WorldWideScience

Sample records for bifunctional nucleases ombbd

  1. Crystallization of recombinant bifunctional nuclease TBN1 from tomato

    International Nuclear Information System (INIS)

    Glycosylated recombinant bifunctional nuclease from tomato has been crystallized and preliminary X-ray diffraction analysis was performed. The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum

  2. Structural analysis of a recombinant plant bifunctional nuclease TBN1

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Lipovová, P.; Podzimek, Tomáš; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    Vol. no 1. Praha : Czech and Slovak Crystallographic Association, 2011. s. 29. ISSN 1211-5894. [Discussions in Structural Molecular Biology /9./. 24.03.2011-26.03.2011, Nové Hrady] R&D Projects: GA ČR GA202/06/0757; GA ČR GA310/09/1407; GA ČR GA521/09/1214 Grant ostatní: AVČR(CZ) Praemium Academiae Institutional research plan: CEZ:AV0Z10100521; CEZ:AV0Z50510513; CEZ:AV0Z40500505 Keywords : bifunctional nuclease * cancer * x-ray analysis Subject RIV: BM - Solid Matter Physics ; Magnetism

  3. Structure analysis of group I plant nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Dohnálek, Jan, E-mail: dohnalek007@gmail.com [Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06 Praha 6 (Czech Republic); Koval’, Tomáš [Institute of Physics AS CR, v.v.i., Na Slovance 2, 182 21 Praha 8 (Czech Republic); Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 3, 121 16 Praha 2 (Czech Republic); Lipovová, Petra [Institute of Chemical Technology, Technická 5, 166 28 Praha 6 (Czech Republic); Podzimek, Tomáš [Institute of Chemical Technology, Technická 5, 166 28 Praha 6 (Czech Republic); Institute of Plant Molecular Biology, Biology Centre, AS CR, v.v.i., Branišovská 31, 37005 České Budějovice (Czech Republic); Matoušek, Jaroslav [Institute of Plant Molecular Biology, Biology Centre, AS CR, v.v.i., Branišovská 31, 37005 České Budějovice (Czech Republic)

    2011-01-01

    Structural properties of plant nuclease TBN1 are studied using synchrotron radiation to explain its specificity, role of glycosylation and to contribute to potential application in cancer treatment. Anticancer drugs attacking nucleic acids of the target cells have so far been based on animal or fungal ribonucleases. Plant nucleases have been proved to exhibit decreased cytotoxic side effects. Tomato bifunctional nuclease 1 with activity against both single-stranded and double-stranded RNA and DNA was produced in tobacco leaves as recombinant protein. The enzyme crystallizes under several different crystallization conditions. The presence of Zn{sup 2+} ions was confirmed by X-ray fluorescence. First crystallographic data were obtained.

  4. Structure analysis of group I plant nucleases

    International Nuclear Information System (INIS)

    Structural properties of plant nuclease TBN1 are studied using synchrotron radiation to explain its specificity, role of glycosylation and to contribute to potential application in cancer treatment. Anticancer drugs attacking nucleic acids of the target cells have so far been based on animal or fungal ribonucleases. Plant nucleases have been proved to exhibit decreased cytotoxic side effects. Tomato bifunctional nuclease 1 with activity against both single-stranded and double-stranded RNA and DNA was produced in tobacco leaves as recombinant protein. The enzyme crystallizes under several different crystallization conditions. The presence of Zn2+ ions was confirmed by X-ray fluorescence. First crystallographic data were obtained

  5. Design of artificial nucleases and studies of their interaction with DNA

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The design of artificial nucleases and nuclease mimics has attracted extensive attention and made great progress due to their significant scientific meanings and potential application in the field of gene medicine and molecular biology. This paper reviews recent progress in the investigation of artificial nuclease,including "bifunctional cooperative catalysis","dinuclear synergistic catalysis","metal-free catalysis" ,and especially,the studies of aza-crown ethers as artificial nucleases and their interaction with DNA.

  6. Crystallization of recombinant bifunctional nuclease TBN1 from tomato

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Lipovová, P.; Podzimek, Tomáš; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    2011-01-01

    Roč. 67, č. 1 (2011), s. 124-128. ISSN 1744-3091 R&D Projects: GA ČR GA310/09/1407; GA ČR GA202/06/0757; GA ČR GA521/09/1214 Grant ostatní: AVČR(CZ) Praemium Academiae Institutional research plan: CEZ:AV0Z10100521; CEZ:AV0Z40500505; CEZ:AV0Z50510513 Keywords : plant endonuclease * anti-tumor properties * crystallization * X-ray diffraction Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.506, year: 2011

