAMP, probably due to differences in orientation on the surface. No enhanced bands from the phosphate or sugar groups were observed. There were general similarities between the SERS spectra of the purine mononucleotides and the pyrimidine mononucleotides, but the spectra were sufficiently different to allow each of them to be distinguished. This method can therefore be used for high sensitivity, label-free identification of mononucleotides.

  7. Plasmonic Enhancement of Raman Signal using Complex Metallic Nanostructures based on DNA Origami

    NASA Astrophysics Data System (ADS)

    Finkelstein, Gleb

    2015-03-01

    DNA-based nanostructures, such as ``DNA origami,'' have recently emerged as one of the leading techniques for precise positioning of nanoscale materials in fields ranging from computer science to biomedical engineering. The origami is composed of a single scaffold DNA strand to which smaller ``staple`` strands are attached through DNA complementarity. The staples help to fold the scaffold strand into the designed structure of a predetermined shape. The resulting templates are highly addressable and have proven to be versatile tools for site-specific placement of various nanocomponents, such as metallic nanoparticles, quantum dots, fluorophores, etc. Building upon massively paralleled assembly mechanism of the origami and its ability to position nanocomponents, one may hope to utilize it for biosensing purposes. One attractive goal is the Raman spectroscopy, which provides a highly specific chemical fingerprint. Unfortunately, the Raman scattering cross section is small; Surface Enhanced Raman Spectroscopy (SERS) enhances the otherwise weak Raman signal by trapping the analyte molecules in the regions of intense electric field produced near rough metallic surfaces. These ``hot spots`` can be understood as resulting from localized surface plasmon modes resonantly exited by the incident laser excitation. We have earlier shown that metallic nanoparticles controllably attached to DNA origami can be further enlarged via an in-solution metallization; this technique allowed us to build metallic structures of complex topology. Recently, we have performed Raman spectroscopy of molecules attached to these metallic assemblies. Specifically, DNA origami is first used to organize the metallic structures, followed by a covalent attachment of Raman-active molecules to the metal. We found that the substrates with four nanoparticles per origami produce a strongly enhanced Raman signal compared to the control samples with only one nanoparticle per origami for the same particle

  8. Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase

    DTIC Science & Technology

    2015-02-05

    Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase...gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase" Name of Candidate: MAJ Jonathan D’ Ambrozio Doctor of Philosophy Degree...gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase" is appropriately acknowledged and, bey9nd brief excerpts, is with

  9. Chemical enhancement techniques of bloodstain patterns and DNA recovery after fire exposure.

    PubMed

    Tontarski, Karolyn L; Hoskins, Kyle A; Watkins, Tani G; Brun-Conti, Leanora; Michaud, Amy L

    2009-01-01

    It is common in forensic casework to encounter situations where the suspect has set a fire to cover up or destroy possible evidence. While bloodstain pattern interpretation, chemical enhancement of blood, and recovery of deoxyribonucleic acid (DNA) from bloodstains is well documented in the literature, very little information is known about the effects of heat or fire on these types of examinations. In this study, a variety of known types of bloodstain patterns were created in a four-room structure containing typical household objects and furnishings. The structure was allowed to burn to flashover and then it was extinguished by firefighters using water. Once the structure cooled over night, the interior was examined using a bright light. The bloodstains were evaluated to see if the heat or fire had caused any changes to the patterns that would inhibit interpretation. Bloodstain patterns remained visible and intact inside the structure and on furnishings unless the surface that held the blood was totally burned away. Additionally, a variety of chemical techniques were utilized to better visualize the patterns and determine the possible presence of blood after the fire. The soot from the fire formed a physical barrier that initially interfered with chemical enhancement of blood. However, when the soot was removed using water or alcohol, the chemicals used, fluorescein, luminol, Bluestar, and Hemastix, performed adequately in most of the tests. Prior to DNA testing, the combined phenolphthalein/tetramethyl benzidine presumptive test for the presence of blood was conducted in the laboratory on samples recovered from the structure in an effort to assess the effectiveness of using this type of testing as a screening tool. Test results demonstrated that reliance on obtaining a positive presumptive result for blood before proceeding with DNA testing could result in the failure to obtain useful typing results. Finally, two DNA recovery methods (swabbing the stain plus

  10. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  12. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    PubMed Central

    1984-01-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation. PMID:6480691

  13. DNA N6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

    PubMed Central

    Zhou, Chenchen; Liu, Yuting; Li, Xiaobing; Zou, Jing; Zou, Shujuan

    2016-01-01

    ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation. PMID:27785372

  14. Fast and sensitive DNA hybridization assays using microwave-accelerated metal-enhanced fluorescence.

    PubMed

    Aslan, Kadir; Malyn, Stuart N; Geddes, Chris D

    2006-09-22

    A new, fast, and sensitive DNA hybridization assay platform based on microwave-accelerated metal-enhanced fluorescence (MAMEF) is presented. Thiolated oligonucleotide anchors were immobilized onto silver nanoparticles on a glass substrate. The hybridization of the complementary fluorescein-labeled DNA target with the surface-bound oligonucleotides was completed within 20 s upon heating with low-power microwaves. In addition, the signal is optically amplified, a consequence of close proximity of the fluorophore to the silvered substrate. In this proof-of-principle methodology, as low as 50 nM of a target DNA was detected, although we envisage far-lower detection limits. Control experiments, where the surface-bound oligonucleotide was omitted, were also performed to determine the extent of non-specific binding. In these studies a significantly reduced non-specific adsorption was found when using microwave heating near to silvered structures as compared to room temperature incubation. These findings suggest that MAMEF could be a most useful alternative to the DNA hybridization assays used today, especially with regard to substantially increasing both the assay rapidity and sensitivity.

  15. DNA sequence-specific binding of CENP-B enhances the fidelity of human centromere function

    PubMed Central

    Fachinetti, Daniele; Han, Joo Seok; McMahon, Moira A.; Ly, Peter; Abdullah, Amira; Wong, Alex J.; Cleveland, Don W.

    2015-01-01

    SUMMARY Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A’s amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to missegregate in cells at rates several fold higher than chromosomes with CENP-B containing centromeres. These data demonstrate a DNA sequence-specific enhancement by CENP-B of the fidelity of epigenetically defined human centromere function. PMID:25942623

  16. B-DNA to Zip-DNA: Simulating a DNA Transition to a Novel Structure with Enhanced Charge-Transport Characteristics

    PubMed Central

    Balaeff, Alexander; Craig, Stephen L.; Beratan, David N.

    2013-01-01

    The forced extension of a DNA segment is studied in a series of steered molecular dynamics simulations, employing a broad range of pulling forces. Throughout the entire force range, the formation of a zipper-like (zip-) DNA structure is observed. In that structure, first predicted by Lohikoski et al., the bases of the DNA strands interdigitate with each other and form a single-base aromatic stack. Similar motifs, albeit only a few base pairs in extent, have been observed in experimental crystal structures. Analysis of the dynamics of structural changes in pulled DNA shows that S-form DNA, thought to be adopted by DNA under applied force, serves as an intermediate between B-DNA and zip-DNA. Therefore, the phase transition plateau observed in force–extension curves of DNA is suggested to reflect the B-DNA to zip-DNA structural transition. Electronic structure analysis of purine bases in zip-DNA indicates a several-fold to order of magnitude increase in the π–π electronic coupling among nearest-neighbor nucleobases, compared to B-DNA. We further observe that zip-DNA does not require base pair complementarity between DNA strands, and we predict that the increased electronic coupling in zip-DNA will result in a much higher rate of charge transfer through an all-purine zip-DNA compared to B-DNA of equal length. PMID:21598926

  17. The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylase.

    PubMed

    Le Meur, Rémy; Culard, Françoise; Nadan, Virginie; Goffinont, Stéphane; Coste, Franck; Guerin, Martine; Loth, Karine; Landon, Céline; Castaing, Bertrand

    2015-10-01

    The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.

  18. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  19. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    NASA Astrophysics Data System (ADS)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-05-01

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  20. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    PubMed Central

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

    2012-01-01

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id+ tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id+ single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id+ fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id+ tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id+ scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients. PMID:23115759

  1. Enhanced non-inflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for HIV DNA vaccines.

    PubMed

    Qiao, Chenmeng; Liu, Jiandong; Yang, Jun; Li, Yan; Weng, Jie; Shao, Yiming; Zhang, Xin

    2016-04-01

    Human immunodeficiency virus (HIV) DNA vaccine can induce cellular and humoral immunity. A safe and effective HIV DNA vaccine is urgent need to prevent the spread of acquired immune deficiency syndrome (AIDS). The major drawback of DNA vaccines is the low immunogenicity, which is caused by the poor delivery to antigen presenting cells and insufficient antigen expression. Sparked by the capability of endosomal/lysosomal escape of the zwitterionic lipid distearoyl phosphoethanol-amine-polycarboxybetaine (DSPE-PCB), we attempted to develop a zwitterionic-based cationic liposome with enhanced immunogenicity of DNA vaccines. The mannosylated zwitterionic-based cationic liposome (man-ZCL) was constructed as a DNA vaccine adjuvant for HIV vaccination. Man-ZCL could complex with DNA antigens to form a tight structure and protect them from nuclei enzyme degradation. Benefited from the capability of the specific mannose receptor mediated antigen processing cells targeting and enhanced endosomal/lysosomal escape, the man-ZCL lipoplexes were supposed to promote antigen presentation and the immunogenicity of DNA vaccines. In vitro and in vivo results revealed that man-ZCL lipoplexes showed enhanced anti-HIV immune responses and lower toxicity compared with CpG/DNA and Lipo2k/DNA, and triggered a Th1/Th2 mixed immunity. An antigen-depot effect was observed in the administration site, and this resulted in enhanced retention of DNA antigens in draining lymph nodes. Most importantly, the man-ZCL could assist to activate T cells through a non-inflammasome pathway. These findings suggested that the man-ZCL could be potentially applied as a safe and efficient DNA adjuvant for HIV vaccines.

  2. Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

    PubMed

    R El-Attar, Laila M; Thomas, Carole; Luke, Jeremy; A Williams, James; Brownlie, Joe

    2015-07-31

    DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

  3. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

    PubMed Central

    Hassan, Sally; Ward, John

    2016-01-01

    ABSTRACT With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26928284

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

    PubMed

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

    2017-01-01

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

  5. Modification by ferrocene: An approach to enhance antioxidant ability of ailanthoidol to protect DNA.

    PubMed

    Zhao, Chao; Liu, Zai-Qun

    2012-08-01

    Ailanthoidol is a benzofuran-type neolignan containing an alcoholic and a phenolic hydroxyl groups and can be synthesized following the description in a previous report. In this work, its antioxidant effect was estimated in the experimental system of 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. It was found that ailanthoidol can scavenge 1.5 radicals in protecting DNA against AAPH-induced oxidation. Moreover, a benzyl group was used to etherize the phenolic hydroxyl group of ailanthoidol, resulting in the formation of (E)-2-(4'-benzyloxy-3'-methoxyphenyl)-5-(3″-hydroxypropenyl)-7-methoxybenzofuran (BBF). However, BBF cannot protect DNA against AAPH-induced oxidation. This result demonstrated that the alcoholic hydroxyl group cannot play the antioxidative role in protecting DNA. Furthermore, a ferrocenyl group was used to substitute the alcoholic hydroxyl group, leading to the formation of (E)-1-ferrocenyl-3-(2'-(4″-hydroxy-3″-methoxyphenyl)-7'-methoxybenzofuran-5'-yl)prop-2-en-1-one (FBF). FBF can scavenge 2.0 radicals in protecting DNA against AAPH-induced oxidation. This result indicated that the antioxidant ability of FBF was higher than that of ailanthoidol. Finally, FBF and ailanthoidol were applied to trap 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(+.)). It was found that FBF can trap 1.92 ABTS(+.), while ailanthoidol can trap 1.58 ABTS(+.). Therefore, the modification of ailanthoidol by ferrocenyl group enhanced radical-scavenging and antioxidative ability of ailanthoidol.

  6. Short communication: Enhancement of topoisomerase I-mediated unwinding of supercoiled DNA by the radioprotector WR-33278

    SciTech Connect

    Holwitt, E.A.; Koda, E.; Swenberg, C.E.

    1990-01-01

    Cellular DNA is one of the critical targets for ionizing radiation. To mitigate the effects of ionizing radiation, the U.S. Army Medical Research and Development Command has synthesized several radioprotective drugs, primarily aminothiol compounds. Many mechanisms responsible for their radioprotective action have been proposed, including radical scavenging, hydrogen atom donation to DNA carbon center radicals, enhancement of DNA repair process, and reduction in the target volume. All of these processes require that the radioprotector or its metabolite be located within molecular distances, less than 50A, from DNA.

  7. Enhanced low-template DNA analysis conditions and investigation of allele dropout patterns.

    PubMed

    Hedell, Ronny; Dufva, Charlotte; Ansell, Ricky; Mostad, Petter; Hedman, Johannes

    2015-01-01

    Forensic DNA analysis applying PCR enables profiling of minute biological samples. Enhanced analysis conditions can be applied to further push the limit of detection, coming with the risk of visualising artefacts and allele imbalances. We have evaluated the consecutive increase of PCR cycles from 30 to 35 to investigate the limitations of low-template (LT) DNA analysis, applying the short tandem repeat (STR) analysis kit PowerPlex ESX 16. Mock crime scene DNA extracts of four different quantities (from around 8-84 pg) were tested. All PCR products were analysed using 5, 10 and 20 capillary electrophoresis (CE) injection seconds. Bayesian models describing allele dropout patterns, allele peak heights and heterozygote balance were developed to assess the overall improvements in EPG quality with altered PCR/CE settings. The models were also used to evaluate the impact of amplicon length, STR marker and fluorescent label on the risk for allele dropout. The allele dropout probability decreased for each PCR cycle increment from 30 to 33 PCR cycles. Irrespective of DNA amount, the dropout probability was not affected by further increasing the number of PCR cycles. For the 42 and 84 pg samples, mainly complete DNA profiles were generated applying 32 PCR cycles. For the 8 and 17 pg samples, the allele dropouts decreased from 100% using 30 cycles to about 75% and 20%, respectively. The results for 33, 34 and 35 PCR cycles indicated that heterozygote balance and stutter ratio were mainly affected by DNA amount, and not directly by PCR cycle number and CE injection settings. We found 32 and 33 PCR cycles with 10 CE injection seconds to be optimal, as 34 and 35 PCR cycles did not improve allele detection and also included CE saturation problems. We find allele dropout probability differences between several STR markers. Markers labelled with the fluorescent dyes CXR-ET (red in electropherogram) and TMR-ET (shown as black) generally have higher dropout risks compared with those

  8. Graphene-based high-efficiency surface-enhanced Raman scattering-active platform for sensitive and multiplex DNA detection.

    PubMed

    He, Shijiang; Liu, Keng-Ku; Su, Shao; Yan, Juan; Mao, Xiuhai; Wang, Dongfang; He, Yao; Li, Lain-Jong; Song, Shiping; Fan, Chunhai

    2012-05-15

    We have developed a surface-enhanced Raman scattering (SERS)-active substrate based on gold nanoparticle-decorated chemical vapor deposition (CVD)-growth graphene and used it for multiplexing detection of DNA. Due to the combination of gold nanoparticles and graphene, the Raman signals of dye were dramatically enhanced by this novel substrate. With the gold nanoparticles, DNA capture probes could be easily assembled on the surface of graphene films which have a drawback to directly immobilize DNA. This platform exhibits extraordinarily high sensitivity and excellent specificity for DNA detection. A detection limit as low as 10 pM is obtained. Importantly, two different DNA targets could be detected simultaneously on the same substrate just using one light source.

  9. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    PubMed

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  10. Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

    PubMed

    Okano, Hiroyuki; Katano, Yuta; Baba, Misato; Fujiwara, Ayako; Hidese, Ryota; Fujiwara, Shinsuke; Yanagihara, Itaru; Hayashi, Tsukasa; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-01-01

    Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.

  11. DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.

    PubMed

    Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C

    2015-02-01

    UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis.

  12. SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers.

    PubMed

    Hass, Matthew R; Liow, Hien-Haw; Chen, Xiaoting; Sharma, Ankur; Inoue, Yukiko U; Inoue, Takayoshi; Reeb, Ashley; Martens, Andrew; Fulbright, Mary; Raju, Saravanan; Stevens, Michael; Boyle, Scott; Park, Joo-Seop; Weirauch, Matthew T; Brent, Michael R; Kopan, Raphael

    2015-08-20

    We developed Split DamID (SpDamID), a protein complementation version of DamID, to mark genomic DNA bound in vivo by interacting or juxtapositioned transcription factors. Inactive halves of DAM (DNA adenine methyltransferase) were fused to protein pairs to be queried. Either direct interaction between proteins or proximity enabled DAM reconstitution and methylation of adenine in GATC. Inducible SpDamID was used to analyze Notch-mediated transcriptional activation. We demonstrate that Notch complexes label RBP sites broadly across the genome and show that a subset of these complexes that recruit MAML and p300 undergo changes in chromatin accessibility in response to Notch signaling. SpDamID differentiates between monomeric and dimeric binding, thereby allowing for identification of half-site motifs used by Notch dimers. Motif enrichment of Notch enhancers coupled with SpDamID reveals co-targeting of regulatory sequences by Notch and Runx1. SpDamID represents a sensitive and powerful tool that enables dynamic analysis of combinatorial protein-DNA transactions at a genome-wide level.

  13. Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI.

    PubMed

    Youngblood, Ben; Shieh, Fa-Kuen; De Los Rios, Stephanie; Perona, John J; Reich, Norbert O

    2006-09-15

    Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.

  14. Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.

    PubMed

    Garrett-Bakelman, Francine E; Sheridan, Caroline K; Kacmarczyk, Thadeous J; Ishii, Jennifer; Betel, Doron; Alonso, Alicia; Mason, Christopher E; Figueroa, Maria E; Melnick, Ari M

    2015-02-24

    DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

  15. Enhancing effect of DNA on chemiluminescence from the decomposition of hydrogen peroxide catalyzed by copper(II).

    PubMed

    Liu, Meilin; Li, Baoxin; Zhang, Zhujun; Lin, Jin-Ming

    2005-02-01

    In the absence of any special luminescence reagent, emission of weak chemiluminescence has been observed during the decomposition of hydrogen peroxide catalyzed by copper(II) in basic aqueous solution. The intensity of the chemiluminescence was greatly enhanced by addition of DNA and was strongly dependent on DNA concentration. Based on these phenomena, a flow-injection chemiluminescence method was established for determination of DNA. The chemiluminescence intensity was linear with DNA concentration in the range 2 x 10(-7)-1 x 10(-5) g L(-1) and the detection limit was 4.1 x 10(-8) g L(-1) (S/N=3). The relative standard deviation was less than 3.0% for 4 x 10(-7) g L(-1) DNA (n=11). The proposed method was satisfactorily applied for determination of DNA in synthetic samples. The possible mechanism of the CL reaction is discussed.

  16. Ferulic acid inhibits gamma radiation-induced DNA strand breaks and enhances the survival of mice.

    PubMed

    Maurya, Dharmendra Kumar; Devasagayam, Thomas Paul Asir

    2013-02-01

    Ferulic acid (FA) is a monophenolic phenylpropanoid occurring in plant products such as rice bran, green tea, and coffee beans. It has been shown to have significant antioxidant effects in many studies. In the present study, we show that intraperitoneal administration of FA at a dose of 50 mg/kg body weight 1 hour prior to or immediately after whole-body γ-irradiation of mice with 4 Gy results in considerable reduction in the micronuclei formation in peripheral blood reticulocytes. Administration of the same amount of FA immediately after 4 Gy γ-irradiation showed significant decrease in the amount of DNA strand breaks in murine peripheral blood leukocytes and bone marrow cells as examined by comet assay. Further, immunostaining of mouse splenic lymphocytes for phspho-γH2AX was carried out, and it was observed that FA inhibits the γH2AX foci formation. Finally, the survival of mice upon 6, 8, and 10 Gy γ-ray exposure was monitored. FA enhances the survival of mice by a factor of 2.5 at a dose of 6 Gy γ-radiation but not at higher doses. In conclusion, FA has protective potential in both pre- and postirradiation exposure scenarios and enhances the survival of mice possibly by decreasing DNA damage as examined by γH2AX foci, micronuclei formation, and comet assay.

  17. Structural mediation on polycation nanoparticles by sulfadiazine to enhance DNA transfection efficiency and reduce toxicity.

    PubMed

    Long, Xingwen; Zhang, Zhihui; Han, Shangcong; Tang, Minjie; Zhou, Junhui; Zhang, Jianhua; Xue, Zhenyi; Li, Yan; Zhang, Rongxin; Deng, Liandong; Dong, Anjie

    2015-04-15

    Reducing the toxicity while maintaining high transfection efficiency is an important issue for cationic polymers as gene carriers in clinical application. In this paper, a new zwitterionic copolymer, polycaprolactone-g-poly(dimethylaminoethyl methyacrylate-co-sulfadiazine methacrylate) (PC-SDZ) with unique pH-sensitivity, was designed and prepared. The incorporation of sulfadiazine into poly(dimethylaminoethyl methacrylate) (PDMAEMA) chains successfully mediates the surface properties including compacter shell structure, lower density of positive charges, stronger proton buffer capability, and enhanced hydrophobicity, which lead to reduction in toxicity and enhancements in stability, cellular uptake, endosome escape, and transfection efficiency for the PC-SDZ2 nanoparticles (NPs)/DNA complexes. Excellent transfection efficiency at the optimal N/P ratio of 10 was observed for PC-SDZ2 NPs/DNA complexes, which was higher than that of the commercial reagent-branched polyethylenimine (PEI). The cytotoxicity was evaluated by CCK8 measurement, and the results showed significant reduction in cytotoxicity even at high concentration of complexes after sulfadiazine modification. Therefore, this work may demonstrate a new way of structural mediation of cationic polymer carriers for gene delivery with high efficiency and low toxicity.

  18. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    SciTech Connect

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; Desireddy, Anil; Artyushkova, Kateryna; Boncella, Amy E.; Atanassov, Plamen; Martinez, Jennifer S.

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

  19. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    DOE PAGES

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; ...

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonicmore » gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.« less

  20. The swine CD81 enhances E2-based DNA vaccination against classical swine fever.

    PubMed

    Li, Wenliang; Mao, Li; Zhou, Bin; Liu, Xia; Yang, Leilei; Zhang, Wenwen; Jiang, Jieyuan

    2015-07-09

    Classical swine fever (CSF) is a highly contagious and economically important viral disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV can induce neutralizing antibodies and protective immunity, and is widely used for novel vaccine development. The objective of this study was to explore whether a tetraspanin molecule CD81 could improve the immune responses of an E2-based DNA vaccine. Plasmids pVAX-CD81, pVAX-E2 and pVAX-CD81-E2 were constructed and the expression of target proteins was confirmed in BHK-21 cells by indirect immunofluorescence assay. BALB/c mice were divided into 5 groups and immunized with different plasmids (pVAX-E2, pVAX-CD81-E2, pVAX-E2+pVAX-CD81, pVAX-CD81 and PBS) three times with two weeks interval. The results showed that the introduction of CD81 promoted higher humoral and cellular immune responses than E2 expression alone (P<0.05). In addition, immunization with pVAX-CD81-E2 induced stronger immune responses than pVAX-E2+pVAX-CD81. Furthermore, four groups of pigs were immunized with pVAX-E2, pVAX-CD81-E2, pVAX-CD81 and PBS, respectively. Humoral and cellular immune responses detection showed similar results with those in mice. Compared to pVAX-E2, pVAX-CD81-E2 induced higher titers of neutralizing antibodies after viral challenge and conferred stronger protection. These results confirmed the capacity of swine CD81 enhancing the humoral and cellular responses with an adjuvant effect on CSFV DNA vaccine. This is the first report demonstrating the adjuvant effect of CD81 to enhance the DNA vaccination for swine pathogen.