  7. X-ray diffraction studies of red tomato nuclease TBN1

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Dohnálek, Jan; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Kolenko, Petr; Hašek, Jindřich

    2010-01-01

    Roč. 17, 1a (2010), b40. ISSN 1211-5894. [Discussions in Structural Molecular Biology /8./. 18.03.2010-20.03.2010, Nové Hrady] R&D Projects: GA ČR GA202/06/0757 Institutional research plan: CEZ:AV0Z10100521; CEZ:AV0Z40500505 Keywords : plant bifunctional nuclease * crystallization * x-ray diffraction analysis Subject RIV: BM - Solid Matter Physics ; Magnetism

  8. Bifunctional alkaline oxygen electrodes

    Science.gov (United States)

    Swette, L.; Kackley, N.; Mccatty, S. A.

    1991-01-01

    The authors describe the identification and testing of electrocatalysts and supports for the positive electrode of moderate-temperature, single-unit, rechargeable alkaline fuel cells. Recent work on Na(x)Pt3O4, a potential bifunctional catalyst, is described, as well as the application of novel approaches to the development of more efficient bifunctional electrode structures. The three dual-character electrodes considered here showed similar superior performance; the Pt/RhO2 and Rh/RhO2 electrodes showed slightly better performance than the Pt/IrO2 electrode. It is concluded that Na(x)Pt3O4 continues to be a promising bifunctional oxygen electrode catalyst but requires further investigation and development.

  9. Tight bifunctional hierarchical catalyst.

    Science.gov (United States)

    Højholt, Karen T; Vennestrøm, Peter N R; Tiruvalam, Ramchandra; Beato, Pablo

    2011-12-28

    A new concept to prepare tight bifunctional catalysts has been developed, by anchoring CoMo(6) clusters on hierarchical ZSM-5 zeolites for simultaneous use in HDS and hydrocracking catalysis. The prepared material displays a significant improved activity in HDS catalysis compared to the impregnated counterpart. PMID:22048337

  10. Cold denaturation of staphylococcal nuclease.

    OpenAIRE

    Griko, Y. V.; Privalov, P L; Sturtevant, J M; Venyaminov SYu

    1988-01-01

    Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70 degrees C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20 degrees C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these t...

  11. Copper complexes as chemical nucleases

    Indian Academy of Sciences (India)

    Akhil R Chakravarty; Pattubala A N Reddy; Bidyut K Santra; Anitha M Thomas

    2002-08-01

    Redox active mononuclear and binuclear copper(II) complexes have been prepared and structurally characterized. The complexes have planar N-donor heterocyclic bases like 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) and dipyridophenazine (dppz) ligands that are suitable for intercalation to B-DNA. Complexes studied for nuclease activity have the formulations [Cu(dpq)2(H2O)] (ClO4)2.H2O (1), [{CuL(H2O)}2(-ox)](ClO4)2 (L = bpy, 2; phen, 3; dpq, 4; and dppz, 5) and [Cu(L)(salgly)] (L = bpy, 6; phen, 7; dpq, 8; and dppz, 9), where salgly is a tridentate Schiff base obtained from the condensation of glycine and salicylaldehyde. The dpq complexes are efficient DNA binding and cleavage active species. The dppz complexes show good binding ability but poor nuclease activity. The cleavage activity of the bis-dpq complex is significantly higher than the bis-phen complex of copper(II). The nuclease activity is found to be dependent on the intercalating nature of the complex and on the redox potential of the copper(II)/copper(I) couple. The ancillary ligand plays a significant role in binding and cleavage activity.

  12. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    OpenAIRE

    Eriksson, Jonas; Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

  13. Bifunctional redox flow battery

    International Nuclear Information System (INIS)

    A new bifunctional redox flow battery (BRFB) system, V(III)/V(II)-L-cystine(O2), was systematically investigated by using different separators. It is shown that during charge, water transfer is significantly restricted with increasing the concentration of HBr when the Nafion 115 cation exchange membrane is employed. The same result can be obtained when the gas diffusion layer (GDL) hot-pressed separator is used. The organic electro-synthesis is directly correlated with the crossover of vanadium. When employing the anion exchange membrane, the electro-synthesis efficiency is over 96% due to a minimal crossover of vanadium. When the GDL hot-pressed separator is applied, the crossover of vanadium and water transfer are noticeably prevented and the electro-synthesis efficiency of over 99% is obtained. Those impurities such as vanadium ions and bromine can be eliminated through the purification of organic electro-synthesized products. The purified product is identified to be L-cysteic acid by IR spectrum. The BRFB shows a favorable discharge performance at a current density of 20 mA cm-2. Best discharge performance is achieved by using the GDL hot-pressed separator. The coulombic efficiency of 87% and energy efficiency of about 58% can be obtained. The cause of major energy losses is mainly associated with the cross-contamination of anodic and cathodic active electrolytes

  14. Adsorption of nuclease p1 on chitosan nano-particles

    Directory of Open Access Journals (Sweden)

    Lu-E Shi

    2009-06-01

    Full Text Available The sorption of nuclease P1 onto chitosan nano-particles is studied in this paper. The effect of some adsorption kinetics factors such as nuclease P1 concentration, chitosan nano-particles solution concentration, adsorption temperature, chitosan nano-particles size, solution pH, etc. is investigated. Adsorption of nuclease P1 onto chitosan nano-particles is fitted into Lagergren first-order equation at initial nuclease P1 concentration of 3.0 mg/mL. The first-order constant for nuclease P1 is 22.98 h-1. When nuclease P1 concentration is controlled into certain region, the adsorption fits into Freundlich isothermal linear equation. A mechanism of adsorption for nuclease P1 is proposed by analyzing IR spectra. The IR spectra shows that the hydrogen bond might be the main force between the hydroxyl group, the NH2 group and the nuclease P1.

  15. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    Directory of Open Access Journals (Sweden)

    Kentaro Ishida

    2015-10-01

    Full Text Available Programmable nucleases, such as zinc finger nucleases (ZFNs, transcription activator like effector nucleases (TALENs, and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9, hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

  16. A sensitive assay for Staphylococcus aureus nucleases

    International Nuclear Information System (INIS)

    A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured [3H] thymidine labelled DNA from E.coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate. (author). 26 refs., 3 figs ., 3 tabs

  17. Eukaryotic zinc-dependent multifunctional nuclease I

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Stránský, Jan; Dušková, Jarmila; Fejfarová, Karla; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

    2014-01-01

    Roč. 70, Supplement /August/ (2014), C211. ISSN 0108-7673. [Congress and General Assembly of the International Union of Crystallography /23./ - IUCr 2014. 05.08.2014-12.08.2014, Montreal] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:86652036 ; RVO:60077344 Keywords : nuclease * tomato * crystal structure Subject RIV: CE - Biochemistry

  18. Genome Engineering With Zinc-Finger Nucleases

    OpenAIRE

    Carroll, Dana

    2011-01-01

    Zinc-finger nucleases (ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools. ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. This article briefly reviews the history of ZFN development and summarizes applications that have been made to genome editing in many different organisms and situations. Considerable progress has been mad...

  19. Zinc dependent nucleases with biomedical potential

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Stránský, Jan; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Fejfarová, Karla; Kolenko, Petr; Dohnálek, Jan

    Prague: Institute of Macromolecular Chemistry AS CR, 2015. s. 9. [ Research Postdoctoral Colloquium. 14.05.2015, Prague] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk LG14009 Institutional support: RVO:61389013 ; RVO:86652036 ; RVO:60077344 Keywords : biomedical applications * nuclease Subject RIV: CE - Biochemistry

  20. Structure analysis of group I plant nucleases

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Kovaľ, Tomáš; Lipovová, P.; Podzimek, Tomáš; Matoušek, Jaroslav

    2011-01-01

    Roč. 18, Part 1 (2011), s. 29-30. ISSN 0909-0495. [International Symposium on Diffraction Structural Biology /3./. Paris - Orsay, 25.05.2010-28.05.2010] R&D Projects: GA ČR GA310/09/1407; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z10100521; CEZ:AV0Z50510513 Keywords : nuclease * protein structure * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.726, year: 2011

  1. Monomeric site-specific nucleases for genome editing.

    Science.gov (United States)

    Kleinstiver, Benjamin P; Wolfs, Jason M; Kolaczyk, Tomasz; Roberts, Alanna K; Hu, Sherry X; Edgell, David R

    2012-05-22

    Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications. PMID:22566637

  2. Venturing into the New Science of Nucleases.

    Science.gov (United States)

    Tolarová, Markéta; McGrath, John A; Tolar, Jakub

    2016-04-01

    Gene editing with zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated proteins system, or meganucleases can, in principle, mediate any genome modification. Recent studies have shown that COL7A1 mutations in cells of patients with recessive dystrophic epidermolysis bullosa can be corrected by homology-directed DNA repair. PMID:27012560

  3. TALE nucleases and next generation GM crops.

    KAUST Repository

    Mahfouz, Magdy M.