  1. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    SciTech Connect

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun

    2012-11-15

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  2. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

  3. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses.

    PubMed

    Smith, Heath A; Rekoske, Brian T; McNeel, Douglas G

    2014-03-26

    Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

  4. FRET enhancement close to gold nanoparticles positioned in DNA origami constructs.

    PubMed

    Aissaoui, Nesrine; Moth-Poulsen, Kasper; Käll, Mikael; Johansson, Peter; Wilhelmsson, L Marcus; Albinsson, Bo

    2017-01-05

    Here we investigate the energy transfer rates of a Förster resonance energy transfer (FRET) pair positioned in close proximity to a 5 nm gold nanoparticle (AuNP) on a DNA origami construct. We study the distance dependence of the FRET rate by varying the location of the donor molecule, D, relative to the AuNP while maintaining a fixed location of the acceptor molecule, A. The presence of the AuNP induces an alteration in the spontaneous emission of the donor (including radiative and non-radiative rates) which is strongly dependent on the distance between the donor and AuNP surface. Simultaneously, the energy transfer rates are enhanced at shorter D-A (and D-AuNP) distances. Overall, in addition to the direct influence of the acceptor and AuNP on the donor decay there is also a significant increase in decay rate not explained by the sum of the two interactions. This leads to enhanced energy transfer between donor and acceptor in the presence of a 5 nm AuNP. We also demonstrate that the transfer rate in the three "particle" geometry (D + A + AuNP) depends approximately linearly on the transfer rate in the donor-AuNP system, suggesting the possibility to control FRET process with electric field induced by 5 nm AuNPs close to the donor fluorophore. It is concluded that DNA origami is a very versatile platform for studying interactions between molecules and plasmonic nanoparticles in general and FRET enhancement in particular.

  5. Targeting hyperactivated DNA-PKcs by KU0060648 inhibits glioma progression and enhances temozolomide therapy via suppression of AKT signaling

    PubMed Central

    Qu, Yanming; Zhang, Mingshan; Wang, Haoran; Zhang, Zhihua; Zhou, Wei; Fan, Xinyi; Yu, Chunjiang; Zhan, Qimin; Song, Yongmei

    2016-01-01

    The overall survival remains undesirable in clinical glioma treatment. Inhibition of DNA-PKcs activity by its inhibitors suppresses tumor growth and enhances chemosensitivity of several tumors to chemotherapy. However, whether DNA-PKcs could be a potential target in glioma therapy remains unknown. In this study, we reported that the hyperactivated DNA-PKcs was profoundly correlated with glioma malignancy and observe a significant association between DNA-PKcs activation and survival of the glioma patients. Our data also found that inhibition of DNA-PKcs by its inhibitor KU0060648 sensitized glioma cells to TMZ in vitro. Specifically, we demonstrated that KU0060648 interrupted the formation of DNA-PKcs/AKT complex, leading to suppression of AKT signaling and resultantly enhanced TMZ efficacy. Combination of KU0060648 and TMZ substantially inhibited downstream effectors of AKT. The in vivo results were similar to those obtained in vitro. In conclusion, this study indicated that inhibition of DNA-PKcs activity could suppress glioma malignancies and increase TMZ efficacy, which was mainly through regulation of the of AKT signaling. Therefore, DNA-PKcs/AKT axis may be a promising target for improving current glioma therapy. PMID:27487130

  6. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA.

    PubMed

    Shen, Li; Gao, Ge; Zhang, Ying; Zhang, He; Ye, Zhiqiang; Huang, Shichao; Huang, Jinyan; Kang, Jiuhong

    2010-10-01

    Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.

  7. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    NASA Astrophysics Data System (ADS)

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  8. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions.

    PubMed

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-11

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  9. Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.

    PubMed

    Zhang, Zhian; Kermekchiev, Milko B; Barnes, Wayne M

    2010-03-01

    PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition, enhance PCR amplification, and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification. In the presence of these enhancer cocktails, the mutant enzymes were able to tolerate at least 25% plasma, serum, or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples, both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial Taq DNA polymerases.

  10. 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)

  11. Hybrid magnetic nanoparticle/nanogold clusters and their distance-dependent metal-enhanced fluorescence effect via DNA hybridization

    NASA Astrophysics Data System (ADS)

    GuThese Authors Contributed Equally To This Study., Xuefan; Wu, Youshen; Zhang, Lingze; Liu, Yongchun; Li, Yan; Yan, Yongli; Wu, Daocheng

    2014-07-01

    To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV)-visible (vis) absorption spectra of the samples were recorded with a UV-2600 spectrophotometer. Fluorescence spectra and the MEF effect were recorded using a spectrophotofluorometer, and lifetimes were determined using a time-correlated single photon counting apparatus. The prepared HMNCs were stable in aqueous solutions and had an average diameter of 87 +/- 3.2 nm, with six to eight AuNPs around a single Fe3O4 nanoparticle. Fluorescein isothiocyanate (FITC) tagged DNA-HMNC conjugates exhibited a significant MEF effect and could accurately detect specific DNA sequences after DNA hybridization. This result indicates their various potential applications in sensors and biomedical fields.To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV

  12. DNA methylation profiling of pediatric B-cell lymphoblastic leukemia with KMT2A rearrangement identifies hypomethylation at enhancer sites.

    PubMed

    Bergmann, Anke K; Castellano, Giancarlo; Alten, Julia; Ammerpohl, Ole; Kolarova, Julia; Nordlund, Jessica; Martin-Subero, Jose Ignacio; Schrappe, Martin; Siebert, Reiner

    2017-03-01

    Deregulation of the epigenome is an important pathogenetic mechanism in acute lymphoblastic leukemia (ALL) with lysine (K)-specific methyltransferase 2A rearrangement (KMT2Ar). We performed array-based DNA methylation profiling of KMT2Ar ALL cells from 26 children in comparison to normal B-cell precursors. Significant changes in DNA methylation in KMT2Ar ALL were identified in 2,545 CpG loci, influenced by age and the translocation partners AFF1 and MLLT1. In KMT2Ar ALL, DNA methylation loss was enriched at enhancers and for certain transcription factor binding sites such as BCL11A, EBF, and MEF2A. In summary, DNA methylation changes in KMT2Ar ALL target enhancers, genes involved in leukemogenesis and normal hematopoiesis, as well as transcription factor networks.

  13. Quantitative Single-Molecule Surface-Enhanced Raman Scattering by Optothermal Tuning of DNA Origami-Assembled Plasmonic Nanoantennas.

    PubMed

    Simoncelli, Sabrina; Roller, Eva-Maria; Urban, Patrick; Schreiber, Robert; Turberfield, Andrew J; Liedl, Tim; Lohmüller, Theobald

    2016-11-22

    DNA origami is a powerful approach for assembling plasmonic nanoparticle dimers and Raman dyes with high yields and excellent positioning control. Here we show how optothermal-induced shrinking of a DNA origami template can be employed to control the gap sizes between two 40 nm gold nanoparticles in a range from 1 to 2 nm. The high field confinement achieved with this optothermal approach was demonstrated by detection of surface-enhanced Raman spectroscopy (SERS) signals from single molecules that are precisely placed within the DNA origami template that spans the nanoparticle gap. By comparing the SERS intensity with respect to the field enhancement in the plasmonic hot-spot region, we found good agreement between measurement and theory. Our straightforward approach for the fabrication of addressable plasmonic nanosensors by DNA origami demonstrates a path toward future sensing applications with single-molecule resolution.

  14. Exploitation of Unintentional Ethernet Cable Emissions Using Constellation Based-Distinct Native Attribute (CB-DNA) Fingerprints to Enhance Network Security

    DTIC Science & Technology

    2015-09-17

    EXPLOITATION OF UNINTENTIONAL ETHERNET CABLE EMISSIONS USING CONSTELLATION BASED-DISTINCT NATIVE ATTRIBUTE (CB-DNA) FINGERPRINTS TO ENHANCE NETWORK... FINGERPRINTS TO ENHANCE NETWORK SECURITY DISSERTATION Presented to the Faculty Graduate School of Engineering and Management Air Force Institute of Technology...EXPLOITATION OF UNINTENTIONAL ETHERNET CABLE EMISSIONS USING CONSTELLATION BASED-DISTINCT NATIVE ATTRIBUTE (CB-DNA) FINGERPRINTS TO ENHANCE NETWORK

  15. Enhancing the Immunogenicity of a Dengue-2 DNA Vaccine With Adjuvants and Anti-FCyRI Antibodies

    DTIC Science & Technology

    2005-10-01

    antibody responses to the vaccine given IM were enhanced slightly by combining it with TT. However, the ELISA and neutralizing antibody levels were...aluminum phosphate, tetanus toxoid and DNA vaccine linked with anti-FcγR antibody . 3. Analyze antibody response by indirect ELISA and plaque reduction...FcγR antibody to the DEN-1 DNA. 3. Analyze antibody response by indirect ELISA and plaque reduction neutralization tests. Perform ELISPOT

  16. The greater negative charge density of DNA in tris-borate buffers does not enhance DNA condensation by multivalent cations.

    PubMed

    Schwinefus, J J; Bloomfield, V A

    2000-12-01

    As indicated by recent measurements of the electrophoretic free solution mobility, DNA appears to have a greater helical charge density in Tris-borate-EDTA (TBE) buffers than in Tris-acetate-EDTA (TAE) buffers. Since electrostatic forces play a major role in DNA packaging processes, we have investigated the condensation of closed circular plasmid DNA using total intensity and dynamic light scattering in Tris-borate, Tris-acetate, and Tris-cacodylate buffers with cobaltic hexa-amine (III) [Co(NH(3))(3+)(6)]. We find that neither the critical concentration of Co(NH(3))(3+)(6) nor the hydrodynamic radii of the resulting condensates vary significantly in the buffer systems studied here despite the prediction that DNA condensation should occur at significantly lower Co(NH(3))(3+)(6) concentrations in Tris-borate buffers. Assuming a persistence length behavior similar to B-DNA in the presence of multivalent cations, a decrease in the attractive counterion correlation pressure decay length in Tris-borate buffers does not account for our observations. It is possible that the binding of multivalent cations to DNA may hinder borate association with the DNA double helix.

  17. Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells.

    PubMed

    Ramos, Alice A; Marques, Filipe; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2013-01-01

    Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

  18. Co-encapsulation of Nigella sativa oil and plasmid DNA for enhanced gene therapy of Alzheimer's disease.

    PubMed

    Doolaanea, Abd Almonem; Mansor, Nur 'Izzati; Mohd Nor, Nurul Hafizah; Mohamed, Farahidah

    2016-01-01

    Alzheimer disease involves genetic and non-genetic factors and hence it is rational to be treated with genetic and non-genetic therapeutic agents. Nigella sativa has multiple therapeutic properties including neuroregeneration. Nigella sativa oil (NSO) was encapsulated in PLGA nanoparticles and pDNA was loaded either by adsorption on chitosan-modified particles or encapsulation within PLGA nanoparticles. The particle size and zeta potential of NSO-pDNA-chitosan-PLGA nanoparticles were highly dependent on the medium and exhibited high burst release. Meanwhile, NSO-pDNA-PLGA nanoparticles were more consistent with lower burst release. The fabricated nanoparticles revealed the expected outcomes of both pDNA and NSO. The pDNA transfected N2a cell while the encapsulated NSO promoted neurite outgrowth that is crucial for neuroregeneration. Results from this study suggest that NSO could be added to the gene delivery carrier to enhance treatment benefits for Alzheimer disease.

  19. Carboplatin enhances the production and persistence of radiation-induced DNA single-strand breaks

    SciTech Connect

    Yang, L.; Douple, E.B.; O`Hara, J.A.; Wang, H.J.

    1995-09-01

    Fluorometric analysis of DNA unwinding and alkaline elution were used to investigate the production and persistence of DNA single-strand breaks (SSBs) in Chinese hamster V79 and xrs-5 cells treated with the chemotherapeutic agent carboplatin in combination with radiation. Carboplatin was administered to cells before irradiation in hypoxic conditions, or the drug was added immediately after irradiation during the postirradiation recovery period in air. The results of DNA unwinding studies suggest that carboplatin enhances the production of radiation-induced SSBs in hypoxic V79 cells and xrs-5 cells by a factor of 1.86 and 1.83, respectively, when combined with radiation compared to the SSBs produced by irradiation alone. Carboplatin alone did not produce a measureable number of SSBs. Alkaline elution profiles also indicated that the rate of elution of SSBs was higher in cells treated with the carboplatin is present after irradiation and during the postirradiation recovery period, the rejoining of radiation-induced SSBs by a factor of 1.46 in V79 cells with 20 Gy irradiation and by a factor of 2.02 in xrs-5 cells with 20 Gy irradiation. When carboplatin is present after irradiation and during the postirradiation recovery period, the rejoining of radiation-induced SSBs is inhibited during this postirradiation incubation period (radiopotentiation) with a relative inhibition factor at 1 h postirradiation of 1.25 in V79 cells and 1.15 in xrs-5 cells. An increased production and persistence of SSBs resulting from the interaction of carboplatin with radiation may be an important step in the mechanism responsible for the potentiated cell killing previously from studies in animal tumors and in cultured cells. 31 refs., 7 figs.

  20. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells.

    PubMed

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga; Zhang, Guang-Yao; Yi, Zong-Chun

    2012-11-15

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes.

  1. Interaction of proflavine with DNA studied by colloid surface enhanced resonance Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Koglin, E.; Séquaris, J.-M.

    1986-03-01

    The interaction of the mutagenic highly fluourescing proflavine (3,6-diaminoacridine: PF) dye with calf thymus DNA has been studied by Surface Enhanced Resonance Raman Scattering (SERRS). Since the Ag-colloids almost completely quenche the strong fluorescence it is possible to obtain excellent vibrational spectra in a wide frequency range providing valuable information about the intercalation. The intercalation does not affect the vibrational frequencies of the proflavine dye. On the other hand, intensity changes are observed in some of the ring- and NH 2-modes of proflavine upon intercalation. This Raman hypochromism is characteristic for ring stacking interactions and in the SERRS spetroscopy for an additional effects of the dye orientation to the surface.

  2. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    PubMed

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms.

  3. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

    SciTech Connect

    Chang, Chung-ke; Wu, Tzong-Huah; Wu, Chu-Ya; Chiang, Ming-hui; Toh, Elsie Khai-Woon; Hsu, Yin-Chih; Lin, Ku-Feng; Liao, Yu-heng; Huang, Tai-huang; Huang, Joseph Jen-Tse

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

  4. DNA Origami Nanoantennas with over 5000-fold Fluorescence Enhancement and Single-Molecule Detection at 25 μM.

    PubMed

    Puchkova, Anastasiya; Vietz, Carolin; Pibiri, Enrico; Wünsch, Bettina; Sanz Paz, María; Acuna, Guillermo P; Tinnefeld, Philip

    2015-12-09

    Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot.

  5. The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress

    PubMed Central

    Kim, Ekaterina R; Selyutina, Anastasia A; Buldakov, Ilya A; Evdokimova, Valentina; Ovchinnikov, Lev P; Sorokin, Alexey V

    2013-01-01

    The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair. PMID:24107631

  6. Cdc14B depletion leads to centriole amplification and its overexpression prevents unscheduled centriole duplication

    SciTech Connect

    Wu, Jun; Plumley, Hyekyung; Rhee, David; Johnson, Dabney K; Dunlap, John; Liu, Yie; Wang, Yisong

    2008-01-01

    Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S-phase arrested cells and proteasome inhibitor Z-L3VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.

  7. Transient Depletion of Kupffer Cells Leads to Enhanced Transgene Expression in Rat Liver Following Retrograde Intrabiliary Infusion of Plasmid DNA and DNA Nanoparticles

    PubMed Central

    Dai, Hui; Jiang, Xuan; Leong, Kam W.

    2011-01-01

    Abstract In this report, we have demonstrated that by temporarily removing Kupffer cells (KCs), the transgene expression levels mediated by retrograde intrabiliary infusion (RII) of plasmid DNA, polyethylenimine-DNA, and chitosan nanoparticles were enhanced by 1,927-, 131-, and 23,450-fold, respectively, in comparison with the respective groups without KC removal. KC removal also led to significantly prolonged transgene expression in the liver that received all three carriers. This increased transgene expression was correlated with significantly reduced serum tumor necrosis factor-α level as an indicator for KC activation. These results suggest that KC activation is a significant contributing factor to the lowered transgene expression by polycation-DNA nanoparticles delivered by RII. More importantly, the combination of RII and transient removal of KCs may be adopted as an effective approach to achieving high and persistent transgene expression in the liver mediated by nonviral nanoparticles. PMID:21091274

  8. Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells.

    PubMed

    Liu, Guanglu; Wang, Yuan; Hu, Yang; Yu, Xiaobo; Zhu, Bin; Wang, Gaoxue

    2016-03-03

    DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH₃⁺ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be "needle-pricking the cells". Transmission electron microscope (TEM) images confirmed that MWCNTs-NH₃⁺ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH₃⁺:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH₃⁺:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry.

  9. Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

    PubMed Central

    Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

    2014-01-01

    The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

  10. Enzymatic on-chip enhancement for high resolution genotyping DNA microarrays.

    PubMed

    Schulze, Holger; Barl, Timo; Vase, Hollie; Baier, Shiromi; Thomas, Peter; Giraud, Gerard; Crain, Jason; Bachmann, Till T

    2012-06-05

    Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant Klebsiella pneumoniae carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination.

  11. Enhancing the sensitivity of localized surface plasmon resonance (LSPR) biosensors using nanorods and DNA aptamers

    NASA Astrophysics Data System (ADS)

    Chuang, Po-Chun; Liao, Pei-Chen; Chen, Yih-Fan

    2015-03-01

    Localized surface plasmon resonance (LSPR) biosensors have drawn much attention for their promising application in point-of-care diagnostics. While surface plasmon resonance (SPR) biosensing systems have been well developed, LSPR systems have the advantages of simpler and more compact setups. The LSPR peak shifts caused by the binding of molecules to the LSPR substrates, however, are usually smaller than 1 nm if no signal amplification mechanism is used. When using nanoparticles to enhance the sensitivity of LSPR biosensors, because of the short field penetration depth, the nanoparticles should be very close to the LSPR substrate to induce significant shifts in the LSPR peak position. In this study, we used DNA aptamers and gold nanorods to significantly increase the change in the LSPR peak position with the concentration of the target molecules. We have successfully used the proposed mechanism to detect 0.1 nM interferongamma (IFN-γ), a biomarker related to the diagnosis of latent tuberculosis infection. The calibration curves obtained in pure buffers and serum-containing buffers show that accurate detection can be achieved even when the sample is from complex biological fluids such as serum. Because of the enhancement in the sensitivity by the proposed sensing scheme, it is possible to use a low-cost spectrometer to build a LSPR biosensing system.

  12. Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.

    PubMed

    Jia, Yimin; Song, Haogang; Gao, Guichao; Cai, Demin; Yang, Xiaojing; Zhao, Ruqian

    2015-11-25

    Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.

  13. From adjacent activation in Escherichia coli and DNA cyclization to eukaryotic enhancers: the elements of a puzzle

    PubMed Central

    Amouyal, Michèle

    2014-01-01

    Deoxyribonucleic acid cyclization, Escherichia coli lac repressor binding to two spaced lac operators and repression enhancement can be successfully used for a better understanding of the conditions required for interaction between eukaryotic enhancers and the machinery of transcription initiation. Chronologically, the DNA looping model has first accounted for the properties initially defining enhancers, i.e., independence of action with distance or orientation with respect to the start of transcription. It has also predicted enhancer activity or its disruption at short distance (site orientation, alignment between promoter and enhancer sites), with high-order complexes of protein, or with transcription factor concentrations close or different from the wild-type situation. In another step, histones have been introduced into the model to further adapt it to eukaryotes. They in fact favor DNA cyclization in vitro. The resulting DNA compaction might explain the difference counted in base pairs in the distance of action between eukaryotic transcription enhancers and prokaryotic repression enhancers. The lac looping system provides a potential tool for analysis of this discrepancy and of chromatin state directly in situ. Furthermore, as predicted by the model, the contribution of operators O2 and O3 to repression of the lac operon clearly depends on the lac repressor level in the cell and is prevented in strains overproducing lac repressor. By extension, gene regulation especially that linked to cell fate, should also depend on transcription factor levels, providing a potential tool for cellular therapy. In parallel, a new function of the O1–O3 loop completes the picture of lac repression. The O1–O3 loop would at the same time ensure high efficiency of repression, inducibility through the low-affinity sites and limitation of the level of repressor through self-repression of the lac repressor. Last, the DNA looping model can be successfully adapted to the enhancer

  14. An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

    PubMed Central

    Iguchi-Ariga, S M; Okazaki, T; Itani, T; Ogata, M; Sato, Y; Ariga, H

    1988-01-01

    We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression. Images PMID:3053161

  15. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

    PubMed Central

    Rasmussen, Kasper D.; Jia, Guangshuai; Johansen, Jens V.; Pedersen, Marianne T.; Rapin, Nicolas; Bagger, Frederik O.; Porse, Bo T.; Bernard, Olivier A.; Christensen, Jesper

    2015-01-01

    DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis. PMID:25886910

  16. Enhanced luminous efficiency and brightness using DNA electron blocking layers in bio-organic light emitting diodes

    NASA Astrophysics Data System (ADS)

    Hagen, Joshua A.

    The biopolymer deoxyribonucleic acid (DNA) has been extracted from salmon (saDNA) and used successfully as an electron blocking layer (EBL) in multiple structures of Organic Light Emitting Diodes (OLED). Water soluble saDNA was complexed with a cationic surfactant hexadecytrimethylammonium chloride (CTMA) which makes the resulting DNA-CTMA molecule water insoluble, and soluble in common organic media such as alcohols. Solutions of DNA-CTMA and butanol make uniform thin films from 20nm to 5 microns in thickness by varying spin coating parameters and molecular weight. The optical properties of DNA-CTMA thin films include high transparency and low optical loss for applications at wavelengths above 400nm. The DNA-CTMA films have an electrical resistivity on the order of 107 O*cm. All of these properties combined made DNA-CTMA a candidate as an EBL in OLEDs, and this resulting device was termed a Bio-organic Light Emitting Diode (BioLED). Enhanced electroluminescent efficiency has been demonstrated in both green and blue emitting BioLEDs. The resulting green and blue BioLEDs showed a maximum luminous efficiency of 8.2 and 0.8 cd/A, respectively. The DNA based BioLEDs were as much as 10x more efficient and 30x brighter than their OLED counterparts. The enhancement in performance is due to the electron blocking action with the 0.9 eV (lowest unoccupied molecular orbital) value, allows hole injection to proceed with a 5.6eV (highest occupied molecular orbital) value. DNA-CTMA has also been successfully deposited in thin film form via molecular beam deposition (MBD). The growth was achieved at 160°C at vacuum levels of 10-5 Torr at a deposition rate of 0.8A/s. MBD grown DNA-CTMA thin films were highly uniform, optically transparent, and adhere to silicon, quartz and glass substrates more strongly than spin coated films. The material deposited was verified as DNA-CTMA through optical absorption, energy dispersive X-ray analysis, and using a DNA indicating fluorescent dye

  17. PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

    The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the

  18. CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

    PubMed

    Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

    2016-05-09

    Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.