    2011-04-01

    Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

  4. Okazaki fragment maturation: nucleases take centre stage

    Institute of Scientific and Technical Information of China (English)

    Li Zheng; Binghui Shen

    2011-01-01

    Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells. Even in yeast, the Okazaki fragment maturation happens approximately a million times during a singte round of DNA replication. Therefore, efficient processing of Okazaki fragments is vital for DNA replication and cell proliferation. During this process,primase-synthesized RNA/DNA primers are removed, and Okazaki fragments are joined into an intact lagging strand DNA. The processing of RNA/DNA primers requires a group of structure-specific nucleases typified by flap endonuclease 1 (FEN1). Here, we summarize the distinct roles of these nucleases in different pathways for removal of RNA/DNA primers. Recent findings reveal that Okazaki fragment maturation is highly coordinated. The dynamic interactions of polymerase δ, FEN1 and DNA ligase I with proliferating cell nuclear antigen allow these enzymes to act sequentially during Okazaki fragment maturation. Such protein-protein interactions may be regulated by post-translational modifications. We also discuss studies using mutant mouse models that suggest two distinct cancer etiological mechanisms arising from defects in different steps of Okazaki fragment maturation.Mutations that affect the efficiency of RNA primer removal may result in accumulation of unligated nicks and DNA double-strand breaks. These DNA strand breaks can cause varying forms of chromosome aberrations, contributing to development of cancer that associates with aneuploidy and gross chromosomal rearrangement. On the other hand, mutations that impair editing out of polymerase o incorporation errors result in cancer displaying a strong mutator phenotype.

  5. Catalytic activity of nuclease P1: Experiment and theory

    International Nuclear Information System (INIS)

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates

  6. Role of recBC nuclease in Escherichia coli transformation.

    OpenAIRE

    Hoekstra, W P; Bergmans, J E; Zuidweg, E.M.

    1980-01-01

    In Escherichia coli transformation with linear donor deoxyribonucleic acid, the recBC pathway is functional, but genetic analysis shows that the recBC nuclease is deleterious to linear deoxyribonucleic acid.

  7. Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies.

    Science.gov (United States)

    Scharenberg, Andrew M; Duchateau, Philippe; Smith, Julianne

    2013-08-01

    Over three years following the discovery of the TAL code, artificial TAL effector DNA binding domains have emerged as the premier platform for building site-specific DNA binding polypeptides for use in biological research. Here, we provide an overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications. We also discuss considerations for choosing TAL effector/mDBD or alternative nuclease technologies for genome engineering projects ranging from basic laboratory gene editing of cultured cell lines to therapeutics. Finally, we highlight how the rapid pace of development of mDBD-based, such as monomeric TALENs (I-TevI-TAL), and more recently RNA-guided nucleases (CRISPR-Cas9) has led to a transition in the field of genome engineering towards development of the next generation of technologies aimed at controlling events that occur after targeted DNA breaks are made. PMID:23888878

  8. Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

    Science.gov (United States)

    Cecere, Paola; Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Nucleases are ubiquitous enzymes that degrade DNA or RNA, thus they can prejudice the good outcome of molecular biology experiments involving nucleic acids. We propose a colorimetric test for the naked-eye detection of nuclease contaminations. The system uses an hybrid nanosensor, based on gold nanoparticles functionalized with DNA probes. Our assay is rapid, instrument-free, simple and low-cost. Moreover, it reaches sensitivity equal or better than those of commercial kits, and presents a lot of advantageous aspects. Therefore, it is very competitive, with a real market potential. This test will be relevant in routine process monitoring in scientific laboratories, and in quality control in clinical laboratories and industrial processes, allowing the simultaneous detection of nucleases with different substrate specificities and large-scale screening.

  9. Bioinformatic analysis of zinc dependent nucleases: post-translational modifications

    Czech Academy of Sciences Publication Activity Database

    Fejfarová, Karla; Koval, Tomáš; Stránský, Jan; Kolenko, Petr; Trundová, Mária; Hašek, Jindřich; Dohnálek, Jan

    Prague: Institute of Macromolecular Chemistry AS CR, 2015. s. 17. [ Research Postdoctoral Colloquium. 14.05.2015, Prague] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:86652036 Keywords : biomedical applications * nuclease Subject RIV: CE - Biochemistry

  10. Bifunctional electrocatalyst for oxygen/air electrodes

    International Nuclear Information System (INIS)

    Highlights: • Nano-Silver powder was prepared by chemical method. • Ag catalyst was characterized by SEM and XRD studies. • Ag was investigated as bi-functional electrocatalyst for oxygen/air electrodes. • Ag shows good electrochemical activity towards OER and ORR reactions. - Abstract: Nano-Silver powder has been studied as bi-functional electrocatalyst for both oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) in alkaline medium. Ag nano-powder has been prepared by a simple wet chemical method with Silver nitrate as precursor and Glucose as reducing agent. X-ray Diffraction and Scanning Electron Microscopy studies were carried out to characterize the Silver catalyst. Electrochemical oxygen evolution characterization shows anodic peak typically at the range between 0.350 and 0.514 V Vs Hg/HgO corresponding to Silver oxidation followed by the onset of oxygen evolution at 0.706 V. Oxygen reduction reaction studies carried out using Rotating Disc Electrode (RDE) confirm the four electron reaction mechanism. Ag catalyst shows promising characteristics for oxygen evolution and oxygen reduction

  11. Antitumor Effects and Cytotoxicity of Recombinant Plant Nucleases

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Podzimek, Tomáš; Pouckova, P.; Stehlík, Jan; Škvor, J.; Souček, J.; Matoušek, Josef

    2009-01-01

    Roč. 18, č. 4 (2009), s. 163-171. ISSN 0965-0407 R&D Projects: GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : Anticarcinogenic and antiproliferative nucleases * Human melanoma * Tumor xenografts * Nicotiana benthamina Subject RIV: FD - Oncology ; Hematology Impact factor: 1.478, year: 2009

  12. Antitumor activity od apoptotic nuclease TBN1 from L. esculentum

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Podzimek, Tomáš; Poučková, P.; Stehlík, Jan; Škvor, J.; Lipovová, P.; Matoušek, Josef

    2010-01-01

    Roč. 57, č. 4 (2010), s. 339-348. ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : anticancerogenic and antiproliferative nuclease * dsRNase * human solid malignant tumors Subject RIV: FD - Oncology ; Hematology Impact factor: 1.449, year: 2010

  13. Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

    OpenAIRE

    Hwang, Woong Y.; Fu, Yanfang; Reyon, Deepak; Maeder, Morgan L.; Tsai, Shengdar Q.; Sander, Jeffry D.; Peterson, Randall T.; Yeh, J.-R. Joanna; Joung, J. Keith

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in ze...

  14. Zinc Finger Nuclease-Mediated Gene Targeting in Plants

    International Nuclear Information System (INIS)

    Zinc finger nucleases were used to facilitate homology driven repair and site-specific transgene integration in transgenic tobacco cell cultures. A target DNA sequence containing a non-functional, partial 3' PAT gene sequence flanked by zinc finger binding sites was stably integrated into BY2 suspension cultures using Agrobacterium-mediated transformation. A transgenic event containing a single integrated copy of the target sequence was used for gene targeting through co-transformation with two different Agrobacterium strains containing: i) donor DNA sequences comprising the 5' partial DNA fragment necessary to correct the non-functional PAT gene flanked by sequences homologous to the pre-integrated target DNA and ii) DNA that encoded a zinc finger nuclease that specifically recognized binding sites within the pre-integrated target. Two gene targeting strategies differing with respect to the distance between the zinc finger binding site and the homologous sequences were used. Gene targeting was demonstrated for both strategies as evidenced by the re-constitution of a functional PAT gene and was confirmed via molecular and biochemical analyses. Sequencing of recombined DNA confirmed that PAT gene reconstitution resulted from homology-driven repair at the zinc finger nuclease cleavage site. However, imperfect recombination resulting from non-homologous processes was also observed. (author)

  15. Nanoscale intimacy in bifunctional catalysts for selective conversion of hydrocarbons

    OpenAIRE

    Zecevic, Jovana; Vanbutsele, Gina; de Jong, Krijn P.; Martens, Johan

    2015-01-01

    The ability to precisely control nanoscale features is increasingly exploited to develop and improve monofunctional catalysts1–4. Striking effects might also be expected in the case of bifunctional catalysts, which play an important role in hydrocracking of fossil and renewable hydrocarbon sources to provide high-quality diesel fuel5–7. Such bifunctional hydrocracking catalysts contain metal sites and acid sites, and for more than 50 years the so-called ‘intimacy criterion’8 has dictated the ...

  16. Nanoparticle Superlattices as Efficient Bifunctional Electrocatalysts for Water Splitting.

    Science.gov (United States)

    Li, Jun; Wang, Yongcheng; Zhou, Tong; Zhang, Hui; Sun, Xuhui; Tang, Jing; Zhang, Lijuan; Al-Enizi, Abdullah M; Yang, Zhongqin; Zheng, Gengfeng