  19. pH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity.

    PubMed

    He, Pan; Takeshima, Shin-nosuke; Tada, Seiichi; Akaike, Toshihiro; Ito, Yoshihiro; Aida, Yoko

    2014-10-29

    To demonstrate the potential of pH-sensitive carbonate apatite (CO₃Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO₃Ap complexes, or pCI-neo/CO₃Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO₃Ap (10 μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO₃Ap (100 μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 μg of pCI-neo-sOVA delivered by CO₃Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO₃Ap nanoparticles for DNA vaccine delivery.

  20. Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline

    SciTech Connect

    Elliott, Althea; Reiners, John J.

    2008-10-15

    p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10 and 25 {mu}M were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations {>=} 50 {mu}M were cytotoxic. Cytostatic concentrations induced transient G{sub 1} and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.

  1. Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

    PubMed

    Tanabe, Maiko; Ishino, Sonoko; Ishino, Yoshizumi; Nishida, Hirokazu

    2014-01-21

    The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

  2. A novel technique with enhanced detection and quantitation of HPV-16 E1- and E2-mediated DNA replication.

    PubMed

    Taylor, Ewan R; Morgan, Iain M

    2003-10-10

    Transient DNA replication assays to detect papillomavirus E1/E2-mediated DNA replication have depended upon Southern blotting. This technique is hazardous (radioactive), labour intensive, semiquantitative, and physically limited in the number of samples that can be processed at any one time. We have overcome these problems by developing a real-time PCR protocol for the detection of E1/E2-mediated transient DNA replication. The results demonstrate detection of replication at levels not seen using Southern blotting demonstrating enhanced sensitivity. This technique is also, by definition, highly quantitative. Therefore, the real-time PCR technique is the optimal method for the detection of E1/E2-mediated DNA replication.

  3. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    SciTech Connect

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B.; Su, Tsann-Long; Yih, Ling-Huei

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  4. Avoiding Ethanol Presence in DNA Samples Enhances the Performance of Ultraviolet Resonance Raman Spectroscopy Analysis.

    PubMed

    Cammisuli, Francesca; Pascolo, Lorella; Morgutti, Marcello; Gessini, Alessandro; Masciovecchio, Claudio; D'Amico, Francesco

    2017-01-01

    Ethanol is an essential chemical reagent in DNA preparation as its use increases the yield of extraction. All methodologies for DNA isolation involve the use of ethanol in order to prevent DNA dissolution in water and to optimize the binding of DNA to chromatographic membranes. In this note, we show how the presence of ethanol traces in DNA aqueous solution affects ultraviolet Raman spectra, leading to possible misinterpretations. We report a simple method to remove the ethanol Raman features from the spectra, based on heating the DNA sample at 80 ℃, followed by a slow cooling procedure.

  5. Scanning electrochemical microscopy of DNA hybridization on DNA microarrays enhanced by HRP-modified SiO2 nanoparticles.

    PubMed

    Fan, Huajun; Wang, Xiaolan; Jiao, Fang; Zhang, Fan; Wang, Qingjiang; He, Pingang; Fang, Yuzhi

    2013-07-02

    Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

  6. Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints.

    PubMed

    Frégeau, C J; Germain, O; Fourney, R M

    2000-03-01

    This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, Hum

  7. 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)

  8. Photonic Crystal Biosensor with In-Situ Synthesized DNA Probes for Enhanced Sensitivity

    SciTech Connect

    Hu, Shuren; Zhao, Y.; Retterer, Scott T; Kravchenko, Ivan I; Weiss, Sharon

    2013-01-01

    We report on a nearly 8-fold increase in multi-hole defect photonic crystal biosensor response by incorporating in-situ synthesis of DNA probes, as compared to the conventional functionalization method employing pre-synthesized DNA probe immobilization.

  9. DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences.

    PubMed Central

    Ariga, H; Imamura, Y; Iguchi-Ariga, S M

    1989-01-01

    We have previously reported that c-myc protein, or protein(s) complexed with c-myc protein, binds to the region upstream of the first exon of the c-myc gene and that this region contains an origin of cellular DNA replication (ori) and also a transcriptional enhancer. Here we show by Southwestern blotting that c-myc protein binds directly to a 7 bp sequence within the above region. Furthermore, we show that the c-myc protein binding sequences are indispensable for both ori and enhancer functions, but that additional sequences are required for maximal ori and enhancer activities. Thus, c-myc protein is a sequence specific factor which is apparently used both in initiation of DNA replication and in regulation of RNA transcription. Images PMID:2686984

  10. Microwave-accelerated metal-enhanced fluorescence: application to detection of genomic and exosporium anthrax DNA in <30 seconds.

    PubMed

    Aslan, Kadir; Zhang, Yongxia; Hibbs, Stephen; Baillie, Les; Previte, Michael J R; Geddes, Chris D

    2007-11-01

    We describe the ultra-fast and sensitive detection of the gene encoding the protective antigen of Bacillus anthracis the causative agent of anthrax. Our approach employs a highly novel platform technology, Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF), which combines the use of Metal-Enhanced Fluorescence to enhance assay sensitivity and focused microwave heating to spatially and kinetically accelerate DNA hybridization. Genomic and exosporium target DNA of Bacillus anthracis spores was detected within a minute in the nanograms per microliter concentration range using low-power focused microwave heating. The MAMEF technology was able to distinguish between B. anthracis and B. cereus, a non-virulent close relative. We believe that this study has set the stage and indeed provides an opportunity for the ultra-fast and specific detection of B. anthracis spores with minimal pre-processing steps using a relatively simple but cost-effective technology that could minimize casualties in the event of another anthrax attack.

  11. Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

    PubMed

    Kathiravan, Mathur Nadarajan; Gim, Geun Ho; Ryu, Jaewon; Kim, Pyung Il; Lee, Chul Won; Kim, Si Wouk

    2015-11-01

    In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

  12. Mechanical constraints on Hin subunit rotation imposed by the Fis-enhancer system and DNA supercoiling during site-specific recombination

    PubMed Central

    Dhar, Gautam; Heiss, John K.; Johnson, Reid C.

    2009-01-01

    Summary Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands re-ligate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions. PMID:19560425

  13. [Polymorphism of DNA nucleotide sequence as a source of enhancement of the discrimination potential of the STR-markers].

    PubMed

    Zemskova, E Yu; Timoshenko, T V; Leonov, S N; Ivanov, P L

    2016-01-01

    The objective of the present pilot investigation was to reveal and to study polymorphism of nucleotide sequence in the alleles of STR loci of human autosomal DNA with special reference to the role of this phenomenon as a source of the differences between homonymous allelic variants. The secondary objection was to evaluate the possibility of using the data thus obtained for the enhancement of the informative value of the forensic medical genotyping of STR loci by means of identification of single nucleotide polymorphisms (SNP) for the purpose of extending their allelic spectrum. The methodological basis of the study was constituted by the comprehensive amplified fragment length polymorphism (AFLP) analysis and amplified fragment sequence polymorphisms (AFSP) analysis of DNA with the use of the PLEX-ID^TM analytical mass-spectrometry platform (Abbot Molecular, USA). The study has demonstrated that polymorphism of DNA nucleotide sequence can be regarded as the possible source of enhancement of the discriminating potential of STR markers. It means that the analysis of polymorphism of DNA nucleotide sequence for genotyping AFLP-type markers of chromosomal DNA can considerably increase the effectiveness of their application as individualizing markers for the purpose of molecular genetic expertises.

  14. Quenching Enhancement of the Singlet Excited State of Pheophorbide-a by DNA in the Presence of the Quinone Carboquone

    PubMed Central

    Díaz-Espinosa, Yisaira; Crespo-Hernández, Carlos E.; Alegría, Antonio E.; García, Carmelo; Arce, Rafael

    2011-01-01

    Changes in the emission fluorescence intensity of pheophorbide-a (PHEO) in the presence of carboquone (CARBOQ) were used to obtain the association constant, the number of CARBOQ molecules interacting with PHEO, and the fluorescence quantum yield of the complex. Excitation spectra of mixtures of PHEO and CARBOQ in ethanol (EtOH) show an unresolved doublet in the red-most excitation band of PHEO, indicating the formation of a loose ground-state complex. The 1:1 CARBOQ–PHEO complex shows a higher fluorescence quantum yield in EtOH (0.41 ± 0.02) than in buffer solution (0.089 ± 0.002), which is also higher than that of the PHEO monomer (0.28). Quenching of the PHEO fluorescence by DNA nucleosides and double-stranded oligonucleotides was also observed and the bimolecular quenching rate constants were determined. The quenching rate constant increase as the oxidation potential of the DNA nucleoside increases. Larger quenching constants were obtained in the presence of CARBOQ suggesting that CARBOQ enhances DNA photo-oxidation, presumably by inhibiting the back–electron-transfer reaction from the photoreduced PHEO to the oxidized base. Thus, the enhanced DNA-base photosensitized oxidation by PHEO in the presence of CARBOQ may be related to the large extent by which this quinone covalently binds to DNA, as previously reported. PMID:21138440

  15. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Radiochemosensitivity in Cancers Proficient in DNA Double-Strand Break Repair.

    PubMed

    Shunkwiler, Lauren; Ferris, Gina; Kunos, Charles

    2013-02-08

    Pharmacologic inhibitors of poly(ADP-ribose) polymerase (PARP) putatively enhance radiation toxicity in cancer cells. Although there is considerable information on the molecular interactions of PARP and BRCA1- and BRCA2-deficient cancers, very little is known of the PARP inhibition effect upon cancers proficient in DNA double-strand break repair after ionizing radiation or after stalled replication forks. In this work, we investigate whether PARP inhibition by ABT-888 (veliparib) augments death-provoking effects of ionizing radiation, or of the topoisomerase I poison topotecan, within uterine cervix cancers cells harboring an unfettered, overactive ribonucleotide reductase facilitating DNA double-strand break repair and contrast these findings with ovarian cancer cells whose regulation of ribonucleotide reductase is relatively intact. Cell lethality of a radiation-ABT-888 combination is radiation and drug dose dependent. Data particularly highlight an enhanced topotecan-ABT-888 cytotoxicity, and corresponds to an increased number of unrepaired DNA double-strand breaks. Overall, our findings support enhanced radiochemotherapy toxicity in cancers proficient in DNA double-strand break repair when PARP is inhibited by ABT-888.

  16. A multifunctional DNA origami as carrier of metal complexes to achieve enhanced tumoral delivery and nullified systemic toxicity.

    PubMed

    Huang, Yanyu; Huang, Wei; Chan, Leung; Zhou, Binwei; Chen, Tianfeng

    2016-10-01

    The use of metal complexes in cancer treatment is hampered by the insufficient accumulation in tumor regions and observable systemic toxicity due to their nonspecificity in vivo. Herein we present a cancer-targeted DNA origami as biocompatible nanocarrier of metal complexes to achieve advanced antitumor effect. The formation of unique tetrahedral nanostructure of DNA cages effectively enhances the interaction between ruthenium polypyridyl complexes (RuPOP) and the cages, thus increasing the drug loading efficacy. Conjugation of biotin to the DNA-based nanosystem (Bio-cage@Ru) enhances its specific cellular uptake, drug retention and cytotoxicity against HepG2 cells. Different from free RuPOP and the cage itself, Bio-cage@Ru translocates to cell nucleus after internalization, where it undergoes self-immolative cleavage in response to DNases, leading to triggered drug release and induction of ROS-mediated cell apoptosis. Moreover, in the nude mice model, the nanosystem specifically accumulates in tumor sites, thus exhibits satisfactory in vivo antitumor efficacy, and alleviates the damage of liver, kidney, lung and heart function of nude mice induced by RuPOP and tumor xenografts. Collectively, this study demonstrates a strategy for construction of biocompatible and cancer-targeted DNA origami with enhanced anticancer efficacy and reduced toxicity for next-generation cancer therapy.

  17. Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation.

    PubMed

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N; Chen, Lung-Chi; Tang, Moon-shong

    2015-10-20

    Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers.

  18. Development of an optical biosensor based on surface-enhanced Raman scattering for DNA analysis

    NASA Astrophysics Data System (ADS)

    Yigit, Tugce; Akdogan, Ebru; Karagoz, Isık. Didem; Kahraman, Mehmet

    2016-03-01

    Rapid, accurate and sensitive DNA analysis is critically important for the diagnostic of genetic diseases. The most common method preferred in practice is fluorescence based microarrays to analyze the DNA. However, there exist some disadvantages related to the above-mentioned method such as the overlapping of the fluorescence emission wavelengths that can diminish in the performance of multiplexing, needed to obtain fluorescence spectra from each dye and photo degradation. In this study, a novel SERS based DNA analysis approach, which is Raman active dye-free and independent of SERS substrate properties, is developed. First, the single strand DNA probe is attached to the SERS substrate and half of the complimentary DNA is attached to gold nanoparticles, as well. We hypothesize that in the presence of target DNA, the complimentary DNA coupled colloids will bind to the SERS substrate surface via hybridization of single strand target DNA. To test this hypothesis, we used UV/Vis spectroscopy, atomic for microscopy (AFM) and dynamic light scattering (DLS). DNA analysis is demonstrated by a peak shift of the certain peak of the small molecules attached to the SERS substrate surface instead of SERS spectrum obtained in the presence of target DNA from the Raman reporter molecules. The degree of peak shifting will be used for the quantification of the target DNA in the sample. Plasmonic properties of SERS substrates and reproducibility issues will not be considerable due to the use of peak shifting instead of peak intensity for the qualitative analysis.

  19. Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

    2015-01-01

    Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

  20. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    NASA Astrophysics Data System (ADS)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  1. Enhanced Genotoxicity of Silver Nanoparticles in DNA Repair Deficient Mammalian Cells

    PubMed Central

    Lim, Hui Kheng; Asharani, P. V.; Hande, M. Prakash

    2012-01-01

    Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure. PMID:22707954

  2. Enhanced genotoxicity of silver nanoparticles in DNA repair deficient Mammalian cells.

    PubMed

    Lim, Hui Kheng; Asharani, P V; Hande, M Prakash

    2012-01-01

    Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure.

  3. Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability.

    PubMed

    Liu, Baoying; Liang, Min-Hui; Kuo, Yu-liang; Liao, Wei; Boros, Imre; Kleinberger, Tami; Blancato, Jan; Giam, Chou-Zen

    2003-08-01

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Naïve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.

  4. Detection of Genomic DNA Damage from Radiated Nasopharyngeal Carcinoma Cells Using Surface-Enhanced Raman Spectroscopy (SERS).

    PubMed

    Ou, Lin; Chen, Yang; Su, Ying; Zou, Changyan; Chen, Zhong

    2016-11-01

    Structural changes and chemical modifications in DNA during interactions with X-ray radiation are still not clear within 48 h of incubation. We investigate genomic DNA from the radiated CNE2 cell line within 48 h of incubation using surface-enhanced Raman spectroscopy (SERS). Multivariate methods including principal component analysis (PCA) and random forest are proposed to explore the statistical significance before and after radiation. Our results show that intensities of several bands change after radiation, which indicates backbone damage and base-unstacking. Biological effects from DNA damage repairing process may be simultaneously stimulated and different from incubation time. Under doses of 10 Gy (with 24 and 48 h of incubation) and 20 Gy (with 48 h of incubation), the relative contents of C against T and A against G deviate obviously from the control level. Statistical results strengthen significantly the idea that modification in DNA bases is associated with the disruption of base-stacking in the DNA duplex. Our findings provide vital information for radiation-induced the DNA damage at the molecular level, which may provide insight into the effect and mechanism of anticarcinogens in tumor therapy.

  5. Effect of acrylamide on hepatocellular DNA repair

    SciTech Connect

    Miller, M.J.; McQueen, C.A.

    1986-01-01

    Acrylamide has recently been reported to induce tumors in laboratory animals. The effect of acrylamide on unscheduled DNA synthesis using the hepatocyte primary culture (HPC)/DNA repair test was examined. Isolated hepatocytes were exposed to acrylamide and (3H)thymidine ( (3H)TdR) for 18 hr. Incorporation of (3H)TdR into DNA was determined by autoradiography. No DNA repair was observed at acrylamide concentrations up to 10(-2) M. These findings were confirmed using density gradients. Acrylamide concentrations exceeding 10(-2) M were cytotoxic to hepatocytes. Because both autoradiography and density gradients measure DNA repair as an endpoint, the ability of acrylamide to inhibit these repair processes was also determined. Acrylamide had no effect on the repair of UV-damaged DNA. These results show that acrylamide is not genotoxic in isolated hepatocytes.

  6. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    NASA Astrophysics Data System (ADS)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  7. Enhancement of HIV-1 DNA vaccine immunogenicity by BCG-PSN, a novel adjuvant.

    PubMed

    Sun, Jing; Hou, Jue; Li, Dingfeng; Liu, Yong; Hu, Ningzhu; Hao, Yanling; Fu, Jingjing; Hu, Yunzhang; Shao, Yiming

    2013-01-07

    Although the importance of DNA vaccines, especially as a priming immunization has been well established in numerous HIV vaccine studies, the immunogenictiy of DNA vaccines is generally moderate. Novel adjuvant is in urgent need for improving the immunogenicity of DNA vaccine. Polysaccharide and nucleic acid fraction extracted by hot phenol method from Mycobacterium bovis bacillus Calmette-Guérin, known as BCG-PSN, is a widely used immunomodulatory product in China clinical practice. In this study, we evaluated whether the BCG-PSN could serve as a novel adjuvant of DNA vaccine to trigger better cellular and humoral immune responses against the HIV-1 Env antigen in Balb/C mouse model. The BCG-PSN was mixed with 10 μg or 100 μg of pDRVI1.0gp145 (HIV-1 CN54 gp145 gene) DNA vaccine and intramuscularly immunized two or three times. We found that BCG-PSN could significantly improve the immunogenicity of DNA vaccine when co-administered with DNA vaccine. Further, at the same vaccination schedule, BCG-PSN co-immunization with 10 μg DNA vaccine could elicit cellular and humoral immune responses which were comparable to that induced by 100 μg DNA vaccine alone. Moreover, our results demonstrate that BCG-PSN can activate TLR signaling pathways and induce Th1-type cytokines secretion. These findings suggest that BCG-PSN can serve as a novel and effective adjuvant for DNA vaccination.

  8. DNA Demethylation of the Foxp3 Enhancer Is Maintained through Modulation of Ten-Eleven-Translocation and DNA Methyltransferases

    PubMed Central

    Nair, Varun Sasidharan; Song, Mi Hye; Ko, Myunggon; Oh, Kwon Ik

    2016-01-01

    Stable expression of Foxp3 is ensured by demethylation of CpG motifs in the Foxp3 intronic element, the conserved non-coding sequence 2 (CNS2), which persists throughout the lifespan of regulatory T cells (Tregs). However, little is known about the mechanisms on how CNS2 demethylation is sustained. In this study, we found that Ten-Eleven-Translocation (Tet) DNA dioxygenase protects the CpG motifs of CNS2 from re-methylation by DNA methyltransferases (Dnmts) and prevents Tregs from losing Foxp3 expression under inflammatory conditions. Upon stimulation of Tregs by interleukin-6 (IL6), Dnmt1 was recruited to CNS2 and induced methylation, which was inhibited by Tet2 recruited by IL2. Tet2 prevented CNS2 re-methylation by not only the occupancy of the CNS2 locus but also by its enzymatic activity. These results show that the CNS2 methylation status is dynamically regulated by a balance between Tets and Dnmts which influences the expression of Foxp3 in Tregs. PMID:27989104

  9. Enhancement of topoisomerase I-mediated unwinding of supercoiled DNA by the radioprotector WR-33278

    SciTech Connect

    Holwitt, E.A.; Koda, E.; Swenberg, C.E. )

    1990-10-01

    The radioprotector WR-33278, the disulfide of WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane), is shown to stimulate eukaryotic topoisomerase I unwinding of negatively supercoiled DNA. This observation suggests the possibility that some protection may be conferred to DNA either by a decrease in its supercoiled state or by altering directly other enzymatic processes. This is the first report of a radioprotective compound stimulating an enzyme involved in DNA structure and synthesis.

  10. Electrosteric stabilization of colloidal TiO2 nanoparticles with DNA and polyethylene glycol for selective enhancement of UV detection sensitivity in capillary electrophoresis analysis.

    PubMed

    Alsudir, Samar; Lai, Edward P C

    2017-03-01

    A new approach to selectively enhance the ultraviolet (UV) detection sensitivity of titania (TiO2), albeit in the presence of silica (SiO2), alumina (Al2O3), and zinc oxide (ZnO), nanoparticles in capillary electrophoresis (CE) analysis was developed. Interactions of Triton X-100 (TX-100), polyethylene glycol (PEG), and deoxyribonucleic acid (DNA) with TiO2 nanoparticles produced larger CE-UV peaks at various enhancement factors. Single-stranded DNA (ssDNA) was a more effective adsorbate than double-stranded DNA (dsDNA) due to its flexible molecular structure that participated in a stronger interaction with TiO2 nanoparticles via its sugar-phosphate backbone. Disaggregation of TiO2 nanoparticles upon DNA binding due to electrosteric stabilization was validated using dynamic light scattering. PEG coating of TiO2-DNA nanoparticles further enhanced the UV detection sensitivity in CE analysis by providing extra electrosteric stabilization. This analytical technique, which involves binding of TiO2 nanoparticles with DNA followed by coating with PEG, has allowed us to achieve progressively an enhancement factor up to 13.0 ± 3.0 - fold in analytical sensitivity for the accurate determination of disaggregated TiO2 nanoparticles. Graphical Abstract Selective enhancement of UV detection sensitivity for TiO2 nanoparticles via electrosteric stabilization using ssDNA and PEG.

  11. Enhanced recognition of hydroxyl radical modified plasmid DNA by circulating cancer antibodies.

    PubMed

    Khan, F; Ali, A; Ali, R

    2005-06-01

    Reactive oxygen species have been implicated in various human diseases which are also responsible for the elimination of invading pathogens. In disease state and inflammatory responses, the excess of these radicals damage cellular macromolecules. DNA is susceptible to attacks by OH-induced damage. Oxidative DNA damage is an important factor in mutagenesis and carcinogenesis. In the present study, purified plasmid Bluescript DNA was modified by hydroxyl radical. Modifications incurred in DNA were characterized by physico-chemical techniques. Sera from patients of cancer were studied for their binding to native and hydroxyl radical modified plasmid DNA. Direct binding ELISA and competition binding results indicated that autoantibodies in cancer showed higher recognition to ROS-plasmid DNA as compared to the native form. Retarded mobility of the immune complex formation between IgG isolated from cancer sera using native and ROS-plasmid DNA as antigens reiterated preferential recognition of modified plasmid DNA by cancer autoantibodies. Therefore, it can be concluded that circulating autoantibodies in cancer sera bind preferentially to ROS-plasmid DNA as compared to native polymer. The data presented in the present communication suggest a role of ROS in the etiology of cancer.