    2015-11-18

    The solar-driven water splitting process is highly attractive for alternative energy utilization, while developing efficient, earth-abundant, bifunctional catalysts for both oxygen evolution reaction and hydrogen evolution reaction has remained as a major challenge. Herein, we develop an ordered CoMnO@CN superlattice structure as an efficient bifunctional water-splitting electrocatalyst, in which uniform Co-Mn oxide (CoMnO) nanoparticles are coated with a thin, continuous nitrogen-doped carbon (CN) framework. The CoMnO nanoparticles enable optimized OER activity with effective electronic structure configuration, and the CN framework serves as an excellent HER catalyst. Importantly, the ordered superlattice structure is beneficial for enhanced reactive sites, efficient charge transfer, and structural stability. This bifunctional superlattice catalyst manifests optimized current densities and electrochemical stability in overall water splitting, outperforming most of the previously reported single- or bifunctional electrocatalysts. Combining with a silicon photovoltaic cell, this CoMnO@CN superlattice bifunctional catalyst enables unassisted solar water splitting continuously for ∼5 days with a solar-to-hydrogen conversion efficiency of ∼8.0%. Our discovery suggests that these transition metal oxide-based superlattices may serve as a unique structure modality for efficient bifunctional water splitting electrocatalysts with scale-up potentials. PMID:26496655

  17. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function

    OpenAIRE

    EFSA Panel on Genetically Modified Organisms (GMO)

    2012-01-01

    The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3) which allows the integration of gene(s) in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3) is used to describe the technique ra...

  18. Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

    Science.gov (United States)

    Mashimo, Tomoji

    2014-01-01

    The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. PMID:24372523

  19. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.

    Science.gov (United States)

    Barzel, A; Paulk, N K; Shi, Y; Huang, Y; Chu, K; Zhang, F; Valdmanis, P N; Spector, L P; Porteus, M H; Gaensler, K M; Kay, M A

    2015-01-15

    Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration. PMID:25363772

  20. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for...

  1. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    OpenAIRE

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commo...

  2. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    OpenAIRE

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing 1, 2 , the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM) 3–6 . As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the c...

  3. DNA targeting specificity of RNA-guided Cas9 nucleases

    OpenAIRE

    Hsu, Patrick D.; Scott, David A.; Weinstein, Joshua A.; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J.; Marraffini, Luciano A.; Bao, Gang; Zhang, Feng

    2013-01-01

    The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches betwee...

  4. Crystal structures of two eukaryotic nucleases involved in RNA metabolism

    DEFF Research Database (Denmark)

    Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich;

    well as the controlled turnover of these in response to changing surrounding conditions is of vital importance to ensure optimal fitness of a cell. Central to both these processes is the degradation of RNA, either as a means of decreasing the level of particular RNAs or as a way to get rid of aberrant...... of very similar nucleases provide a visualisation of the catalytic cycle of the S. pombe Pop2p protein. In addition, structural comparison of S. pombe Pop2p to other similar proteins identifies Pop2p and deadenylase specific regions near the active site. Contrary to Pop2p Rrp6p is solely a nuclear...

  5. Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1

    Directory of Open Access Journals (Sweden)

    Zhang Xiaobo

    2008-04-01

    Full Text Available Abstract Background Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN was obtained from a thermophilic bacteriophage GBSV1 for the first time. Results After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60°C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, β-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% – 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 μM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively. Conclusion Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.

  6. Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

    KAUST Repository

    Wibowo, Anjar

    2011-06-06

    Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

  7. Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Angen, Øystein; Jensen, J.; Lavritsen, D. T.

    2001-01-01

    Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluated...... with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C-t value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5' nuclease assay represents a test with 100......% sensitivity and 100% specificity, A high degree of reproducibility of the test was demonstrated. If samples with C-t values of less than or equal to 30 are considered positive, the detection limit of the assay was 1 CPU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30...

  8. Oligomeric structure diversity within the GIY-YIG nuclease family.

    Science.gov (United States)

    Ibryashkina, Elena M; Sasnauskas, Giedrius; Solonin, Alexander S; Zakharova, Marina V; Siksnys, Virginijus

    2009-03-20

    The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5'-CCGC/GG-3' ("/" marks the cleavage position) and cuts it to generate 3'-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family. PMID:19361436

  9. Novel Bifunctional Natriuretic Peptides as Potential Therapeutics*

    Science.gov (United States)

    Dickey, Deborah M.; Burnett, John C.; Potter, Lincoln R.

    2008-01-01

    Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics. PMID:18940797

  10. Novel bifunctional natriuretic peptides as potential therapeutics.

    Science.gov (United States)

    Dickey, Deborah M; Burnett, John C; Potter, Lincoln R

    2008-12-12

    Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics. PMID:18940797

  11. Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Angen, Øystein; Jensen, J.; Lavritsen, D. T.

    2001-01-01

    , nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae, is the...... equal to 30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.......Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluated...

  12. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end......A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for...

  13. Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases.

    Science.gov (United States)

    Hwang, Woong Y; Fu, Yanfang; Reyon, Deepak; Gonzales, Andrew P W; Joung, J Keith; Yeh, Jing-Ruey Joanna

    2015-01-01

    In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms. PMID:25981483

  14. Bifunctional mesoporous silicas with clearly distinguished localization of grafted groups

    Science.gov (United States)

    Roik, N. V.; Belyakova, L. A.

    2013-12-01

    Bifunctional mesoporous silicas with clearly distinguished localization of grafted groups on the surface of particles and inside their pores were obtained by means of sol-gel synthesis with postsynthetic vapor-phase treatment in vacuum. It was found that the synthesized materials have the hexagonally ordered porous structure typical of MCM-41 type silica.

  15. Single flexible nanofiber to simultaneously realize electricity-magnetism bifunctionality

    International Nuclear Information System (INIS)

    In order to develop new-typed multifunctional composite nanofibers, PANI/Fe3O4/PVP flexible bifunctional composite nanofibers with simultaneous electrical conduction and magnetism have been successfully fabricated via a facile electrospinning technology. Polyvinyl pyrrolidone (PVP) is used as a matrix to construct composite nanofibers containing different amounts of polyaniline (PANI) and Fe3O4 nanoparticles (NPs). The bifunctional composite nanofibers simultaneously possess excellent electrical conductivity and magnetic properties. The electrical conductivity reaches up to the order of 10-3 S·cm-1. The electrical conductivity and saturation magnetization of the composite nanofibers can be respectively tuned by adding various amounts of PANI and Fe3O4 NPs. The obtained electricity-magnetism bifunctional composite nanofibers are expected to possess many potential applications in areas such as electromagnetic interference shielding, special coating, microwave absorption, molecular electronics and future nanomechanics. More importantly, the design concept and construct technique are of universal significance to fabricate other bifunctional one-dimensional nanostructures. (author)

  16. Nanoscale intimacy in bifunctional catalysts for selective conversion of hydrocarbons

    NARCIS (Netherlands)

    Zecevic, Jovana; Vanbutsele, Gina; de Jong, Krijn P.; Martens, Johan A.

    2015-01-01

    The ability to control nanoscale features precisely is increasingly being exploited to develop and improve monofunctional catalysts(1-4). Striking effects might also be expected in the case of bifunctional catalysts, which are important in the hydrocracking of fossil and renewable hydrocarbon source

  17. Single flexible nanofiber to simultaneously realize electricity-magnetism bifunctionality

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Ming; Sheng, Shujuan; Ma, Qianli; Lv, Nan; Yu, Wensheng; Wang, Jinxian; Dong, Xiangting; Liu, Guixia, E-mail: wenshengyu2009@sina.com, E-mail: dongxiangting888@163.com [Key Laboratory of Applied Chemistry and Nanotechnology at Universities of Jilin Province, Changchun University of Science and Technology, Changchun (China)

    2016-03-15

    In order to develop new-typed multifunctional composite nanofibers, PANI/Fe{sub 3}O{sub 4}/PVP flexible bifunctional composite nanofibers with simultaneous electrical conduction and magnetism have been successfully fabricated via a facile electrospinning technology. Polyvinyl pyrrolidone (PVP) is used as a matrix to construct composite nanofibers containing different amounts of polyaniline (PANI) and Fe{sub 3}O{sub 4} nanoparticles (NPs). The bifunctional composite nanofibers simultaneously possess excellent electrical conductivity and magnetic properties. The electrical conductivity reaches up to the order of 10{sup -3} S·cm{sup -1}. The electrical conductivity and saturation magnetization of the composite nanofibers can be respectively tuned by adding various amounts of PANI and Fe{sub 3}O{sub 4} NPs. The obtained electricity-magnetism bifunctional composite nanofibers are expected to possess many potential applications in areas such as electromagnetic interference shielding, special coating, microwave absorption, molecular electronics and future nanomechanics. More importantly, the design concept and construct technique are of universal significance to fabricate other bifunctional one-dimensional nanostructures. (author)

  18. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    Directory of Open Access Journals (Sweden)

    Leśniewicz Krzysztof

    2012-10-01

    Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

  19. AdnAB: a new DSB-resecting motor–nuclease from mycobacteria

    OpenAIRE

    Sinha, Krishna Murari; Unciuleac, Mihaela-Carmen; Glickman, Michael S.; Shuman, Stewart

    2009-01-01

    The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase–nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase–nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is trig...

  20. Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells.