  12. Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

    NASA Astrophysics Data System (ADS)

    Manera, M. G.; Spadavecchia, J.; Taurino, A.; Rella, R.

    2010-03-01

    The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV-vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA-Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA-DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T)15ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality.

  13. BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage

    PubMed Central

    Artati, Anna; Adamski, Jerzy

    2016-01-01

    Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease. PMID:27959944

  14. BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage.

    PubMed

    Storch, Katja; Dickreuter, Ellen; Artati, Anna; Adamski, Jerzy; Cordes, Nils

    2016-01-01

    Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

  15. Surface-enhanced localized surface plasmon resonance biosensing of avian influenza DNA hybridization using subwavelength metallic nanoarrays

    NASA Astrophysics Data System (ADS)

    Kim, Shin Ae; Byun, Kyung Min; Kim, Kyujung; Jang, Sung Min; Ma, Kyungjae; Oh, Youngjin; Kim, Donghyun; Kim, Sung Guk; Shuler, Michael L.; Kim, Sung June

    2010-09-01

    We demonstrated enhanced localized surface plasmon resonance (SPR) biosensing based on subwavelength gold nanoarrays built on a thin gold film. Arrays of nanogratings (1D) and nanoholes (2D) with a period of 200 nm were fabricated by electron-beam lithography and used for the detection of avian influenza DNA hybridization. Experimental results showed that both nanoarrays provided significant sensitivity improvement and, especially, 1D nanogratings exhibited higher SPR signal amplification compared with 2D nanohole arrays. The sensitivity enhancement is associated with changes in surface-limited reaction area and strong interactions between bound molecules and localized plasmon fields. Our approach is expected to improve both the sensitivity and sensing resolution and can be applicable to label-free detection of DNA without amplification by polymerase chain reaction.

  16. Enhanced Immunogenicity of an HIV-1 DNA Vaccine Delivered with Electroporation via Combined Intramuscular and Intradermal Routes

    PubMed Central

    McKay, Paul F.; Fiserova, Anezka; Klein, Katja; Cope, Alethea; Rogers, Paul; Swales, Julie; Seaman, Michael S.; Combadiere, Behazine

    2014-01-01

    ABSTRACT It is accepted that an effective prophylactic HIV-1 vaccine is likely to have the greatest impact on viral transmission rates. As previous reports have implicated DNA-priming, protein boost regimens to be efficient activators of humoral responses, we sought to optimize this regimen to further augment vaccine immunogenicity. Here we evaluated single versus concurrent intradermal (i.d.) and intramuscular (i.m.) vaccinations as a DNA-priming strategy for their abilities to elicit humoral and cellular responses against a model HIV-1 vaccine antigen, CN54-gp140. To further augment vaccine-elicited T and B cell responses, we enhanced cellular transfection with electroporation and then boosted the DNA-primed responses with homologous protein delivered subcutaneously (s.c.), intranasally (i.n.), i.m., or transcutaneously (t.c.). In mice, the concurrent priming regimen resulted in significantly elevated gamma interferon T cell responses and high-avidity antigen-specific IgG B cell responses, a hallmark of B cell maturation. Protein boosting of the concurrent DNA strategy further enhanced IgG concentrations but had little impact on T cell reactivity. Interestingly protein boosting by the subcutaneous route increased antibody avidity to a greater extent than protein boosting by either the i.m., i.n., or t.c. route, suggesting that this route may be preferential for driving B cell maturation. Using an alternative and larger animal model, the rabbit, we found the concurrent DNA-priming strategy followed by s.c. protein boosting to again be capable of eliciting high-avidity humoral responses and to also be able to neutralize HIV-1 pseudoviruses from diverse clades (clades A, B, and C). Taken together, we show that concurrent multiple-route DNA vaccinations induce strong cellular immunity, in addition to potent and high-avidity humoral immune responses. IMPORTANCE The route of vaccination has profound effects on prevailing immune responses. Due to the insufficient

  17. Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers

    PubMed Central

    Jusuk, Ija; Vietz, Carolin; Raab, Mario; Dammeyer, Thorben; Tinnefeld, Philip

    2015-01-01

    Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer). Conjugation to single stranded DNA and immobilization via DNA hybridization on a DNA origami 12 helix bundle in aqueous solution allowed photophyiscal studies of eYFP at the single-molecule level and at close to physiological conditions. The benefit of improved photophysical properties for localization-based super-resolution microscopy is demonstrated and quantitatively characterized by imaging 12 helix bundle DNA origami nanorulers with binding sites at designed distances of 160 and 100 nm and by imaging microtubules in fixed mammalian Vero cells. PMID:26373229

  18. Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers.

    PubMed

    Jusuk, Ija; Vietz, Carolin; Raab, Mario; Dammeyer, Thorben; Tinnefeld, Philip

    2015-09-16

    Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer). Conjugation to single stranded DNA and immobilization via DNA hybridization on a DNA origami 12 helix bundle in aqueous solution allowed photophyiscal studies of eYFP at the single-molecule level and at close to physiological conditions. The benefit of improved photophysical properties for localization-based super-resolution microscopy is demonstrated and quantitatively characterized by imaging 12 helix bundle DNA origami nanorulers with binding sites at designed distances of 160 and 100 nm and by imaging microtubules in fixed mammalian Vero cells.

  19. Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers

    NASA Astrophysics Data System (ADS)

    Jusuk, Ija; Vietz, Carolin; Raab, Mario; Dammeyer, Thorben; Tinnefeld, Philip

    2015-09-01

    Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer). Conjugation to single stranded DNA and immobilization via DNA hybridization on a DNA origami 12 helix bundle in aqueous solution allowed photophyiscal studies of eYFP at the single-molecule level and at close to physiological conditions. The benefit of improved photophysical properties for localization-based super-resolution microscopy is demonstrated and quantitatively characterized by imaging 12 helix bundle DNA origami nanorulers with binding sites at designed distances of 160 and 100 nm and by imaging microtubules in fixed mammalian Vero cells.

  20. Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant.

    PubMed

    Li, Na; Yu, Yun-Zhou; Yu, Wei-Yuan; Sun, Zhi-Wei

    2011-03-01

    Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.

  1. Single-Particle Spectroscopic Study on Fluorescence Enhancement by Plasmon Coupled Gold Nanorod Dimers Assembled on DNA Origami.

    PubMed

    Zhang, Taishi; Gao, Nengyue; Li, Shuang; Lang, Matthew J; Xu, Qing-Hua

    2015-06-04

    Metal-enhanced fluorescence has attracted much attention due to its scientific importance and lots of potential applications. Plasmon coupled metal nanoparticles have been demonstrated to further improve the enhancement effects. Conventional studies of metal-enhanced fluorescence on the bulk systems are complicated by the ensemble average effects over many critical factors with large variations. Here, fluorescence enhancement of ATTO-655 by a plasmon coupled gold nanorod dimer fixed on a DNA origami nanobreadboard was studied on the single-particle level. A series of gold nanorod dimers with linear orientation and different gap distances ranging from 6.1 to 26.0 nm were investigated to explore the plasmon coupling effect on fluorescence enhancement. The results show that the dimer with the smallest gap (6.1 nm) gives the highest enhancement (470-fold), and the enhancement gradually decreases as the gap distance increases and eventually approaches that from a monomer (120-fold). This trend is consistent with the numerical calculation results. This study indicates that plasmon coupling in gold nanorod dimers offers further increased excitation efficiency to achieve large fluorescence enhancement.

  2. Molecular and cellular interactions between intoplicine, DNA, and topoisomerase II studied by surface-enhanced Raman scattering spectroscopy.

    PubMed

    Morjani, H; Riou, J F; Nabiev, I; Lavelle, F; Manfait, M

    1993-10-15

    The surface-enhanced Raman scattering spectra of the new antitumoral agent, intoplicine (RP 60475, NSC 645008), and those of its complexes with DNA and topoisomerase II in vitro and in K562 cancer cells were obtained. Intoplicine was found to unwind DNA and to inhibit purified calf thymus topoisomerase II via a stabilization of the ternary cleavable complex. The intensity of the surface-enhanced Raman scattering spectrum of intoplicine was not modified by the addition of plasmid pBR322 or calf thymus DNA. In the complex of this antitumor agent with topoisomerase II, the signal of intoplicine was completely abolished, indicating that at least some portion of intoplicine binds to an internal part of the enzyme. During the formation of the ternary complex, intoplicine was released from the interior of the protein and formed hydrogen bonds via its hydroxyl and/or amino groups. Similar modifications of the intoplicine spectra were found by microsurface-enhanced Raman scattering spectroscopy of the compound in the nucleus of treated K562 cells. In contrast, intoplicine was found to be in a free form in the cytoplasm.

  3. The mouse albumin enhancer contains a negative regulatory element that interacts with a novel DNA-binding protein.

    PubMed Central

    Herbst, R S; Boczko, E M; Darnell, J E; Babiss, L E

    1990-01-01

    The far-upstream mouse albumin enhancer (-10.5 to -8.43 kilobases) has both positive and negative regulatory domains which contribute to the rate and tissue specificity of albumin gene transcription. (R. S. Herbst, N. Friedman, J. E. Darnell, Jr., and L. E. Babiss, Proc. Natl. Acad. Sci. USA 86:1553-1557). In this work, the negative regulatory region has been functionally localized to sequences -8.7 to -8.43 kilobases upstream of the albumin gene cap site. In the absence of the albumin-modulating region (in which there are binding sites for the transcription factor C/EBP), the negative region can suppress a neighboring positive-acting element, thereby interfering with albumin enhancer function. The negative region is also capable of negating the positive action of the heterologous transthyretin enhancer in an orientation-independent fashion. Within this negative-acting region we can detect two DNA-binding sites, both of which are recognized by a protein present in all cell types tested. This DNA-binding activity is not competed for by any of a series of known DNA-binding sites, and hence this new protein is a candidate for a role in suppressing the albumin gene in nonhepatic cells. Images PMID:2370857

  4. Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.

    PubMed Central

    Hromas, R; Pauli, U; Marcuzzi, A; Lafrenz, D; Nick, H; Stein, J; Stein, G; Van Ness, B

    1988-01-01

    The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer. Images PMID:2830597

  5. A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

    PubMed

    Morris, Gavin; Fanucchi, Sylvia

    2016-04-05

    Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

  6. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    PubMed

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  7. Effect of β-carotene on catechol-induced genotoxicity in vitro: evidence of both enhanced and reduced DNA damage.

    PubMed

    Åsgård, R; Hellman, B

    2013-09-01

    Intake of antioxidants from the diet has been recognized to have beneficial health effects, but the potential benefit of taking antioxidants such as β-carotene as supplements is controversial. The aim of the present study was to evaluate the potential protective effects of a physiologically relevant concentration (2 μM) of β-carotene on the DNA damaging effects of catechol in mouse lymphoma L5178Y cells. Two different exposure protocols were used: simultaneous exposure to β-carotene and catechol for 3 h; and exposure to catechol for 3 h after 18 h pre-treatment with the vitamin. DNA damage was evaluated using the comet assay (employing one procedure for general damage, and another procedure, which also included oxidative DNA damage). Independent of exposure protocol and procedure for comet assay, β-carotene did not increase the basal level of DNA damage. However, at the highest concentration of catechol (1 mM), β-carotene was found to clearly increase the level of catechol-induced DNA damage, especially in the pre-treated cells. Interestingly, an opposite effect was observed at lower concentrations of catechol, but the β-carotene related reduction of catechol-induced genotoxicity was significant (P < 0.05) only for the procedure including oxidative damage induced by 0.5 mM catechol. Taken together our results indicate that β- carotene can both reduce and enhance the DNA damaging effects of a genotoxic agent such as catechol. This indicates that it is the level of catechol-induced DNA damage that seems to determine whether β-carotene should be regarded as a beneficial or detrimental agent when it comes to its use as a dietary supplement.

  8. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    PubMed

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  9. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

    PubMed Central

    Kitts, P A; Ayres, M D; Possee, R D

    1990-01-01

    Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location. Images PMID:2216760

  10. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

    PubMed Central

    Hendry, P; McCall, M J; Santiago, F S; Jennings, P A

    1992-01-01

    Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate. PMID:1280808

  11. Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells.

    PubMed

    Pazmandi, Kitti; Agod, Zsofia; Kumar, Brahma V; Szabo, Attila; Fekete, Tunde; Sogor, Viktoria; Veres, Agota; Boldogh, Istvan; Rajnavolgyi, Eva; Lanyi, Arpad; Bacsi, Attila

    2014-12-01

    Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.

  12. Ultra-small rhenium nanoparticles immobilized on DNA scaffolds: An excellent material for surface enhanced Raman scattering and catalysis studies.

    PubMed

    Anantharaj, S; Sakthikumar, K; Elangovan, Ayyapan; Ravi, G; Karthik, T; Kundu, Subrata

    2016-12-01

    Highly Sensitive and ultra-small Rhenium (Re) metal nanoparticles (NPs) were successfully stabilized in water by the staging and fencing action of the versatile biomolecule DNA that resulted in two distinct aggregated chain-like morphologies with average grain sizes of 1.1±0.1nm and 0.7±0.1nm for the very first time within a minute of reaction time. Re NPs are formed by the borohydride reduction of ammonium perrhenate (NH4ReO4) in the presence of DNA at room temperature (RT) under stirring. The morphologies were controlled by carefully monitoring the molar ratio of NH4ReO4 and DNA. The synthesized material was employed in two potential applications: as a substrate for surface enhanced Raman scattering (SERS) studies and as a catalyst for the reduction of aromatic nitro compounds. SERS study was carried out by taking methylene blue (MB) as the probe and the highest SERS enhancement factor (EF) of 2.07×10(7) was found for the aggregated chain-like having average grain size of 0.7±0.1nm. Catalytic reduction of 4-nitro phenol (4-NP), 2-nitro phenol (2-NP) and 4-nitroaniline (4-NA) with a rate constant value of 6×10(-2)min(-1), 33.83×10(-2)min(-1) and 37.4×10(-2)min(-1) have testified the excellent catalytic performance of our Re NPs immobilized on DNA. The overall study have revealed the capability of DNA in stabilizing the highly reactive Re metal at nanoscale and made them applicable in practice. The present route can also be extended to prepare one dimensional (1-D), self-assembled NPs of other reactive metals, mixed metals or even metal oxides for specific applications in water based solutions.

  13. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest.

    PubMed

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B; Su, Tsann-Long; Yih, Ling-Huei

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

  14. Microsatellite DNA markers: evaluating their potential for estimating the proportion of hatchery-reared offspring in a stock enhancement programme.

    PubMed

    Bravington, M V; Ward, R D

    2004-05-01

    We describe a statistical method for estimating the effectiveness of a stock enhancement programme using nuclear DNA loci. It is based on knowing the population allele frequencies and the genotypes of the hatchery parents (mother only, or mother and father), and on determining the probability that a wild-born animal will by chance have a genotype consistent with hatchery origin. We show how to estimate the proportion of released animals in the wild population, and its standard error. The method is applied to a data set of eight microsatellite loci in brown tiger prawns (Penaeus esculentus), prior to the start of a possible enhancement programme. We conclude that, for this particular data set, the effectiveness of such an enhancement programme could be quantified accurately if both maternal and paternal genotypes are known, but not if maternal genotypes only are known. Full paternal genotyping would require offspring genotyping and thus would be expensive, but a partly typed paternal genotype from a mass homogenate of offspring would be almost as effective and much cheaper. The experiment would become feasible based on maternal genotypes alone, if a further three typical microsatellite loci could be found to add to the existing panel of eight. The methods detailed should be of interest to any enhancement project that relies on nuclear DNA markers to provide tags.

  15. Enhanced Emission of Highly Labeled DNA Oligomers near Silver Metallic Surfaces

    PubMed Central

    Malicka, Joanna; Gryczynski, Ignacy; Lakowicz, Joseph R.

    2009-01-01

    Fluorescein is a widely used fluorescent probe in DNA analysis. One difficulty with fluorescein is its self-quenching due to resonance energy transfer between the residues, which results in decreased intensities with increasing labeling density. We examined the emission spectral properties of DNA oligomers labeled with one or five fluorescein residues. The emission intensity of the more highly labeled oligomer was decreased due to self-quenching. The self-quenching was mostly eliminated when this oligomer was held ~90 Å from the surface of metallic silver particles. The intensities increased 7- and 19-fold for the oligomers with one or five fluoresceins, respectively. The increased intensity did not result in increased photobleaching. These results suggest the use of substrates coated with silver particles for increased sensitivity on DNA arrays or for DNA analysis. PMID:14632044

  16. Enhancement of X-ray-induced breaks in DNA bound to molecules containing platinum: a possible application to hadrontherapy.

    PubMed

    Kobayashi, K; Frohlich, H; Usami, N; Takakura, K; Le Sech, C

    2002-01-01

    Complexes made of DNA and chloroterpyridine platinum (PtTC) bound to plasmid DNA were placed in aqueous solution and irradiated with monochromatic X rays tuned to the resonant photoabsorption energy of the L(III) shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) induced by irradiation on a supercoiled DNA plasmid was measured by the production of the circular-nicked and linear forms. To distinguish the contribution of the direct effects of ionization from the indirect effects due to a free radical attack, experiments were also performed in the presence of a hydroxyl free radical scavenger, dimethyl sulfoxide (DMSO). An enhancement of the number of SSBs and DSBs was observed when the plasmids contained the platinum intercalating molecules. A quantitative analysis was made to evaluate the respective contributions of the direct effects (Auger effect) and the indirect effects (free radical attack) to the number of DNA strand breaks. Even when off-resonant X rays were used, the strand break efficiency remained higher than expected based upon the absorption cross section, suggesting that the platinum bound to DNA might be increasing the yield of strand breaks. A mechanism is suggested that involves photoelectrons generated from the ionization of water which efficiently ionize platinum atoms. If this mechanism is correct, then heavy atoms, with a large cross section for ionization by electrons that are bound to the DNA, should behave as a radiosensitizer. This observation may provide insight into understanding the effects of new radiotherapy protocols, related chemotherapeutic agents such as cisplatin, and conventional radiotherapy for the treatment of tumors. A possible way to deliver the dose selectively in a well-defined volume, which uses the properties of the linear energy transfer of atomic ions interacting with matter, is suggested.

  17. 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.

  18. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide.

    PubMed

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-12-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  19. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-09-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  20. The Establishment of an Assay to Measure DNA Polymerase-Catalyzed Repair of UVB-Induced DNA Damage in Skin Cells and Screening of DNA Polymerase Enhancers from Medicinal Plants

    PubMed Central

    Ikeoka, Sawako; Nakahara, Tatsuo; Iwahashi, Hiroyasu; Mizushina, Yoshiyuki

    2016-01-01

    An in vitro assay method was established to measure the activity of cellular DNA polymerases (Pols) in cultured normal human epidermal keratinocytes (NHEKs) by modifying Pol inhibitor activity. Ultraviolet (UV) irradiation enhanced the activity of Pols, especially DNA repair-related Pols, in the cell extracts of NHEKs. The optimal ultraviolet B (UVB) exposure dose and culture time to upregulate Pols activity was 100 mJ/cm2 and 4-h incubation, respectively. We screened eight extracts of medicinal plants for enhancement of UVB-exposed cellular Pols activity using NHEKs, and found that rose myrtle was the strongest Pols enhancer. A Pols’ enhancement compound was purified from an 80% ethanol extract of rose myrtle, and piceatannol was isolated by spectroscopic analysis. Induction of Pol activity involved synergy between UVB irradiation and rose myrtle extract and/or piceatannol. Both the extract and piceatannol reduced UVB-induced cyclobutane pyrimidine dimer production, and prevented UVB-induced cytotoxicity. These results indicate that rose myrtle extract and piceatannol, its component, are potential photo-protective candidates for UV-induced skin damage. PMID:27153062

  1. Apoptosis and age-dependant induction of nuclear and mitochondrial etheno-DNA adducts in Long-Evans Cinnamon (LEC) rats: enhanced DNA damage by dietary curcumin upon copper accumulation.

    PubMed

    Nair, Jagadeesan; Strand, Susanne; Frank, Norbert; Knauft, Jutta; Wesch, Horst; Galle, Peter R; Bartsch, Helmut

    2005-07-01

    Long-Evans Cinnamon (LEC) rats, a model for human Wilson's disease, develop chronic hepatitis and liver tumors owing to accumulation of copper and induced oxidative stress. Lipid peroxidation (LPO)-induced etheno-DNA adducts in nuclear- and mitochondrial-DNA along with apoptosis was measured in LEC rat liver. Levels of etheno-DNA adducts (1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine) increased with age reaching a peak at 8 and 12 weeks in nuclear and mitochondrial DNA, respectively. This is the first demonstration that etheno-DNA adducts are also formed in mitochondrial DNA. Apoptosis was assessed by TUNEL+ cells in liver sections. CD95L RNA expression was also measured by in situ hybridization in the same sections. The highest nuclear DNA adduct levels coincided with a reduced apoptotic rate at 8 weeks. Mitochondrial-DNA adducts peaked at 12 weeks that coincided with the highest apoptotic rate, suggesting a link of etheno-DNA adducts in mitochondrial DNA to apoptosis. The DNA damage in liver was further enhanced and sustained by 0.5% curcumin in the diet. Treatment for 2 weeks elevated etheno-DNA adducts 9- to 25-fold in nuclear DNA and 3- to 4-fold in mitochondrial-DNA, providing a plausible explanation as to why in our earlier study [Frank et al. (2003) Mutat. Res., 523-524, 127-135], curcumin failed to prevent liver tumors in LEC rats. Our results also confirm the reported in vitro DNA damaging potential of curcumin in the presence of copper ions by reactive oxygen species. LPO-induced adduct formation in nuclear and mitochondrial DNA appear as early lesions in LEC rat liver carcinogenesis and are discussed in relation to apoptotic events in the progression of malignant disease.

  2. Application of exonuclease III-aided target recycling in flow cytometry: DNA detection sensitivity enhanced by orders of magnitude.

    PubMed

    Lu, Jie; Paulsen, Ian T; Jin, Dayong

    2013-09-03

    DNA-functionalized microspheres in conjugation with flow cytometry detection are widely used for high-throughput nucleic acid assays. Although such assays are rapid and capable of simultaneous analysis of multiple nucleic acid analytes in a single test, the intrinsic limitation in sensitivity remains challenging. Here we report a simple, highly sensitive, and reproducible method based on Exonuclease III-aided target recycling technique applied for DNA quantification in flow cytometry. By loading a high density of Cy5-labeled probe DNA on microspheres (15 μm), we achieved hitherto unreported DNA detection limit of 3.2 pM in flow cytometry bead assay, enhancing the sensitivity by a factor of over 56.8 compared to the conventional direct hybridization bead assay. Furthermore, we evaluated multiplexing capability by simultaneous detections of two target DNAs with FAM and Cy5 reporter conjugated probes. Therefore, the novel Exonuclease III-amplified flow cytometry bead assay has great potential for the rapid, sensitive, and accurate detection and quantification of nucleic acids in clinical diagnosis and biomedical research.

  3. Crowding Activates ClpB and Enhances Its Association with DnaK for Efficient Protein Aggregate Reactivation

    PubMed Central

    Martín, Ianire; Celaya, Garbiñe; Alfonso, Carlos; Moro, Fernando; Rivas, Germán; Muga, Arturo

    2014-01-01

    Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner. PMID:24806934

  4. Development of an efficient process intensification strategy for enhancing Pfu DNA polymerase production in recombinant Escherichia coli.