    Science.gov (United States)

    Xu, Jian; Teng, Da; Jiang, Fei; Zhang, Yuewei; El-Ashram, Saeed A; Wang, Hui; Sun, Zhenhong; He, Jinyan; Shen, Junjun; Wu, Wenxue; Li, Jinxiang

    2015-02-01

    Mycoplasma gallisepticum can infect a wide variety of birds including the commercial poultry. M. gallisepticum MGA_0676 is a putative lipoprotein, which is similar to bacterial thermostable nucleases. But the possible pathogenic effect of M. gallisepticum MGA_0676 has not been investigated so far. In the present study, we cloned the MGA_0676 gene after deletion of the amino-terminal signal sequence and mutagenesis of the Mycoplasma TGA tryptophan codons to TGG and expressed recombinant MGA_0676 protein in Escherichia coli. We identified and characterized MGA_0676 as a Ca(2+)-dependent cytotoxic nuclease of M. gallisepticum with a staphylococcal nuclease (SNc) region that displays the hallmarks of nucleases. Membrane protein immunoblot analysis and immunogold electron microscopy revealed that MGA_0676 locates on the membrane surface of M. gallisepticum. Furthermore, apoptosis assay using annexin V-FITC and propidium iodide (annexin V/PI) indicated that MGA_0676 played significant roles in apoptosis induction and pathological damages in chicken cells. Moreover, confocal microscopy showed that MGA_0676 localizes in the nuclei of host cells. Besides, after the SNc region was deleted, MGA_0676 lost its ability of nuclear localization, nuclease activity, and cytotoxicity, which revealed that the SNc region is essential for nuclear translocation and induction of apoptosis in chicken cells. The above results suggest that MGA_0676 is an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M. gallisepticum. PMID:25363559

  1. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

    Science.gov (United States)

    Chang, Howard H.Y.; Lieber, Michael R.

    2016-01-01

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. PMID:27198222

  2. Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

    OpenAIRE

    Foley, Jonathan E.; Reyon, Deepak; Yeh, Jing-Ruey Joanna; Maeder, Morgan L.; Sander, Jeffry D.; Peterson, Randall Theodore; Joung, Jae Keith

    2009-01-01

    Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-jo...

  3. Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae

    OpenAIRE

    Moon, Andrea F.; Gaudu, Philippe; Pedersen, Lars C.

    2014-01-01

    The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to ...

  4. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

    OpenAIRE

    Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

    2013-01-01

    The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases th...

  5. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

    Czech Academy of Sciences Publication Activity Database

    Kašpárek, Petr; Krausová, Michaela; Hanečková, Radka; Kříž, Vítězslav; Žbodáková, Olga; Kořínek, Vladimír; Sedláček, Radislav

    2014-01-01

    Roč. 588, č. 21 (2014), 3982-3988. ISSN 0014-5793 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA14-33952S; GA MŠk(CZ) LM2011032 Institutional support: RVO:68378050 Keywords : Gene targeting * Homologous recombination * Rosa26 locus * Transgenic * Transcription activator-like effector nucleases * Zinc-finger nucleases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.169, year: 2014

  6. High performance bi-functional quantum cascade laser and detector

    Science.gov (United States)

    Schwarz, Benedikt; Ristanic, Daniela; Reininger, Peter; Zederbauer, Tobias; MacFarland, Donald; Detz, Hermann; Andrews, Aaron Maxwell; Schrenk, Werner; Strasser, Gottfried

    2015-08-01

    An improved bi-functional quantum cascade laser and detector emitting and detecting around 6.8 μ m is demonstrated. The design allows a significantly higher laser performance, showing that bi-functional designs can achieve a comparable pulsed performance to conventional quantum cascade lasers. In particular, the device has a threshold current density of 3 kA / cm 2 , an output power of 0.47 W , and a total wall-plug efficiency of 4.5% in pulsed mode. Optimized electron extraction and the prevention of thermal backfilling allow higher duty cycles, operation up to 10%, with 15 mW average output power at room temperature without optimization of the laser cavity or coatings. At zero bias, the device has a responsivity of around 40 mA / W and a noise equivalent power of 80 pW / √{ Hz } at room temperature, which in on-chip configuration outperforms conventional uncooled discrete detectors.

  7. Investigation of new bifunctional agents. D-Penicillamine

    International Nuclear Information System (INIS)

    Somatostatin inhibits the release of growth hormone (somatotropin) from the Anterior Pituitary. The main use of derivatives of somatostatin is to diagnose growth hormone problems and to use against some forms of cancer which involve growth hormone. Also somatostatin suppresses gastric acid secretion, gallbladder contractions, and pancreatic enzyme secretion. The aim of the current study is to investigate new bifunctional agents for labeling with 99mTc. Therefore