    PubMed

    Hu, Jian-Hua; Wang, Feng; Liu, Chun-Zhao

    2015-04-01

    An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-β-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5% in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5% under the controlled dissolved oxygen concentration of 30% in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.

  5. Micron-sized surface enhanced Raman scattering reporter/fluorescence probe encoded colloidal microspheres for sensitive DNA detection.

    PubMed

    You, Lijun; Li, Ruimin; Dong, Xu; Wang, Fang; Guo, Jia; Wang, Changchun

    2017-02-15

    A new type of optical probes, featuring surface enhanced Raman scattering (SERS) and fluorescence spectra dual-mode encoding, has been reported in this article. Based on the uniform micrometer-sized melamine resin/Ag nanoparticles (MRM/Ag-NPs) composite microspheres, the SERS reporters and fluorescent probes were successfully fixed onto the different layers of the MEM/Ag-NPs microspheres, which supported the sensitive DNA detecton. The two spectroscopic methods commonly considered to be contradictive to each other, yet the optical signals were separable in the experiments. The dual-encoding strategy and single microsphere detecton method put the number of available independent codes to be rough the multiple of those available in the two optical detection channels, which increases far more rapidly than the summation of the two channels. As a proof of cencept, the utility of this dual spectrum mode SERS-fluoresecence encoded microsphere (SFEM) was demonstrated in a specific DNA detection using complimentary ssDNA functionalized magnetic beads as the DNA capturing and separation agents. Excellent encoding results were demonstrated from the decoding of the SERS and fluorescence signals of the SFEM. The method appears to be general in scope and we expect that the SERS-fluoresecence encoded microspheres system is applicable to multiplex bioassays of a variety of biomolecules.

  6. Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

    PubMed

    Romanczuk, H; Wormington, W M

    1989-02-01

    Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

  7. Quantum dot monolayer for surface plasmon resonance signal enhancement and DNA hybridization detection.

    PubMed

    Ghrera, Aditya Sharma; Pandey, Manoj Kumar; Malhotra, Bansi Dhar

    2016-06-15

    We report results of studies relating to the fabrication of a surface plasmon resonance (SPR)-based nucleic acid sensor for quantification of DNA sequence specific to chronic myelogeneous leukemia (CML). The SPR disk surface has been modified with octadecanethiol self-assembled monolayer followed by deposition of the tri-n-octylphosphine oxide capped cadmium selenide quantum dots (QD) Langmuir monolayer. The deposition is performed via Langmuir-Blodgett (LB) technique. For the sensor chip preparation, covalent immobilization of the thiol-terminated DNA probe sequence (pDNA) using displacement reaction is accomplished. This integrated SPR chip has been used to detect target complementary DNA concentration by monitoring the change in coupling angle via hybridization. It is revealed that this biosensor exhibits high sensitivity (0.7859 m(0)pM(-1)) towards complementary DNA and can be used to detect it in the concentration range, 180 pM to 5 pM with detection limit as 4.21 pM. The results of kinetic studies yield the values of hybridization and dissociation rate constants as 9.6 × 10(4) M(-1) s(-1) and 2.3 × 10(-2) s(-1), respectively, with the equilibrium constant for hybridization as 4.2 × 10(6) M(-1).

  8. Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins.

    PubMed

    Otten, Gillis R; Schaefer, Mary; Doe, Barbara; Liu, Hong; Srivastava, Indresh; Megede, Jan zur; Kazzaz, Jina; Lian, Ying; Singh, Manmohan; Ugozzoli, Mildred; Montefiori, David; Lewis, Mark; Driver, David A; Dubensky, Thomas; Polo, John M; Donnelly, John; O'Hagan, Derek T; Barnett, Susan; Ulmer, Jeffrey B

    2005-07-01

    DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.

  9. Histidine-Based Lipopeptides Enhance Cleavage of Nucleic Acids: Interactions with DNA and Hydrolytic Properties.

    PubMed

    Bélières, M; Déjugnat, C; Chouini-Lalanne, N

    2015-12-16

    Interaction studies and cleavage activity experiments were carried out between plasmid DNA and a series of histidine-based lipopeptides. Specific fluorescent probes (ethidium bromide, Hoechst 33342, and pyrene) were used to monitor intercalation, minor groove binding, and self-assembly of lipopeptides, respectively. Association between DNA and lipopeptides was thus evidenced, highlighting the importance of both histidine and hydrophobic tail in the interaction process. DNA cleavage in the presence of lipopeptides was then detected by gel electrophoresis and quantified, showing the importance of histidine and the involvement of its side-chain imidazole in the hydrolysis mechanism. These systems could then be developed as synthetic nucleases while raising concern of introducing histidine in the design of lipopeptide-based transfection vectors.

  10. An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

    PubMed

    Chen, Jia; Huang, Yong; Vdovenko, Marina; Sakharov, Ivan Yu; Su, Guifa; Zhao, Shulin

    2015-06-01

    An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods.

  11. Comparison of the performance of the borax buffer-based HRP-enhanced reagent and the 'Lumi-Phos 530' chemiluminescence systems in the detection of biotinylated DNA.

    PubMed

    Cercek, B; Roby, K; Siaw, M

    1995-01-01

    A comparison of two chemiluminescence methods, the borax buffer-based HRP-enhanced reagent and Lumi-Phos 530, applied to the detection of a biotinylated 30-mer DNA slot blotted onto a nylon membrane, is presented. A streptavidin-HRP and streptavidin-ALP mediated detection system was used. The HRP-enhanced system is up to 15-fold greater with respect to the signal/background ratios than the Lumi-Phos 530 system at 0.5 microgram biotinylated DNA with at least a two-fold improvement in detection sensitivity for 0.5 ng biotinylated DNA.

  12. Immunostimulant patches containing Escherichia coli LT enhance immune responses to DNA- and recombinant protein-based Alzheimer's disease vaccines.

    PubMed

    Davtyan, Hayk; Ghochikyan, Anahit; Hovakimyan, Armine; Petrushina, Irina; Yu, Jianmei; Flyer, David; Madsen, Peter Juul; Pedersen, Lars Ostergaard; Cribbs, David H; Agadjanyan, Michael G

    2014-03-15

    Immunotherapeutic approaches to treating Alzheimer's disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the Escherichia coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer's disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.

  13. DNA vaccines against infectious agents: recent strategies for enhancing immune responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There have been widespread efforts to develop plasmid DNA vaccines against animal and human pathogens, and for use as therapies in the treatment of cancers, autoimmune diseases, and allergies. The impetus for this research activity was based on early promising results in laboratory animals that sho...

  14. GITRL as a genetic adjuvant enhances enterovirus 71 VP1 DNA vaccine immunogenicity.

    PubMed

    Yuan, Jing; Tang, Xinyi; Yin, Kai; Tian, Jie; Rui, Ke; Ma, Jie; Mao, Chaoming; Chen, Jianguo; Lu, Liwei; Xu, Huaxi; Wang, Shengjun

    2015-05-01

    VP1 protein is the immunodominant capsid protein of enterovirus 71 (EV71) which is responsible for large outbreaks of hand, foot and mouth disease. It has been reported that glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and its ligand (GITRL) are involved in modulating both innate and adaptive immune responses. In this study, a DNA vaccine vector encoding EV71 VP1 gene and mGITRL gene (pIRES/VP1/mGITRL) was constructed. And female Balb/c mice were immunized intramuscularly with the DNA vaccine. Compared with the groups immunized with pIRES, pIRES/VP1, pIRES/mGITRL and PBS, the inoculation of pIRES/VP1/mGITRL induced a higher levels of EV71 VP1-specific antibody and specific antibody-forming cells. However, significantly higher levels of CD4(+)Th1, Th2 and CD8(+)IFN-γ(+)T cells were found in the pIRES/VP1/mGITRL group compared with control groups. Our results demonstrate that a novel DNA vaccine, expressing VP1 and mGITRL, could effectively elicit both humoral and cell-mediated immune responses against EV71 VP1 in mice. Thus, the mGITRL may be used as molecular adjuvant for EV71 DNA vaccine.

  15. Enhancement of Radiation Therapy in Prostate Cancer by DNA-PKcs Inhibitor

    DTIC Science & Technology

    2013-07-01

    Nakagawa K, et al. (2006). Heterogeneous expression of DNA-dependent protein kinase in esophageal cancer and normal epithelium . Int J Mol Med 18, 441... glandular structure. (B) H&E stained tissue section from an irradiated tumor. The irradiated area is highly necrotic (black dash arrow) displaying loss

  16. Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

    SciTech Connect

    Shen, Yan Nan; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun-Ran; Kim, Su-Hyeon; Hwang, Sang-Gu; Park, Sang Jun; Kim, Chun-Ho; Lee, Kee-Ho

    2014-01-17

    Highlights: •HAS2 may be a promising target for the radiosensitization of human cancer. •HAS2 is elevated (up to ∼10-fold) in irradiated radioresistant and -sensitive cancer cells. •HAS2 knockdown sensitizes cancer cells to radiation. •HAS2 knockdown potentiates irradiation-induced DNA damage and apoptotic death. •Thus, the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. -- Abstract: Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.

  17. Genetically enhanced cellulase production in Pseudomonas cellulosa using recombinant DNA technology

    DOEpatents

    Dees, H. Craig

    1999-01-01

    An enhanced strain of Pseudomonas celllulosa was obtained by introducing a recombinant genetic construct comprising a heterologous cellulase gene operably connected to a promoter into ATCC 55702, mutagenizing the transformants by treatment with MNNG, and selecting a high cellulase producing transformant. The transformant, designated Pseudomonas cellulosa ATCC XXXX, exhibits enhanced levels of cellulase production relative to the untransformed Pseudomonas cellulosa strain #142 ATCC 55702.

  18. Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

    PubMed

    Zhang, Yonghong; Liang, Shuang; Li, Xiujin; Wang, Liyue; Zhang, Jianlou; Xu, Jian; Huo, Shanshan; Cao, Xuebin; Zhong, Zhenyu; Zhong, Fei

    2015-07-09

    Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation.

  19. Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

    PubMed

    Rius, Maria; Stresemann, Carlo; Keller, Daniela; Brom, Manuela; Schirrmacher, Esther; Keppler, Dietrich; Lyko, Frank

    2009-01-01

    The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

  20. Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

    PubMed

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2015-02-01

    Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3μg of plasmid DNA was mixed with 70-80μL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen.

  1. Enhancing cell nucleus accumulation and DNA cleavage activity of anti-cancer drug via graphene quantum dots.

    PubMed

    Wang, Chong; Wu, Congyu; Zhou, Xuejiao; Han, Ting; Xin, Xiaozhen; Wu, Jiaying; Zhang, Jingyan; Guo, Shouwu

    2013-10-04

    Graphene quantum dots (GQDs) maintain the intrinsic layered structural motif of graphene but with smaller lateral size and abundant periphery carboxylic groups, and are more compatible with biological system, thus are promising nanomaterials for therapeutic applications. Here we show that GQDs have a superb ability in drug delivery and anti-cancer activity boost without any pre-modification due to their unique structural properties. They could efficiently deliver doxorubicin (DOX) to the nucleus through DOX/GQD conjugates, because the conjugates assume different cellular and nuclear internalization pathways comparing to free DOX. Also, the conjugates could enhance DNA cleavage activity of DOX markedly. This enhancement combining with efficient nuclear delivery improved cytotoxicity of DOX dramatically. Furthermore, the DOX/GQD conjugates could also increase the nuclear uptake and cytotoxicity of DOX to drug-resistant cancer cells indicating that the conjugates may be capable to increase chemotherapy efficacy of anti-cancer drugs that are suboptimal due to the drug resistance.

  2. Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.

    PubMed

    Sihi, Sayantani; Bakshi, Sankar; Sengupta, Dibyendu Narayan

    2015-02-01

    DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

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

    PubMed Central

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

    1989-01-01

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

  4. Modulating chromatin structure and DNA accessibility by deacetylase inhibition enhances the anti-cancer activity of silver nanoparticles.

    PubMed

    Igaz, Nóra; Kovács, Dávid; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M; Kiricsi, Mónika

    2016-10-01

    Histone deacetylase (HDAC) inhibitors are considered as novel therapeutic agents inducing cell cycle arrest and apoptotic cell death in various cancer cells. Inhibition of deacetylase activity results in a relaxed chromatin structure thereby rendering the genetic material more vulnerable to DNA targeting agents that could be exploited by combinational cancer therapy. The unique potential of silver nanoparticles (AgNPs) in tumor therapy relies on the generation of reactive radicals which trigger oxidative stress, DNA damage and apoptosis in cancer cells. The revolutionary application of AgNPs as chemotherapeutical drugs seems very promising, nevertheless the exact molecular mechanisms of AgNP action in combination with other anti-cancer agents have yet to be elucidated in details before clinical administrations. As a step towards this we investigated the combinational effect of HDAC inhibition and AgNP administration in HeLa cervical cancer cells. We identified synergistic inhibition of cancer cell growth and migration upon combinational treatments. Here we report that the HDAC inhibitor Trichostatin A enhances the DNA targeting capacity and apoptosis inducing efficacy of AgNPs most probably due to its effect on chromatin condensation. These results point to the potential benefits of combinational application of HDAC inhibitors and AgNPs in novel cancer medication protocols.

  5. 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.

  6. Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN-deficient human fibroblasts.

    PubMed

    Saha, Bidisha; Cypro, Alexander; Martin, George M; Oshima, Junko

    2014-06-01

    Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.

  7. Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN-deficient human fibroblasts

    PubMed Central

    Saha, Bidisha; Cypro, Alexander; Martin, George M; Oshima, Junko

    2014-01-01

    Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS. PMID:24308646

  8. Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification.

    PubMed

    Yamato, K; Egawa, N; Endo, S; Ui-Tei, K; Yamada, T; Saigo, K; Hyodo, I; Kiyono, T; Nakagawa, I

    2011-08-01

    Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.

  9. N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

    PubMed

    Suk, Jung Soo; Boylan, Nicholas J; Trehan, Kanika; Tang, Benjamin C; Schneider, Craig S; Lin, Jung-Ming G; Boyle, Michael P; Zeitlin, Pamela L; Lai, Samuel K; Cooper, Mark J; Hanes, Justin

    2011-11-01

    For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

  10. N-acetylcysteine Enhances Cystic Fibrosis Sputum Penetration and Airway Gene Transfer by Highly Compacted DNA Nanoparticles.

    PubMed

    Suk, Jung Soo; Boylan, Nicholas J; Trehan, Kanika; Tang, Benjamin C; Schneider, Craig S; Lin, Jung-Ming G; Boyle, Michael P; Zeitlin, Pamela L; Lai, Samuel K; Cooper, Mark J; Hanes, Justin

    2011-11-01

    For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK30PEG10k). We found that CK30PEG10k/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK30PEG10k/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

  11. Enhancing immune responses to a DNA vaccine encoding Toxoplasma gondii GRA14 by calcium phosphate nanoparticles as an adjuvant.

    PubMed

    Ahmadpour, Ehsan; Sarvi, Shahabeddin; Hashemi Soteh, Mohammad Bagher; Sharif, Mehdi; Rahimi, Mohammad Taghi; Valadan, Reza; Tehrani, Mohsen; Khalilian, Alireza; Montazeri, Mahbobeh; Fasihi-Ramandi, Mahdi; Daryani, Ahmad

    2017-03-09

    Several approaches have been used to improve the immunogenicity of DNA vaccines. In the current study, we constructed the plasmid encoding T. gondii dense granule 14 (GRA14) and investigated the immunological properties of calcium phosphate nanoparticles (CaPNs) as nano-adjuvant to enhance the protective efficacy of pcGRA14. BALB/c mice intramuscularly injected three times at two-week intervals and the immune responses were evaluated using lymphocyte proliferation assay, cytokine and antibody measurements, survival times, and parasite load of mice challenged with the virulent T. gondii RH strain. The results showed that the immune responses were induced in mice receiving pcGRA14 DNA vaccine. Interestingly, pcGRA14 coated with nanoparticles led to statistically significant enhancements of cellular and humoral immune responses against Toxoplasma infection (P<0.05). After challenge with RH strain of T. gondii, immunized mice with pcGRA14 showed prolong survival time compared to control groups (P<0.05). In addition, pcGRA14 coated with nano-adjuvant exhibited the lowest parasitic load in the infected mice tissues. For the first time, our data indicate that the pcGRA14 coated with CaPN was more effective for stimulation of immune responses and should be considered as an adjuvant in the design of vaccines against toxoplasmosis.

  12. The medicinal fungus Antrodia cinnamomea regulates DNA repair and enhances the radiosensitivity of human esophageal cancer cells

    PubMed Central

    Liu, Yu-Ming; Liu, Yu-Kuo; Wang, Ling-Wei; Huang, Yu-Chuen; Huang, Pin-I; Tsai, Tung-Hu; Chen, Yu-Jen

    2016-01-01

    This study investigated the adjunctive effects of Antrodia cinnamomea mycelial fermentation broth (AC-MFB), a Taiwanese medicinal fungus, in enhancing the radiosensitivity of esophageal cancer cells. Human CE81T/VGH squamous and BE3 adenocarcinoma esophageal cancer cells were used in this study. A colony formation assay showed that pretreatment with AC-MFB decreased the survival of irradiated esophageal cancer cells, with a maximum sensitizer enhancement ratio of 1.91% and 37% survival. A DNA histogram study showed that AC-MFB pretreatment enhanced cell cycle arrest at the G2/M phase, the most radiosensitive phase. An immunofluorescence assay and a Western blotting assay showed that AC-MFB delayed the abrogation of γ-H2AX, upregulated p21 expression, and attenuated the radiation-induced phosphorylation of ataxia telangiectasia-mutated kinase and checkpoint kinase 2. An in vivo validation study showed that AC-MFB treatment tended to have a synergistic effect with radiation on the tumor growth delay of CE81T/VGH cells in BALB/c mice. These data suggest that this edible fungus product could enhance the effect of radiotherapy against esophageal cancer. PMID:27826196

  13. Investigating and correcting plasma DNA sequencing coverage bias to enhance aneuploidy discovery.

    PubMed

    Chandrananda, Dineika; Thorne, Natalie P; Ganesamoorthy, Devika; Bruno, Damien L; Benjamini, Yuval; Speed, Terence P; Slater, Howard R; Bahlo, Melanie

    2014-01-01

    Pregnant women carry a mixture of cell-free DNA fragments from self and fetus (non-self) in their circulation. In recent years multiple independent studies have demonstrated the ability to detect fetal trisomies such as trisomy 21, the cause of Down syndrome, by Next-Generation Sequencing of maternal plasma. The current clinical tests based on this approach show very high sensitivity and specificity, although as yet they have not become the standard diagnostic test. Here we describe improvements to the analysis of the sequencing data by reducing GC bias and better handling of the genomic repeats. We show substantial improvements in the sensitivity of the standard trisomy 21 statistical tests, which we measure by artificially reducing read coverage. We also explore the bias stemming from the natural cleavage of plasma DNA by examining DNA motifs and position specific base distributions. We propose a model to correct this fragmentation bias and observe that incorporating this bias does not lead to any further improvements in the detection of fetal trisomy. The improved bias corrections that we demonstrate in this work can be readily adopted into existing fetal trisomy detection protocols and should also lead to improvements in sub-chromosomal copy number variation detection.

  14. Monodispersed mesoporous silica nanoparticles with very large pores for enhanced adsorption and release of DNA.

    PubMed

    Gao, Fei; Botella, Pablo; Corma, Avelino; Blesa, Jose; Dong, Lin

    2009-02-12

    Silica nanoparticles with controlled diameter (approximately 70-300 nm) and with uniform pores of 20 nm are prepared by a low temperature (10 degrees C) synthetic method in the presence of a dual surfactant system. While a triblock copolymer (Pluronic F127) acts as supramolecular template and coassembles with hydrolyzed silica species to develop a partially ordered mesophase with face-centered cubic symmetry, a fluorocarbon surfactant with high surface activity (FC-4) surrounds the silica particles through S+X-I+ interactions, thereby limiting their growth. The final textural properties of this material are achieved by means of a subsequent hydrothermal treatment to yield high pore volume mesoporous silica nanoparticles with the largest pore entrance size (17 nm) and cavity diameter (20 nm) reported up to now. After surface modification with aminopropyl groups, the nanoparticles are able to encapsulate inside the pores molecules of the firefly luciferase plasmid DNA (pGL3-Control, 5256 pb), leading to stable conjugates with up to 0.07 microg DNA m(-2), which is the highest content achieved with silica-based materials. Furthermore, plasmid DNA becomes protected from enzymatic degradation when conjugated with the mesostructured nanoparticles.

  15. DNA-binding domain fusions enhance the targeting range and precision of Cas9

    PubMed Central

    Bolukbasi, Mehmet Fatih; Gupta, Ankit; Oikemus, Sarah; Derr, Alan G.; Garber, Manuel; Brodsky, Michael H.; Zhu, Lihua Julie; Wolfe, Scot A.

    2015-01-01

    The CRISPR-Cas9 system is commonly employed in biomedical research; however, the precision of Cas9 is sub-optimal for gene therapy applications that involve editing a large population of cells. Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site within the human genome over a large cohort of treated cells. Here, we demonstrate that fusing a programmable DNA-binding domain (pDBD) to Cas9 combined with the attenuation of Cas9’s inherent DNA binding affinity produces a Cas9-pDBD chimera with dramatically improved precision and increased targeting range. Because the specificity and affinity of this framework is easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus – characteristics that are ideal for gene therapy applications. PMID:26480473

  16. Cimetidine synergizes with Praziquantel to enhance the immune response of HBV DNA vaccine via activating cytotoxic CD8(+) T cell.

    PubMed

    Xie, Xiaoping; Geng, Shuang; Liu, Hu; Li, Chaofan; Yang, Yuqin; Wang, Bin

    2014-01-01

    Previously, we have reported that either CIM or PZQ, 2 clinical drugs, could be used to develop as adjuvants on HBV DNA vaccine to elicit both humoral and cellular immune responses. Here, we demonstrate that combinations of CIM and PZQ as adjuvants for a HBV DNA vaccine, could induce much stronger antigen specific CD4(+) and CD8(+) T cell responses compared either with CIM or PZQ alone. The synergistic effects of CIM plus PZQ to HBV DNA vaccine were observed on a higher IgG2a/IgG1 ratio, an increase of HBsAg-specific CD4(+) T cells capable of producing IFN-γ or IL-17A and a robust IFN-γ-, IL-17A-, or TNF-α-producing CD8(+) T cells to HBsAg. Most importantly, the antigen-specific CTL response was also elevated significantly, which is critical for the eradication of hepatitis B virus (HBV) infected cells. Using an HBsAg transgenic mouse model, the expression of HBsAg in the hepatic cells was also significantly reduced after immunized with pCD-S 2 in the presence of 0.5% CIM and 0.25% PZQ. Further investigations demonstrated that the synergistic effects of combination of CIM and PZQ were dependent on enhanced cytotoxic CD8(+) T cells, which was correlated with impaired activities of regulatory T cells. Therefore, combinations of CIM and PZQ have great potential to be used as effective adjuvants on DNA-based vaccinations for the treatment of chronic hepatitis B.

  17. Detection of deoxyribonucleic acid (DNA) targets using polymerase chain reaction (PCR) and paper surface-enhanced Raman spectroscopy (SERS) chromatography.

    PubMed

    Hoppmann, Eric P; Yu, Wei W; White, Ian M

    2014-01-01

    Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjet-fabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5' Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

  18. Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

    PubMed

    Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

    2017-01-01

    Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

  19. Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage

    PubMed Central

    Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

    2017-01-01

    Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2’-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy. PMID:28045991

  20. Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against gamma-radiation-induced epigenetic modifications.

    PubMed

    Batra, Vipen; Sridhar, Swathi; Devasagayam, Thomas Paul Asir

    2010-02-12

    Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that gamma-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to gamma-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C(1)) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P<0.01), while hepatic homocysteine levels decreased (P<0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P<0.01), it showed a decrease in response to high doses of gamma-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C(1) flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P<0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under gamma-radiation stress might be a very dynamic

  1. Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

    PubMed

    Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

    2011-03-07

    For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis.

  2. Enhanced replication of UV-damaged Simian virus 40 DNA in carcinogen-treated mammalian cells

    SciTech Connect

    Maga, J.A.

    1983-01-01

    The replication of UV-damaged Simian virus 40 (SV40) in carcinogen-treated monkey cells has been studied to elucidate the mechanism of carcinogen-enhanced reactivation. Carcinogen enhanced reactivation is the observed increase in UV-irradiated virus survival in host cells treated with low doses of carcinogen compared to UV-irradiated virus survival in untreated hosts. Carcinogen treatment of monkey kidney cells with either N-acetoxy-2-acetylaminofluorene (AAAF) or UV radiation leads to an enhanced capacity to replicate UV-damaged virus during the first round of infection. To further define the mechanism leading to enhanced replication, a detailed biochemical analysis of replication intermediates in carcinogen-treated cells was performed. Several conclusions can be drawn. First enhanced replication can be observed in the first four rounds of replication after UV irradiation of viral templates. The second major finding is that the relaxed circular intermediate model proposed for the replication of UV-damaged templates in untreated cells appears valid for replication of UV-damaged templates in carcinogen-treated cells. Possible mechanisms and the supporting evidence are discussed and future experiments outlined.

  3. DNA repair in spermatocytes and spermatids of the mouse

    SciTech Connect

    Sega, G.A.

    1982-01-01

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced.

  4. Enhanced gene expression of systemically administered plasmid DNA in the liver with therapeutic ultrasound and microbubbles.

    PubMed

    Raju, Balasundar I; Leyvi, Evgeniy; Seip, Ralf; Sethuraman, Shriram; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight

    2013-01-01

    Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 μg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p < 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into

  5. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

    PubMed Central

    Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  6. Conjugation of benzylvanillin and benzimidazole structure improves DNA binding with enhanced antileukemic properties.

    PubMed

    Al-Mudaris, Zena A; Majid, Aman S A; Ji, Dan; Al-Mudarris, Ban A; Chen, Shih-Hsun; Liang, Po-Huang; Osman, Hasnah; Jamal Din, Shah Kamal Khan; Abdul Majid, Amin M S

    2013-01-01

    Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the

  7. Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

    PubMed Central

    Al-Mudarris, Ban A.; Chen, Shih-Hsun; Liang, Po-Huang; Osman, Hasnah; Jamal Din, Shah Kamal Khan; Abdul Majid, Amin M. S.

    2013-01-01

    Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the

  8. Enhancement of Radiation Therapy in Prostate Cancer by DNA-PKcs Inhibitor

    DTIC Science & Technology

    2012-07-01

    modulate cell proliferation, survival, and apoptosis by coordinating PI3K , Akt, and the ASK1 pathway [25, 26]. Our previous work indicated that loss...of S6K almost totally attenuated in DAB2IP overexpressed PCa cells . Notably, DAB2IP functions as a scaffold protein to inhibit the PI3K -Akt pathway ... via a direct protein interaction with PI3K through its PR domain [25]. Autophagy-defective cells also show an increase in DNA-DSB in response to

  9. Charge enhancement of single-stranded DNA in negative electrospray ionization using the supercharging reagent meta-nitrobenzyl alcohol.

    PubMed

    Brahim, Bessem; Alves, Sandra; Cole, Richard B; Tabet, Jean-Claude

    2013-12-01

    Charge enhancement of single-stranded oligonucleotide ions in negative ESI mode is investigated. The employed reagent, meta-nitrobenzyl alcohol (m-NBA), was found to improve total signal intensity (Itot), increase the highest observed charge states (zhigh), and raise the average charge states (zavg) of all tested oligonucleotides analyzed in negative ESI. To quantify these increases, signal enhancement ratios (SER1%) and charge enhancement coefficients (CEC1%) were introduced. The SER1%, (defined as the quotient of total oligonucleotide ion abundances with 1% m-NBA divided by total oligonucleotide abundance without m-NBA) was found to be greater than unity for every oligonucleotide tested. The CEC1% values (defined as the average charge state in the presence of 1% m-NBA minus the average charge state in the absence of m-NBA) were found to be uniformly positive. Upon close inspection, the degree of charge enhancement for longer oligonucleotides was found to be dependent upon thymine density (i.e., the number and the location of phospho-thymidine units). A correlation between the charge enhancement induced by the presence of m-NBA and the apparent gas-phase acidity (largely determined by the sequence of thymine units but also by the presence of protons on other nucleobases) of multiply deprotonated oligonucleotide species, was thus established. Ammonium cations appeared to be directly involved in the m-NBA supercharging mechanism, and their role seems to be consistent with previously postulated ESI mechanisms describing desorption/ionization of single-stranded DNA into the gas phase.

  10. Charge Enhancement of Single-Stranded DNA in Negative Electrospray Ionization Using the Supercharging Reagent Meta-nitrobenzyl Alcohol

    NASA Astrophysics Data System (ADS)

    Brahim, Bessem; Alves, Sandra; Cole, Richard B.; Tabet, Jean-Claude

    2013-12-01

    Charge enhancement of single-stranded oligonucleotide ions in negative ESI mode is investigated. The employed reagent, meta-nitrobenzyl alcohol (m-NBA), was found to improve total signal intensity (Itot), increase the highest observed charge states (zhigh), and raise the average charge states (zavg) of all tested oligonucleotides analyzed in negative ESI. To quantify these increases, signal enhancement ratios (SER1%) and charge enhancement coefficients (CEC1%) were introduced. The SER1%, (defined as the quotient of total oligonucleotide ion abundances with 1 % m-NBA divided by total oligonucleotide abundance without m-NBA) was found to be greater than unity for every oligonucleotide tested. The CEC1% values (defined as the average charge state in the presence of 1 % m-NBA minus the average charge state in the absence of m-NBA) were found to be uniformly positive. Upon close inspection, the degree of charge enhancement for longer oligonucleotides was found to be dependent upon thymine density (i.e., the number and the location of phospho-thymidine units). A correlation between the charge enhancement induced by the presence of m-NBA and the apparent gas-phase acidity (largely determined by the sequence of thymine units but also by the presence of protons on other nucleobases) of multiply deprotonated oligonucleotide species, was thus established. Ammonium cations appeared to be directly involved in the m-NBA supercharging mechanism, and their role seems to be consistent with previously postulated ESI mechanisms describing desorption/ionization of single-stranded DNA into the gas phase.

  11. Distance-dependent quenching and enhancing of electrochemiluminescence from a CdS:Mn nanocrystal film by Au nanoparticles for highly sensitive detection of DNA.

    PubMed

    Shan, Yun; Xu, Jing-Juan; Chen, Hong-Yuan

    2009-02-28

    The quenching of electrochemiluminescence (ECL) from a CdS:Mn nanocrystal film by proximal Au nanoparticles was observed as a result of Förster energy transfer, while an enhancement of ECL takes place after hybridization with target DNA due to the energy transfer of ECL excited surface plasmon resonances in AuNPs to the CdS:Mn NCs at large separation, based on which an ultrasensitive and specific DNA biosensor was constructed.

  12. 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.

  13. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-04-30

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  14. 5-color multiplexed microwave-accelerated metal-enhanced fluorescence: detection and analysis of multiple DNA sequences from within one sample well within a few seconds.

    PubMed

    Dragan, Anatoliy; Geddes, Chris D

    2014-11-01

    We present a potentially highly sensitive and selective bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA "rapid catch and signal" (DNA-RCS) technology developed for the detection of different DNA sequences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes attached to the probe-DNAs, which hybridizes with the pre-formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF-surface and anchor/probe DNA hybridization, i.e. "rapid catch" of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed.

  15. Specific detection of RT activity in culture supernantants of retrovirus-producing cells, using synthetic DNA as competitor in polymerase enhanced reverse transcriptase assay.

    PubMed

    Voisset, C; Tönjes, R R; Breyton, P; Mandrand, B; Paranhos-Baccalà, G

    2001-05-01

    The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

  16. Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

    PubMed

    Cherif, Mahamoud Sama; Shuaibu, Mohammed Nasir; Kodama, Yukinobu; Kurosaki, Tomoaki; Helegbe, Gideon Kofi; Kikuchi, Mihoko; Ichinose, Akitoyo; Yanagi, Tetsuo; Sasaki, Hitoshi; Yui, Katsuyuki; Tien, Nguyen Huy; Karbwang, Juntra; Hirayama, Kenji

    2014-04-07

    We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

  17. NAD(+) administration decreases doxorubicin-induced liver damage of mice by enhancing antioxidation capacity and decreasing DNA damage.

    PubMed

    Wang, Ban; Ma, Yingxin; Kong, Xiaoni; Ding, Xianting; Gu, Hongchen; Chu, Tianqing; Ying, Weihai

    2014-04-05

    One of the major obstacles for cancer treatment is the toxic side effects of anti-cancer drugs. Doxorubicin (DOX) is one of the most widely used anti-cancer drugs, which produces significant toxic side effects on the heart and such organs as the liver. Because NAD(+) can decrease cellular or tissue damage under multiple conditions, we hypothesized that NAD(+) administration may decrease DOX-induced hepatotoxicity. In this study we tested this hypothesis by using a mouse model, showing that NAD(+) administration can significantly attenuate DOX-induced increase in serum glutamate oxaloacetate transaminase activity and decrease in liver weight. The NAD(+) administration also attenuated the DOX-induced increases in the levels of double-strand DNA (dsDNA) damage, TUNEL signals, and active caspase-3. Furthermore, our data has suggested that the NAD(+) administration could produce protective effects at least partially by restoring the antioxidation capacity of the liver, because NAD(+) administration can attenuate the decreases in both the GSH levels and the glutathione reductase activity of the DOX-treated liver, which could play a significant role in the DOX-induced hepatotoxicity. This finding has provided the first evidence indicating that NAD(+) is capable of increasing the antioxidation capacity of tissues. Collectively, our study has found that NAD(+) can significantly decrease DOX-induced liver damage at least partially by enhancing antioxidation capacity and decreasing dsDNA damage. Because it can also selectively decrease tumor cell survival, NAD(+) may have significant merits over antioxidants for applying jointly with DOX to decrease the toxic side effects of DOX.

  18. Spectroscopic studies of micelle-enhanced ligand exchange of gallium (III)/4-(2-pyridylazo) resorcinol complex by calf thymus DNA.

    PubMed

    Romeika, Jennifer M; Spurgeon, Charina L; Yan, Fei

    2014-01-03

    The effect of cationic micelles of cetyltrimethylammonium bromide (CTAB) on the interaction of gallium (III) with 4-(2-pyridylazo) resorcinol (PAR) under varying conditions has been studied spectrophotometrically. At pH 6.0, CTAB (0.05% w/v) markedly enhanced the absorption intensity of gallium (III)-PAR complex. Furthermore, the introduction of CTAB provided unique selectivity for the ligand exchange of Ga(III)-PAR by calf thymus dsDNA over calf thymus ssDNA. This phenomenon offers a novel spectrophotometric sensing strategy for direct detection of dsDNA.

  19. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

    PubMed Central

    Waxman, A B; Einarsson, O; Seres, T; Knickelbein, R G; Warshaw, J B; Johnston, R; Homer, R J; Elias, J A

    1998-01-01

    Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation. PMID:9576762

  20. Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

    PubMed Central

    Poecheim, Johanna; Heuking, Simon; Brunner, Livia; Barnier-Quer, Christophe; Collin, Nicolas; Borchard, Gerrit

    2015-01-01

    Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

  1. Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation.

    PubMed Central

    Révet, B; Brahms, S; Brahms, G

    1995-01-01

    Electron microscopic visualization indicates that the transcription activator NRI (NTRC) binds with exceptional selectivity and efficiency to a sequence-induced superhelical (spiral) segment inserted upstream of the glnA promoter, accounting for its observed ability to substitute for the natural glnA enhancer. The cooperative binding of NRI to the spiral insert leads to protein oligomerization which, at higher concentration, promotes selective coating of the entire superhelical segment with protein. Localization of NRI at apical loops is observed with negatively supercoiled plasmid DNA. With a linear plasmid, bending of DNA is observed. We confirm that NRI is a DNA-bending protein, consistent with its high affinity for spiral DNA. These results prove that spiral DNA without any homology to the NRI-binding sequence site can substitute for the glnA enhancer by promoting cooperative activator binding to DNA and facilitating protein oligomerization. Similar mechanisms might apply to other prokaryotic and eukaryotic activator proteins that share the ability to bend DNA and act efficiently as multimers. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7638226

  2. Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

    PubMed

    Wang, Yuan-Yuan; Chang, Xue-Lian; Tao, Zhi-Yong; Wang, Xiao-Li; Jiao, Yu-Meng; Chen, Yong; Qi, Wen-Juan; Xia, Hui; Yang, Xiao-Di; Sun, Xin; Shen, Ji-Long; Fang, Qiang

    2015-07-01

    Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt‑TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

  3. Clinical characteristics, DNA repair, and complementation groups in xeroderma pigmentosum patients from Egypt.

    PubMed

    Hashem, N; Bootsma, D; Keijzer, W; Greene, A; Coriell, L; Thomas, G; Cleaver, J E

    1980-01-01

    Xeroderma pigmentosum (XP) has been reported to be unusually frequent among Middle Eastern populations. This report describes the first survey of DNA repair characteristics among Egyptians. Sixteen XP patients were contacted, and biopsies from eight were analyzed for unscheduled DNA synthesis, strand breakage during pyrimidine dimer excision, and complementation groups. The patients were equally distributed between Complementation Groups A and C. Unscheduled synthesis and strand breaks were significantly higher in Group C than in Group A cells. Central nervous system disorders were found in all of the Group A patients and in none of the Group C patients. No clinical symptoms were observed in the heterozygotes. A 2-month-old sib of an XP patient was free of symptoms, but unscheduled synthesis and strand breakage in cultures from this sib were the same as in the related XP homozygote. From the relative frequencies of each complementation group found in various parts of the world, we offer a hypothesis concerning the relative sizes and roles for gene products specified by the alleles or genes corresponding to each complementation group.

  4. Enhanced radiation-induced cytotoxic effect by 2-ME in glioma cells is mediated by induction of cell cycle arrest and DNA damage via activation of ATM pathways.

    PubMed

    Zou, Huichao; Zhao, Shiguang; Zhang, Jianhua; Lv, Gongwei; Zhang, Xu; Yu, Hongwei; Wang, Huibo; Wang, Ligang

    2007-12-14

    Glioblastoma multiform is the most common malignant primary brain tumor in adults, but there remains no effective therapeutic approach. 2-methoxyestradiol (2-ME), which is a naturally occurring metabolite of 17beta-estradiol, was shown to enhance radiotherapeutic effect in certain tumors; however, whether 2-ME can also enhance the sensitivity of glioma cells to radiotherapy remains unknown. The present study, therefore, was to address this issue using two human glioma cell lines (T98G and U251MG). These cells were irradiated with and without 2-ME and then clonogenic assay, apoptosis assay, DNA damage, and cell cycle change were examined. Results showed that 2-ME significantly enhances radiation-induced cell death in both glioma cells, shown by decreasing cell viability and increasing apoptotic cell death. No such radiosensitizing effect was observed if cells pre-treated with Estrodiol, suggesting the specifically radiosensitizing effect of 2-ME rather than a general effect of estrodials. The enhanced radio-cytotoxic effect in glioma cells by 2-ME was found to be associated with its enhancement of G(2)/M arrest and DNA damage, and phosphorylated ATM protein kinases as well as cell cycle checkpoint protein Chk2. Furthermore, inhibition of ATM by ATM inhibitor abolished 2-ME-activated Chk2 and enhanced radio-cytotoxic effects. These results suggest that 2-ME enhancement of the sensitivity of glioma cell lines to radiotherapy is mediated by induction of G2/M cell cycle arrest and increased DNA damage via activation of ATM kinases.

  5. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  6. DNA-only cascade: a universal tool for signal amplification, enhancing the detection of target analytes.

    PubMed

    Bone, Simon M; Hasick, Nicole J; Lima, Nicole E; Erskine, Simon M; Mokany, Elisa; Todd, Alison V

    2014-09-16

    Diagnostic tests performed in the field or at the site of patient care would benefit from using a combination of inexpensive, stable chemical reagents and simple instrumentation. Here, we have developed a universal "DNA-only Cascade" (DoC) to quantitatively detect target analytes with increased speed. The DoC utilizes quasi-circular structures consisting of temporarily inactivated deoxyribozymes (DNAzymes). The catalytic activity of the DNAzymes is restored in a universal manner in response to a broad range of environmental and biological targets. The present study demonstrates DNAzyme activation in the presence of metal ions (Pb(2+)), small molecules (deoxyadenosine triphosphate) and nucleic acids homologous to genes from Meningitis-causing bacteria. Furthermore, DoC efficiently discriminates nucleic acid targets differing by a single nucleotide. When detection of analytes is orchestrated by functional nucleic acids, the inclusion of DoC reagents substantially decreases time for detection and allows analyte quantification. The detection of nucleic acids using DoC was further characterized for its capability to be multiplexed and retain its functionality following long-term exposure to ambient temperatures and in a background of complex medium (human serum).

  7. A Family of Potent Ru(II) Photosensitizers with Enhanced DNA Intercalation: Bimodal Photokillers.

    PubMed

    Pefkianakis, Eleftherios K; Theodossiou, Theodossis A; Toubanaki, Dimitra K; Karagouni, Evdokia; Falaras, Polycarpos; Papadopoulos, Kyriakos; Vougioukalakis, Georgios C

    2015-01-01

    A new family of Ru(II)-based photosensitizers was synthesized and systematically characterized. The ligands employed to coordinate the ruthenium metal center were the commercially available 2,2'-bipyridine and a pyridine-quinoline hybrid bearing an anthracene moiety. The complexes obtained carry either PF6- or Cl(-) counterions. These counterions determine the complexes' hydrophobic or hydrophilic character, respectively, therefore dictating their solubility in biologically related media. All photosensitizers exhibit characteristic, relatively strong and wide UV-Vis absorption spectral profiles. Their high efficiency in generating cytotoxic singlet oxygen was established (up to ΦΔ ~0.8). Moreover, the interaction of these photosensitizers with double-stranded DNA was studied fluoro- and photospectroscopically and their binding affinities were found to be of the order of 3 × 10(7)  M(-1) . All complexes are photocytotoxic to DU145 human prostate cancer cells. The highest light-induced toxicity was conferred by the photosensitizers bearing Cl(-) counterions, probably due to the looser ionic "chaperoning" of Cl(-) , in comparison to PF6-, leading to higher cell internalization.

  8. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication

    PubMed Central

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M.; Knapman, Alison; Davies, Rachel

    2016-01-01

    ABSTRACT Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication. IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification

  9. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication.

    PubMed

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M; Knapman, Alison; Davies, Rachel; Androphy, Elliot J; Parish, Joanna L

    2017-03-01

    Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of

  10. Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

    PubMed

    Gargett, Tessa; Grubor-Bauk, Branka; Garrod, Tamsin J; Yu, Wenbo; Miller, Darren; Major, Lee; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

    2014-04-01

    The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

  11. The study of naphthoquinones and their complexes with DNA by using Raman spectroscopy and surface enhanced Raman spectroscopy: new insight into interactions of DNA with plant secondary metabolites.

    PubMed

    Vaverkova, Veronika; Vrana, Oldrich; Adam, Vojtech; Pekarek, Tomas; Jampilek, Josef; Babula, Petr

    2014-01-01

    Naphthoquinones represent the group of plant secondary metabolites with cytotoxic properties based on their ability to generate reactive oxygen species and interfere with the processes of cell respiration. Due to this fact, the possible cytotoxic mechanisms on cellular and subcellular levels are investigated intensively. There are many targets of cytotoxic action on the cellular level; however, DNA is a critical target of many cytotoxic compounds. Due to the cytotoxic properties of naphthoquinones, it is necessary to study the processes of naphthoquinones, DNA interactions (1,4-naphthoquinone, binapthoquinone, juglone, lawsone, plumbagin), especially by using modern analytical techniques. In our work, the Raman spectroscopy was used to determine the possible binding sites of the naphthoquinones on the DNA and to characterize the bond of naphthoquinone to DNA. Experimental data reveals the relationships between the perturbations of structure-sensitive Raman bands and the types of the naphthoquinones involved. The modification of DNA by the studied naphthoquinones leads to the nonspecific interaction, which causes the transition of B-DNA into A-DNA conformation. The change of the B-conformation of DNA for all measured DNA modified by naphthoquinones except plumbagin is obvious.

  12. The Study of Naphthoquinones and Their Complexes with DNA by Using Raman Spectroscopy and Surface Enhanced Raman Spectroscopy: New Insight into Interactions of DNA with Plant Secondary Metabolites

    PubMed Central

    Vrana, Oldrich; Adam, Vojtech

    2014-01-01

    Naphthoquinones represent the group of plant secondary metabolites with cytotoxic properties based on their ability to generate reactive oxygen species and interfere with the processes of cell respiration. Due to this fact, the possible cytotoxic mechanisms on cellular and subcellular levels are investigated intensively. There are many targets of cytotoxic action on the cellular level; however, DNA is a critical target of many cytotoxic compounds. Due to the cytotoxic properties of naphthoquinones, it is necessary to study the processes of naphthoquinones, DNA interactions (1,4-naphthoquinone, binapthoquinone, juglone, lawsone, plumbagin), especially by using modern analytical techniques. In our work, the Raman spectroscopy was used to determine the possible binding sites of the naphthoquinones on the DNA and to characterize the bond of naphthoquinone to DNA. Experimental data reveals the relationships between the perturbations of structure-sensitive Raman bands and the types of the naphthoquinones involved. The modification of DNA by the studied naphthoquinones leads to the nonspecific interaction, which causes the transition of B-DNA into A-DNA conformation. The change of the B-conformation of DNA for all measured DNA modified by naphthoquinones except plumbagin is obvious. PMID:25045679

  13. Base pair sensitivity and enhanced ON/OFF ratios of DNA-binding: donor-acceptor-donor fluorophores.

    PubMed

    Wilson, James N; Wigenius, Jens; Pitter, Demar R G; Qiu, Yanhua; Abrahamsson, Maria; Westerlund, Fredrik

    2013-10-10

    The photophysical properties of two recently reported live cell compatible, DNA-binding dyes, 4,6-bis(4-(4-methylpiperazin-1-yl)phenyl)pyrimidin-2-ol, 1, and [1,3-bis[4-(4-methylpiperazin-1-yl)phenyl]-1,3-propandioato-κO, κO']difluoroboron, 2, are characterized. Both dyes are quenched in aqueous solutions, while binding to sequences containing only AT pairs enhances the emission. Binding of the dyes to sequences containing only GC pairs does not produce a significant emission enhancement, and for sequences containing both AT and GC base pairs, emission is dependent on the length of the AT pair tracts. Through emission lifetime measurements and analysis of the dye redox potentials, photoinduced electron transfer with GC pairs is implicated as a quenching mechanism. Binding of the dyes to AT-rich regions is accompanied by bathochromic shifts of 26 and 30 nm, respectively. Excitation at longer wavelengths thus increases the ON/OFF ratio of the bound probes significantly and provides improved contrast ratios in solution as well as in fluorescence microscopy of living cells.

  14. Chromium(VI) at plausible drinking water concentrations is not genotoxic in the in vivo bone marrow micronucleus or liver unscheduled DNA synthesis assays

    SciTech Connect

    Mirsalis, J.C.; Hamilton, C.M.; O`Loughlin, K.G.

    1996-12-31

    This study examines the potential in vivo genotoxicity of Chromium(VI) in drinking water at concentrations ranging from the relevant human exposure level of 1 mg/l to the upper limit of palatability in rodents (20mg/l).

  15. Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

    PubMed Central

    Gagnon, David; Lehoux, Michaël

    2015-01-01

    ABSTRACT The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the

  16. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system.

    PubMed

    Meerak, Jomkhwan; Wanichwecharungruang, Supason P; Palaga, Tanapat

    2013-01-21

    DNA vaccines are a promising new generation of vaccines that can elicit an immune response using DNA encoding the antigen of interest. The efficacy of these vaccines, however, still needs to be improved. In this study, we investigated the effect of autophagy on increasing the efficacy of a candidate DNA vaccine against Mycobacterium tuberculosis (MTB), a causative agent of tuberculosis. Low molecular weight chitosan was used to encapsulate plasmid DNA containing a gene encoding MTB Antigen 85B (Ag85B), a secreted fibronectin-binding protein. To induce autophagy upon DNA vaccination, the kinase defective mTOR (mTOR-KD) was transfected into cells, and autophagy was detected based on the presence of LC3II. To investigate whether autophagy enhances an immune response upon DNA vaccination, we coencapsualted the Ag85B-containing plasmid with a plasmid encoding mTOR-KD. Plasmids encapsulated by chitosan particles were used for primary subcutaneous immunization and for intranasal boost in mice. After the boost vaccination, sera from the mice were measured for humoral immune response. The DNA vaccine with the autophagy-inducing construct elicited significantly higher Ag85B-specific antibody levels than the control group treated with the Ag85B plasmid alone or with the Ag85B plasmid plus the wild type mTOR construct. Upon in vitro stimulation of splenocytes from mice immunized with recombinant Ag85B, the highest levels of secreted IFN-γ and IL-2 were detected in mice immunized with the autophagy-inducing plasmid, while no differences in IL-4 levels were detected between the groups, suggesting that the DNA vaccine regimen with autophagy induction induced primarily a Th1 immune response. Furthermore, the enhanced proliferation of CD4+ T cells from mice receiving the autophagy-inducing vaccine was observed in vitro. Based on the evidence presented, we conclude that incorporating an autophagy-inducing element into a DNA vaccine may help to improve immune response.

  17. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation

    PubMed Central

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    2016-01-01

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents. PMID:27833761

  18. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation.

    PubMed

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    2016-01-01

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents.

  19. Surface-enhanced Raman spectroscopy of genomic DNA from in vitro grown tomato (Lycopersicon esculentum Mill.) cultivars before and after plant cryopreservation

    NASA Astrophysics Data System (ADS)

    Muntean, Cristina M.; Leopold, Nicolae; Tripon, Carmen; Coste, Ana; Halmagyi, Adela

    2015-06-01

    In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm-1. Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2‧-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm-1, being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy.

  20. Surface-enhanced Raman spectroscopy of genomic DNA from in vitro grown tomato (Lycopersicon esculentum Mill.) cultivars before and after plant cryopreservation.

    PubMed

    Muntean, Cristina M; Leopold, Nicolae; Tripon, Carmen; Coste, Ana; Halmagyi, Adela

    2015-06-05

    In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm(-1). Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2'-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm(-1), being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy.

  1. Surface-enhanced Raman spectroscopy for DNA detection by the self-assembly of Ag nanoparticles onto Ag nanoparticle-graphene oxide nanocomposites.

    PubMed

    Lin, Tsung-Wu; Wu, Hong-Yi; Tasi, Ting-Ti; Lai, Ying-Huang; Shen, Hsin-Hui

    2015-07-28

    A novel surface-enhanced Raman scattering (SERS) sensing system which operates by the self-assembly of Ag nanoparticles (AgNPs) onto the nanocomposite of AgNPs and graphene oxide (AgNP-GO) in the presence of two complementary DNAs has been developed. In this system, AgNP-GO serves as a SERS-active substrate. The AgNPs with the modification of non-fluorescent 4-mercaptobenzoic acid (4-MBA) act as highly efficient Raman probes for DNA hybridization. When probe DNAs on AgNP-GO are complementary to target DNAs on AgNPs functionalized with 4-MBA, the DNA hybridization occurring directs the self-assembly of AgNPs onto AgNP-GO, leading to the creation of SERS hot spots. Due to the fact that partial 4-MBA molecules are located in the region of the hot spots, their SERS signals are greatly enhanced, indicating successful DNA hybridization. It is noteworthy that the size of AgNPs contributes significantly to the enhancement of SERS activity. The detection limit of the target DNAs at the pM level can be achieved through the self-assembly of large sized AgNPs onto AgNP-GO. More importantly, the AgNP-AgNP-GO system shows reproducible SERS signals in proportion to the logarithm of the target DNA concentrations spanning from 10(-6) to 10(-12) M and the excellent capability for multiplex DNA detection.

  2. Internal-Modified Dithiol DNA-Directed Au Nanoassemblies: Geometrically Controlled Self-Assembly and Quantitative Surface-Enhanced Raman Scattering Properties

    NASA Astrophysics Data System (ADS)

    Yan, Yuan; Shan, Hangyong; Li, Min; Chen, Shu; Liu, Jianyu; Cheng, Yanfang; Ye, Cui; Yang, Zhilin; Lai, Xuandi; Hu, Jianqiang

    2015-11-01

    In this work, a hierarchical DNA-directed self-assembly strategy to construct structure-controlled Au nanoassemblies (NAs) has been demonstrated by conjugating Au nanoparticles (NPs) with internal-modified dithiol single-strand DNA (ssDNA) (Au-B-A or A-B-Au-B-A). It is found that the dithiol-ssDNA-modified Au NPs and molecule quantity of thiol-modified ssDNA grafted to Au NPs play critical roles in the assembly of geometrically controlled Au NAs. Through matching Au-DNA self-assembly units, geometrical structures of the Au NAs can be tailored from one-dimensional (1D) to quasi-2D and 2D. Au-B-A conjugates readily give 1D and quasi-2D Au NAs while 2D Au NAs can be formed by A-B-Au-B-A building blocks. Surface-enhanced Raman scattering (SERS) measurements and 3D finite-difference time domain (3D-FDTD) calculation results indicate that the geometrically controllable Au NAs have regular and linearly “hot spots”-number-depended SERS properties. For a certain number of NPs, the number of “hot spots” and accordingly enhancement factor of Au NAs can be quantitatively evaluated, which open a new avenue for quantitative analysis based on SERS technique.

  3. Internal-Modified Dithiol DNA-Directed Au Nanoassemblies: Geometrically Controlled Self-Assembly and Quantitative Surface-Enhanced Raman Scattering Properties.

    PubMed

    Yan, Yuan; Shan, Hangyong; Li, Min; Chen, Shu; Liu, Jianyu; Cheng, Yanfang; Ye, Cui; Yang, Zhilin; Lai, Xuandi; Hu, Jianqiang

    2015-11-19

    In this work, a hierarchical DNA-directed self-assembly strategy to construct structure-controlled Au nanoassemblies (NAs) has been demonstrated by conjugating Au nanoparticles (NPs) with internal-modified dithiol single-strand DNA (ssDNA) (Au-B-A or A-B-Au-B-A). It is found that the dithiol-ssDNA-modified Au NPs and molecule quantity of thiol-modified ssDNA grafted to Au NPs play critical roles in the assembly of geometrically controlled Au NAs. Through matching Au-DNA self-assembly units, geometrical structures of the Au NAs can be tailored from one-dimensional (1D) to quasi-2D and 2D. Au-B-A conjugates readily give 1D and quasi-2D Au NAs while 2D Au NAs can be formed by A-B-Au-B-A building blocks. Surface-enhanced Raman scattering (SERS) measurements and 3D finite-difference time domain (3D-FDTD) calculation results indicate that the geometrically controllable Au NAs have regular and linearly "hot spots"-number-depended SERS properties. For a certain number of NPs, the number of "hot spots" and accordingly enhancement factor of Au NAs can be quantitatively evaluated, which open a new avenue for quantitative analysis based on SERS technique.

  4. Surface-enhanced Raman scattering detection of DNA derived from the West Nile virus genome using magnetic capture of Raman-active gold nanoparticles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A model paramagnetic nanoparticle (MNP) assay is demonstrated for surface-enhanced Raman scattering (SERS) detection of DNA oligonucleotides derived from the West Nile virus (WNV) genome. Detection is based on the capture of WNV target sequences by hybridization with complementary oligonucleotide pr...

  5. Cloning and mapping of a novel human cDNA homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene

    SciTech Connect

    Isomura, Minoru; Okui, Keiko; Nakamura, Yusuke

    1996-02-15

    This article reports on the isolation and localization to human chromosome 7q34 of a human cDNA clone that encodes a protein which is homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene. The structure and expression of this gene is also discussed. 12 refs., 3 figs.

  6. Virus-Surface-Mimicking Surface Clustering of AuNPs onto DNA-Entrapped Polymeric Nanoparticle for Enhanced Cellular Internalization and Nanocluster-Induced NIR Photothermal Therapy.

    PubMed

    Jia, Hui-Zhen; Chen, Wei-Hai; Wang, Xuli; Lei, Qi; Yin, Wei-Na; Wang, Yan; Zhuo, Ren-Xi; Feng, Jun; Zhang, Xian-Zheng

    2015-12-01

    Virus-surface-mimicking decoration of deoxyribonucleic acid (DNA)-entrapped polymeric nanoparticle with AuNPs is demonstrated to lead to enhanced cellular uptake, improved gene transfection, and particularly efficient near-infrared photothermal therapy that cannot be achieved by both of them separately. This hybrid nanosystem represents a novel paradigm of multipurpose organic-inorganic nanoplatform, especially for cancer treatments.

  7. p16(INK4A) enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction.

    PubMed

    Al-Khalaf, Huda H; Nallar, Shreeram C; Kalvakolanu, Dhananjaya V; Aboussekhra, Abdelilah

    2017-02-20

    p16(INK4A) and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16(INK4A) and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16(INK4A) enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16(INK4A) and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16(INK4A) mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16(INK4A) . Furthermore, the association between p16(INK4A) and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16(INK4A) and p53.

  8. Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

    PubMed Central

    Holgado, María Pía; Falivene, Juliana; Gherardi, María Magdalena

    2014-01-01

    Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses. PMID:25215887

  9. Synthesis and evaluation of DNA-targeted spatially separated bis(aniline mustards) as potential alkylating agents with enhanced DNA cross-linking capability.

    PubMed

    Gourdie, T A; Prakash, A S; Wakelin, L P; Woodgate, P D; Denny, W A

    1991-01-01

    DNA-targeted separated bis-mustards were synthesized by attaching aniline mono-mustards at the 4- and 9-positions of the DNA-intercalating ligand 9-aminoacridine-4-carboxamide, with the intention of improving the low cross-link to monoadduct ratio found with most alkylating agents. The geometry of these compounds requires that, when the acridine binds to DNA by intercalation, one alkylating moiety is delivered to each DNA groove. Gel electrophoretic studies show that only one arm of these compounds (probably that attached to the 9-position) alkylates DNA, such alkylation occurring specifically in the major groove at the N7 of guanines. Cell-line studies confirm that the mode of cytotoxicity of these compounds (unlike that of untargeted aniline bis-mustards of comparable reactivity) is due to bulky DNA monoadduct formation. It is concluded that more information is required about the exact orientation of the initial monoadducts before ligands with specific DNA cross-linking ability can be designed.

  10. Enhanced DNA repair of bleomycin-induced 3'-phosphoglycolate termini at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-11-01

    The anti-tumour agent, bleomycin, cleaves DNA to give 3'-phosphoglycolate and 5'-phosphate termini. The removal of 3'-phosphoglycolate to give 3'-OH ends is a very important step in the DNA repair of these lesions. In this study, next-generation DNA sequencing was utilised to investigate the repair of these 3'-phosphoglycolate termini at the transcription start sites (TSSs) of genes in HeLa cells. The 143,600 identified human TSSs in HeLa cells comprised 82,596 non-transcribed genes and 61,004 transcribed genes; and the transcribed genes were divided into quintiles of 12,201 genes comprising the top 20%, 20-40%, 40-60%, 60-80%, 80-100% of expressed genes. Repair of bleomycin-induced 3'-phosphoglycolate termini was enhanced at actively transcribed genes. The top 20% and 20-40% quintiles had a very similar level of enhanced repair, the 40-60% quintile was intermediate, while the 60-80% and 80-100% quintiles were close to the low level of enhancement found in non-transcribed genes. There were also interesting differences regarding bleomycin repair on the sense and antisense strands of DNA at TSSs. The sense strand had highly enhanced repair between 0 and 250bp relative to the TSS, while for the antisense strand highly enhanced repair was between 150 and 450bp. Repair of DNA damage is a major mechanism of resistance to anti-tumour drugs and this study provides an insight into this process in human tumour cells.

  11. Adaptation and Impairment of DNA Repair Function in Pollen of Betula verrucosa and Seeds of Oenothera biennis from Differently Radionuclide-contaminated Sites of Chernobyl

    PubMed Central

    Boubriak, I. I.; Grodzinsky, D. M.; Polischuk, V. P.; Naumenko, V. D.; Gushcha, N. P.; Micheev, A. N.; McCready, S. J.; Osborne, D. J.

    2008-01-01

    Background and Aims The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to α-, β- and γ-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. Methods Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. Key Results Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at γ/β-emitter sites has now recovered this ability. At a site with high levels of combined α- and γ/β-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same γ/β-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined α- and γ/β-contaminated site do not show this improvement. Conclusions Chronic irradiation at γ/β-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function. PMID:17981881

  12. Two-dimensional micro-bubble actuator array to enhance the efficiency of molecular beacon based DNA micro-biosensors.

    PubMed

    Deng, Peigang; Lee, Yi-Kuen; Cheng, Ping

    2006-02-15

    Two-dimensional micro-bubble actuator arrays were developed and studied in detail to enhance the hybridization kinetics of a DNA micro-biosensor. The hybridization between a molecular beacon, a kind of oligonucleotide probe, and its complement was investigated in a millimeter-sized PDMS based reaction chamber, where various 2D micro-heater arrays were distributed on the bottom for micro-bubble generation. The hybridization assay without the micro-bubble actuation revealed that the fluorescence increased fast at the beginning and slowed down after that. However, a uniform fluorescence increase was observed when periodic micro-bubble agitation was introduced in the static hybridization solution. A comparison of hybridization assays with and without micro-bubble agitation revealed that the hybridization time could be effectively shortened by 33% with 10 cycles of micro-bubble agitation from a 2 x 1 bubble actuator array, and by 43% with 10 cycles of micro-bubble agitation from a 2 x 2 bubble actuator array.

  13. Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots.

    PubMed

    Atkinson, C; Emery, V C; Griffiths, P D

    2014-02-01

    Newborn screening for congenital cytomegalovirus (CCMV) using dried blood spots (DBS) has been proposed because many developed countries have DBS screening programmes in place for other diseases. The aim of this study was to develop a rapid, single tube nested polymerase chain reaction (PCR) method for enhanced detection of CMV from DBS compared to existing (single target) real time PCRs. The new method was compared with existing real time PCRs for sensitivity and specificity. Overall sensitivity of the single target PCR assays in both asymptomatic and symptomatic infants with laboratory confirmed congenital CMV was 69% (CMV PCR or culture positive before day 21 of life). In contrast, the single tube nested assay had an increased sensitivity of 81% with100% specificity. Overall the assay detected CMV from a DBS equivalent to an original blood sample which contained 500IU/ml. In conclusion this single tube nested methodology allows simultaneous amplification and detection of CMV DNA in 1.5h removing the associated contamination risk of a two step nested PCR. Owing to its increased sensitivity, it has the potential to be used as a screening assay and ultimately allow early identification and intervention for children with congenital CMV.

  14. A Protective Mechanism of Visible Red Light in Normal Human Dermal Fibroblasts: Enhancement of GADD45A-Mediated DNA Repair Activity.

    PubMed

    Kim, Yeo Jin; Kim, Hyoung-June; Kim, Hye Lim; Kim, Hyo Jeong; Kim, Hyun Soo; Lee, Tae Ryong; Shin, Dong Wook; Seo, Young Rok

    2017-02-01

    The phototherapeutic effects of visible red light on skin have been extensively investigated, but the underlying biological mechanisms remain poorly understood. We aimed to elucidate the protective mechanism of visible red light in terms of DNA repair of UV-induced oxidative damage in normal human dermal fibroblasts. The protective effect of visible red light on UV-induced DNA damage was identified by several assays in both two-dimensional and three-dimensional cell culture systems. With regard to the protective mechanism of visible red light, our data showed alterations in base excision repair mediated by growth arrest and DNA damage inducible, alpha (GADD45A). We also observed an enhancement of the physical activity of GADD45A and apurinic/apyrimidinic endonuclease 1 (APE1) by visible red light. Moreover, UV-induced DNA damages were diminished by visible red light in an APE1-dependent manner. On the basis of the decrease in GADD45A-APE1 interaction in the activating transcription factor-2 (ATF2)-knockdown system, we suggest a role for ATF2 modulation in GADD45A-mediated DNA repair upon visible red light exposure. Thus, the enhancement of GADD45A-mediated base excision repair modulated by ATF2 might be a potential protective mechanism of visible red light.

  15. Nicotinamide enhances repair of arsenic and ultraviolet radiation-induced DNA damage in HaCaT keratinocytes and ex vivo human skin.

    PubMed

    Thompson, Benjamin C; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

  16. Enhanced thermal stability and mismatch discrimination of mutation-carrying DNA duplexes and their kinetic and thermodynamic properties in microchannel laminar flow.

    PubMed

    Nagata, Maria Portia B; Yamashita, Kenichi; Miyazaki, Masaya; Nakamura, Hiroyuki; Maeda, Hideaki

    2009-07-01

    This article reports the enhancement of thermal stability involving normal duplex and mutation-carrying DNA duplexes in microchannel laminar flow. The application of an in-house temperature-controllable microchannel-type flow cell is demonstrated for improved discrimination of mismatch base pairs such as A-G and T-G that are difficult to distinguish due to the rather small thermal destabilizations. Enhancement in thermal stability is reflected by an increased thermal melting temperature achieved in microchannel laminar flow as compared with batch reactions. To examine the kinetics and thermodynamics of duplex-coil equilibrium of DNA oligomers, denaturation-renaturation hysteresis curves were measured. The influence of microchannel laminar flow on DNA base mismatch analysis was described from the kinetic and thermodynamic perspectives. An increasing trend was observed for association rate constant as flow rate increased. In contrast, an apparent decrease in dissociation rate constant was observed with increasing flow rate. The magnitudes of the activation energies of dissociation were nearly constant for both the batch and microchannel laminar flow systems at all flow rates. In contrast, the magnitudes of activation energies of association decreased as flow rate increased. These results clearly show how microchannel laminar flow induces change in reaction rate by effecting change in activation energy. We anticipate, therefore, that this approach based on microchannel laminar flow system holds great promise for improved mismatch discrimination in DNA analyses, particularly on single-base-pair mismatch, by pronouncedly enhancing thermal stability.

  17. In vivo stage- and tissue-specific DNA-protein interactions at the D. melanogaster alcohol dehydrogenase distal promoter and adult enhancer.

    PubMed Central

    Jackson, J R; Benyajati, C

    1992-01-01

    We performed a high resolution analysis of the chromatin structure within the regions required for distal transcription of the Drosophila melanogaster alcohol dehydrogenase gene (Adh). Using dimethyl sulfate, DNase I, and micrococcal nuclease as structural probes, and comparing chromatin structure in tissues isolated from several developmental stages, we have identified several sites of stage- and tissue-specific DNA-protein interactions that correlate with distal transcription initiation. Most were within previously identified cis-acting elements and/or in vitro protein binding sites of the adult enhancer (AAE) and distal promoter, including the TATA box. We also detected a novel stage-specific DNA-protein interaction at the Adf-2a binding site where a non-histone protein was bound to the DNA on the surface of a positioned nucleosome previously identified between the distal promoter and adult enhancer. In addition to footprints, we have also revealed stage- and tissue-specific DNA helix deformations between many of the non-histone protein binding sites. These helix distortions suggest there are interactions among the adjacently bound proteins that result in bending or kinking of the intervening DNA. The distal promoter and AAE have an accessible chromatin conformation in fat body prior to the third larval instar and many of the regulatory proteins that bind in these regions are also available before distal transcription begins. Nevertheless, the timing of DNA-protein interactions in the distal promoter and AAE suggest these proteins do not bind individually or assemble progressively as they and their binding sites become available. Instead, there appears to be a coordinated assembly of a large cooperative complex of proteins interacting with the distal promoter, the positioned nucleosome, the enhancer of the distal promoter (the AAE), and each other. Images PMID:1437559

  18. PAMAM-Lys, a Novel Vaccine Delivery Vector, Enhances the Protective Effects of the SjC23 DNA Vaccine against Schistosoma japonicum Infection

    PubMed Central

    Wang, Xiaoting; Dai, Yang; Zhao, Song; Tang, Jianxia; Li, Hongjun; Xing, Yuntian; Qu, Guoli; Li, Xinsong; Dai, Jianrong; Zhu, Yinchang; Zhang, Xueguang

    2014-01-01

    Background Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection. Methodology/Principal Findings Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ. Conclusion/Significance Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection. PMID:24497955

  19. Enhanced in vivo gene expression mediated by listeriolysin O incorporated anionic LPDII: Its utility in cytotoxic T lymphocyte-inducing DNA vaccine.

    PubMed

    Sun, Xun; Provoda, Chester; Lee, Kyung-Dall

    2010-12-01

    Enhanced in vivo gene expression using non-viral vectors is a critical issue in gene therapy in general. Among the many potential utilities of non-viral vector-mediated gene delivery, its application in DNA-based vaccination is an attractive approach with several practical advantages over conventional vaccination. We have previously shown that the endosomolytic bacterial protein listeriolysin O (LLO) is capable of facilitating transfection in vitro using the LPDII (anionic liposome-polycation-DNA complexes) delivery system. In the present study we have extended and investigated the DNA delivery of LLO-containing LPDII to in vivo and evaluated its utility in DNA vaccination in mice. We further investigated the ability of this non-viral gene delivery system to elicit an immune response to a model antigen ovalbumin (OVA), particularly focusing on the OVA-specific CD8(+) cytotoxic T lymphocyte (CTL) response, after delivery of a plasmid containing the OVA cDNA. A DNA prime and protein boost protocol was employed to generate cytotoxic T cell responses. Our results show that increased in vitro and in vivo transfection efficiencies were observed when LLO was incorporated into LPDII. This LLO-LPDII formulation produced an enhanced functional antigen-specific CD8(+) T cell response in vivo compared to the heat-inactivated LLO-containing LPDII (HI-LLO-LPDII) formulation. Furthermore, a significantly higher CTL frequency was observed in the splenocytes isolated from the mice primed with LLO-LPDII by an enzyme-linked immunosorbent spot assay. Interferon-γ production upon specific stimulation by OVA-specific CD8(+) peptide was also significantly stronger with the inclusion of LLO into LPDII. These findings suggest that the LLO-containing LPDII system possesses noteworthy potential as a candidate carrier for DNA vaccine delivery.

  20. HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.

    PubMed

    Wagner, S; Green, M R

    1993-10-15

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  1. Enhanced suppression of tumor growth using a combination of NK4 plasmid DNA-PEG engrafted cationized dextran complex and ultrasound irradiation.

    PubMed

    Hosseinkhani, H; Kushibiki, T; Matsumoto, K; Nakamura, T; Tabata, Y

    2006-05-01

    This investigation aims to determine experimentally whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of NK4 plasmid DNA and suppressing tumor growth. NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow for polyionic complexation with NK4 plasmid DNA. The cationized dextran was additionally modified with poly(ethylene glycol) (PEG) molecules giving PEG engrafted cationized dextran. Significant suppression of tumor growth was observed when PEG engrafted cationized dextran-NK4 plasmid DNA complexes were intravenously injected into mice carrying a subcutaneous Lewis lung carcinoma tumor mass with subsequent US irradiation when compared with the cationized dextran-NK4 plasmid DNA complex and naked NK4 plasmid DNA with or without US irradiation. We conclude that complexation with PEG-engrafted cationized dextran in combination with US irradiation is a promising way to target the NK4 plasmid DNA to the tumor for gene expression.

  2. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

    PubMed Central

    Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

    2017-01-01

    Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

  3. Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation.

    PubMed

    Yang, Lina; Liu, Yuanyuan; Sun, Chao; Yang, Xinrui; Yang, Zhen; Ran, Juntao; Zhang, Qiuning; Zhang, Hong; Wang, Xinyu; Wang, Xiaohu

    2015-11-01

    Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G2/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5-50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of

  4. Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

    PubMed

    Saribas, A Sami; Mun, Sarah; Johnson, Jaslyn; El-Hajmoussa, Mohammad; White, Martyn K; Safak, Mahmut

    2014-01-20

    JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

  5. The dual topoisomerase inhibitor A35 preferentially and specially targets topoisomerase 2α by enhancing pre-strand and post-strand cleavage and inhibiting DNA religation

    PubMed Central

    Bi, Chongwen; Li, Yangbiao; Liu, Jingbo; Ye, Cheng; He, Hongwei; Li, Liang

    2015-01-01

    DNA topoisomerases play a key role in tumor proliferation. Chemotherapeutics targeting topoisomerases have been widely used in clinical oncology, but resistance and side effects, particularly cardiotoxicity, usually limit their application. Clinical data show that a decrease in topoisomerase (top) levels is the primary factor responsible for resistance, but in cells there is compensatory effect between the levels of top1 and top2α. Here, we validated cyclizing-berberine A35, which is a dual top inhibitor and preferentially targets top2α. The impact on the top2α catalytic cycle indicated that A35 could intercalate into DNA but did not interfere with DNA-top binding and top2α ATPase activity. A35 could facilitate DNA-top2α cleavage complex formation by enhancing pre-strand and post-strand cleavage and inhibiting religation, suggesting this compound can be a topoisomerase poison and had a district mechanism from other topoisomerase inhibitors. TARDIS and comet assays showed that A35 could induce cell DNA breakage and DNA-top complexes but had no effect on the cardiac toxicity inducer top2β. Silencing top1 augmented DNA break and silencing top2α decreased DNA break. Further validation in H9c2 cardiac cells showed A35 did not disturb cell proliferation and mitochondrial membrane potency. Additionally, an assay with nude mice further demonstrated A35 did not damage the heart. Our work identifies A35 as a novel skeleton compound dually inhibits topoisomerases, and predominantly and specially targets top2α by interfering with all cleavage steps and its no cardiac toxicity was verified by cardiac cells and mice heart. A35 could be a novel and effective targeting topoisomerase agent. PMID:26462155

  6. /sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications

    SciTech Connect

    Reddy, M.V.; Randerath, K.

    1987-12-01

    Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A /sup 32/P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10/sup 7/ to 10/sup 8/ nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are /sup 32/P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed (/sup 32/P) phosphate transfer from (..gamma..-/sup 32/P)ATP. /sup 32/P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P/sub 1/. The latter observation has been utilized to enhance the /sup 32/P-assay's sensitivity to 1 adduct in 10/sup 10/ nucleotides for a 10-..mu..g DNA sample by postincubation of DNA digests with nuclease P/sup 1/ before /sup 32/P-labeling. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and humans.

  7. Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

    NASA Astrophysics Data System (ADS)

    Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

    2015-02-01

    A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

  8. Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase.

    PubMed

    Aarattuthodiyil, Suja; Byrd, Alicia K; Raney, Kevin D

    2014-10-01

    Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.

  9. Cooperative effect of BI-69A11 and celecoxib enhances radiosensitization by modulating DNA damage repair in colon carcinoma.

    PubMed

    Pal, Ipsita; Dey, Kaushik Kumar; Chaurasia, Madhuri; Parida, Sheetal; Das, Subhayan; Rajesh, Y; Sharma, Kulbhushan; Chowdhury, Tamohan; Mandal, Mahitosh

    2016-05-01

    Amplification of PI3K-Akt pathway promotes radioresistance in various cancers including colorectal carcinoma. Local recurrence in colon cancer causes poor prognosis affecting overall survival of cancer-affected patient population. To avoid local recurrence, pre-operative or post-operative additional radiotherapy is given. However, main concern regarding radiotherapy is to increase the radiosensitivity of malignant cell without hampering the activities of normal cells. In this context, addition of two or more than two chemotherapeutic drugs as a radiosensitizer is a common practice in radiation biology. BI-69A11 earlier showed potential apoptosis-inducing effect in melanoma and colon carcinoma. Celecoxib showed anti-cancer effects in both COX-2 dependent and independent pathways and used to act as a radiosensitizing enhancer. Here, we suggest that the combination of BI-69A11 and celecoxib inhibits the phosphorylation of ataxia telangiectasia mutated (ATM) kinase and DNA-PK responsible for ionizing radiation (IR)-induced double-strand break (DSB) repair. Moreover, the combinatorial effect of BI-69A11 and celecoxib attenuates the IR-induced G2/M cell cycle arrest. Furthermore, this combination also impairs IR-induced activation of Akt and downstream targets of ATM. This might lead to induced activation of apoptotic pathway after triple therapy treatment modulating pro-apoptotic and anti-apoptotic proteins. This activation of apoptotic pathway also showed the interdependence of PUMA and BAD in triple combination-treated colon cancer cells in a p53 independent manner. This study reveals the therapeutic potential of the triple combination therapy in prevention of radioresistance. Besides, it also demonstrates the cytotoxic effects of triple combination therapy in colon cancer. This study shows utility and potential implication on safety of the patients undergoing radiation therapy.

  10. Overexpression of the scaffold WD40 protein WRAP53β enhances the repair of and cell survival from DNA double-strand breaks

    PubMed Central

    Rassoolzadeh, H; Böhm, S; Hedström, E; Gad, H; Helleday, T; Henriksson, S; Farnebo, M

    2016-01-01

    Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells. PMID:27310875

  11. Camptothecin enhances the frequency of oligonucleotide-directed gene repair in mammalian cells by inducing DNA damage and activating homologous recombination.

    PubMed

    Ferrara, Luciana; Kmiec, Eric B

    2004-01-01

    Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.

  12. Enhancing capacitive DNA biosensor performance by target overhang with application on screening test of HLA-B*58:01 and HLA-B*57:01 genes.

    PubMed

    Thipmanee, Orawan; Numnuam, Apon; Limbut, Warakorn; Buranachai, Chittanon; Kanatharana, Proespichaya; Vilaivan, Tirayut; Hirankarn, Nattiya; Thavarungkul, Panote

    2016-08-15

    A highly sensitive label-free DNA biosensor based on PNA probes immobilized on a gold electrode was used to detect a hybridization event. The effect of a target DNA overhang on the hybridization efficiency was shown to enhance the detected signal and allowed detection at a very low concentration. The sensors performances were investigated with a complementary target that had the same length as the probe, and the signal was compared to the target DNAs with different lengths and overhangs. A longer target DNA overhang was found to provide a better response. When the overhang was on the electrode side the signal enhancement was greater than when the overhang was on the solution side due to the increased thickness of the sensing surface, hence produced a larger capacitance change. Using conformationally constrained acpcPNA probes, double stranded DNA was detected sensitively and specifically without any denaturing step. When two acpcPNA probes were applied for the screening test for the double stranded HLA-B*58:01 and HLA-B*57:01 genes that are highly similar, the method differentiated the two genes in all samples. Both purified and unpurified PCR products gave comparable results. This method would be potentially useful as a rapid screening test without the need for purification and denaturation of the PCR products.

  13. Identification of the 187 bp EphA7 Genomic DNA as the Dorsal Midline-Specific Enhancer of the Diencephalon and Mesencephalon

    PubMed Central

    Kim, Yujin; Park, Eunjeong; Park, Soochul

    2015-01-01

    EphA7 is a key molecule in regulating the development of the dien- and mesencephalon. To get insight into the mechanism of how EphA7 gene expression is regulated during the dorsal specification of the dien- and mesencephalon, we investigated the cis-acting regulatory sequence driving EphA7 to the dorsal midline of the dien- and mesencephalon. Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb. Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7. Importantly, our results indicate that the 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon. PMID:26537192

  14. Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments

    PubMed Central

    Lee, Wonbae; von Hippel, Peter H.; Marcus, Andrew H.

    2014-01-01

    DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems. PMID:24627223

  15. Rapid renaturation of complementary DNA strands mediated by cationic detergents: a role for high-probability binding domains in enhancing the kinetics of molecular assembly processes.

    PubMed Central

    Pontius, B W; Berg, P

    1991-01-01

    The rate of renaturation for complementary DNA strands can be enhanced greater than 10(4)-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein A1 protein. Under optimal conditions, renaturation rates are greater than 2000-fold faster than reactions run in 1 M NaCl at 68 degrees C. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stabilizing properties. The reaction is also extremely rapid in the presence of up to a 10(6)-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover, this work provides further support for a model relating the existence of flexible, weakly interacting, repeating domains to their function in rapid molecular assembly processes in vitro and in vivo. PMID:1896475

  16. Two design strategies for enhancement of multilayer–DNA-origami folding: underwinding for specific intercalator rescue and staple-break positioning

    PubMed Central

    Ke, Yonggang; Bellot, Gaëtan; Voigt, Niels V.; Fradkov, Elena; Shih, William M.

    2012-01-01

    Single-layer DNA origami is an efficient method for programmable self-assembly of nanostructures approximating almost any desired two-dimensional shape from ~5 MDa of DNA building material. In this method, a 7 kilobase single “scaffold” strand is assembled with hundreds of oligodeoxyribonucleotide “staple” strands to form a parallel array of double helices. Multiple layers of such DNA sheets also can be designed to assemble into a stack, enabling construction of solid three-dimensional shapes with considerably greater mechanical rigidity than two-dimensional shapes; however, the folding yield often is much lower and the required folding times are much longer. Here we introduce two strategies for designing multi-layer DNA origami that demonstrate potential for boosting assembly yield: (1) individual base pairs can be inserted between crossovers, allowing for greater bowing of helices at positions away from crossovers and therefore reduced electrostatic repulsion. At the same time, this underwinding of double helices increases a destabilizing torsional strain energy but then also increases affinity for intercalators, and binding of such intercalators can relieve this stress. We also have exploited this enhanced affinity for intercalators to PEGylate the surface of the nanostructures in a noncovalent fashion using PEG-tris-acridine. (2) Positioning of staple-strand breaks in the DNA origami such that each staple strand includes a 14 nucleotide (nt) continuous segment that binds to a complementary 14 nt continuous segment of the scaffold can greatly improve folding yields. PMID:24653832

  17. Core-shell magnetite-silica composite nanoparticles enhancing DNA damage induced by a photoactive platinum-diimine complex in red light.

    PubMed

    Zhang, Zhigang; Chai, Aiyun

    2012-12-01

    Lack of solubility under physiological conditions poses an additional risk for toxicity and side effects for intravenous delivery of the photodynamic therapeutic agent in vivo. Employing magnetite-silica composite nanoparticles as carriers of the photodynamic therapeutic agents may be a promising way to solve the problem. In this study, core-shell magnetite-silica composite nanoparticles were prepared by a sol-gel method, and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering, then they were used as carriers of a photoactive platinum diimine complex. The interactions of the photosensitizer-loaded magnetic composite nanoparticles with DNA in red light were monitored by agarose-gel electrophoresis. The results suggest that high doses of magnetite-silica composite nanoparticles might facilitate the transformation of covalently closed circular (ccc)-DNA band to open circular (oc)-DNA band though they are harmless to DNA at their low concentrations, therefore enhancing the extent of DNA damage caused by the metal complex in red light.

  18. Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer

    PubMed Central

    AL-ATTAS*, ASMAA; ASSIDI*, MOURAD; AL-MAGHRABI, JAUDAH; DALLOL, ASHRAF; SCHULTEN, HANS-JUERGEN; ABU-ELMAGD, MUHAMMAD; CHAUDHARY, ADEEL; ABUZENADAH, ADEL; BUDOWLE, BRUCE; BUHMEIDA, ABDELBASET; AL-QAHTANI, MOHAMMED

    2016-01-01

    Background/Aim: To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. Materials and Methods: FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide’s image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. Results: High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. Conclusion: This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists’ knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine. *These Authors contributed equally to this manuscript. PMID:27566658

  19. Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA

    PubMed Central

    Zhu, Fujie; Liu, Xiao; Sun, Zhenhong; Yu, Cuilian; Liu, Liping; Yang, Shifa; Li, Bing; Wei, Kai; Zhu, Ruiliang

    2016-01-01

    Bordetella avium is the causative agent of bordetellosis, which remains to be the cause of severe losses in the turkey industry. Given the lack of vaccines that can provide good protection, developing a novel vaccine against B. avium infection is crucial. In this study, we constructed a eukaryotic expression plasmid, which expressed the outer membrane protein A (ompA) of B. avium, to prepare a B. avium recombinant ompA-DNA vaccine. Three concentrations (low, middle, and high) of Taishan Pinus massoniana pollen polysaccharides (TPPPS), a known immunomodulator, were used as adjuvants, and their immune conditioning effects on the developed DNA vaccine were examined. The pure ompA-DNA vaccine, Freund’s incomplete adjuvant ompA-DNA vaccine, and the empty plasmid served as the controls. The chickens in each group were separately inoculated with these vaccines three times at 1, 7, and 14 days old. Dynamic changes in antibody production, cytokine secretion, and lymphocyte count were then determined from 7 to 49 days after the first inoculation. Protective rates of the vaccines were also determined after the third inoculation. Results showed that the pure DNA vaccine obviously induced the production of antibodies, the secretion of cytokines, and the increase in CD4+ and CD8+ T lymphocyte counts in peripheral blood, as well as provided a protective rate of 50% to the B. avium-challenged chickens. The chickens inoculated with the TPPPS adjuvant ompA-DNA vaccine and Freund’s adjuvant ompA-DNA vaccine demonstrated higher levels of immune responses than those inoculated with pure ompA-DNA vaccine, whereas only the ompA-DNA vaccine with 200 mg/mL TPPPS completely protected the chickens against B. avium infection. These findings indicate that the B. avium ompA-DNA vaccine combined with TPPPS is a potentially effective B. avium vaccine. PMID:26870023

  20. Gene Gun Bombardment with DNA-Coated Golden Particles Enhanced the Protective Effect of a DNA Vaccine Based on Thioredoxin Glutathione Reductase of Schistosoma japonicum

    PubMed Central

    Cao, Yan; Zhao, Bin; Han, Yanhui; Zhang, Juan; Li, Xuezhen; Qiu, Chunhui; Wu, Xiujuan; Hong, Yang; Ai, Dezhou; Lin, Jiaojiao; Fu, Zhiqiang

    2013-01-01

    Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% (P < 0.01) of worm reduction and 40.38–44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects. PMID:23509820

  1. Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

    PubMed

    Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R; Jauch, Ralf

    2014-10-01

    Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

  2. Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis.

    PubMed

    Shima, Jun; Kuwazaki, Seigo; Tanaka, Fumiko; Watanabe, Hajime; Yamamoto, Hideki; Nakajima, Ryoichi; Tokashiki, Tadaaki; Tamura, Hiromi

    2005-06-25

    Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.

  3. A series of oxyimine-based macrocyclic dinuclear zinc(II) complexes enhances phosphate ester hydrolysis, DNA binding, DNA hydrolysis, and lactate dehydrogenase inhibition and induces apoptosis.

    PubMed

    Anbu, Sellamuthu; Kamalraj, Subban; Varghese, Babu; Muthumary, Johnpaul; Kandaswamy, Muthusamy

    2012-05-21

    A symmetrical macrocyclic dizinc(II) complex (1) has been synthesized by using the ligand (L(1)) [μ-11,24-dimethyl-4,7,16,19-tetraoxa-3,8,15,20-tetraazatricyclo-[20.3.1.1(10,13)] heptacosa-1(25),2,7,9,11,13(27),14,20,22(26),23-decaene-26,27-diol]. A series of unsymmetrical macrocyclic dizinc(II) complexes (2-6) has been synthesized by Schiff base condensation of bicompartmental mononuclear complex [ZnL] [μ-3,16-dimethyl-8,11-dioxa-7,12-diazadicyclo-[1.1(14,18)] heptacosa-1,3,5(20),6,12,14,16,18(19)-octacaene-19,20-diolato)zinc(II)] with various diamines like 1,2-diamino ethane (L(2)), 1,3-diamino propane (L(3)), 1,4-diamino butane (L(4)), 1,2-diamino benzene (L(5)), and 1,8-diamino naphthalene (L(6)). The ligand L(1) and all the zinc(II) complexes were structurally characterized. To corroborate the consequence of the aromatic moiety in comparison to the aliphatic moiety present in the macrocyclic ring on the phosphate ester hydrolysis, DNA binding and cleavage properties have been studied. The observed first order rate constant values for the hydrolysis of 4-nitrophenyl phosphate ester reaction are in the range from 2.73 × 10(-2) to 9.86 × 10(-2) s(-1).The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence, and circular dichroism spectroscopy. The DNA binding constant values of the complexes were found in the range from 1.80 × 10(5) to 9.50 × 10(5) M(-1), and the binding affinities are in the following order: 6 > 5 > 1 > 2 > 3 > 4. All the dizinc(II) complexes 1-6 effectively promoted the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 6 under physiological conditions give the observed rate constant (k(obs)) of 4.42 ± 0.2 h(-1), which shows a 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. The comparison of the dizinc(II) complexes 1-6 with the monozinc(II) complex [ZnL] indicates

  4. Overcoming RNA inhibition in the fluorescent polymerase chain reaction assay to enhance detection of bovine DNA in cattle feeds.

    PubMed

    Sawyer, Mary; Rensen, Gabriel; Smith, Wayne; Yee, Melanie; Wong, Alice; Osburn, Bennie; Cullor, James

    2004-01-01

    The practice of incorporating mammalian protein in ruminant feeds was banned in the United States in 1997 as a measure to avoid transmission of bovine spongiform encephalopathy (BSE). A sensitive means of identifying the banned additives in feeds would be by detection of species-specific DNA using the polymerase chain reaction (PCR). However, problems may arise in the PCR due to the presence of inhibitory substances. Using human DNA as an internal PCR control, inhibitory substances were evident in the DNA extraction products of cattle feeds. The results of heating experiments excluded enzymes as a cause of inhibition, and spectrophotometric calculations suggested the possibility of RNA contamination. Co-electrophoresis of untreated and RNAse digested extracts confirmed the presence of RNA in the undigested product. Seven cattle feeds were spiked with predetermined amounts of bovine meat and bone meal (BMBM). The DNA extracted products were treated with RNAse and the bovine specific mitochondrial DNA (B-mtDNA) was amplified by PCR. The minimum level of detection of B-mtDNA was influenced by RNAse treatment and feed composition. RNAse treatment decreased false-negative results overall by 75%. False-negative results were decreased 100% in the higher BMBM concentrations and 50% in the lower BMBM concentrations. Also, each cattle feed was spiked to attain a 2% wt/wt concentration with each swine, fish, sheep, or poultry product, or cattle dried blood. Amplification of B-mtDNA occurred only with the cattle dried blood and only in three feeds in which B-mtDNA was detected at the only level tested (2%). A commercial immunochromotographic assay (Neogen) detected the spiked BMBM in only one of the seven feeds and only at the upper concentration (1%).

  5. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.

    PubMed

    Chung, Woo-Hyun; Zhu, Zhu; Papusha, Alma; Malkova, Anna; Ira, Grzegorz

    2010-05-13

    The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2-4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Delta exo1Delta mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Delta exo1Delta, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Delta exo1Delta cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.

  6. TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia

    PubMed Central

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R.; Kantarjian, Hagop M.; Liang, Shoudan; Estecio, Marcos R.; Godley, Lucy A.; Issa, Jean-Pierre J.

    2015-01-01

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites. PMID:25972343

  7. A modular system of DNA enhancer elements mediates tissue-specific activation of transcription by high dietary zinc in C. elegans

    PubMed Central

    Roh, Hyun Cheol; Dimitrov, Ivan; Deshmukh, Krupa; Zhao, Guoyan; Warnhoff, Kurt; Cabrera, Daniel; Tsai, Wendy; Kornfeld, Kerry

    2015-01-01

    Zinc is essential for biological systems, and aberrant zinc metabolism is implicated in a broad range of human diseases. To maintain homeostasis in response to fluctuating levels of dietary zinc, animals regulate gene expression; however, mechanisms that mediate the transcriptional response to fluctuating levels of zinc have not been fully defined. Here, we identified DNA enhancer elements that mediate intestine-specific transcriptional activation in response to high levels of dietary zinc in C. elegans. Using bioinformatics, we characterized an evolutionarily conserved enhancer element present in multiple zinc-inducible genes, the high zinc activation (HZA) element. The HZA was consistently adjacent to a GATA element that mediates expression in intestinal cells. Functional studies using transgenic animals demonstrated that this modular system of DNA enhancers mediates tissue-specific transcriptional activation in response to high levels of dietary zinc. We used this information to search the genome and successfully identified novel zinc-inducible genes. To characterize the mechanism of enhancer function, we demonstrated that the GATA transcription factor ELT-2 and the mediator subunit MDT-15 are necessary for zinc-responsive transcriptional activation. These findings define new mechanisms of zinc homeostasis and tissue-specific regulation of transcription. PMID:25552416

  8. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    SciTech Connect

    Ganesan, Shanthi Nteeba, Jackson Keating, Aileen F.

    2014-12-01

    7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity.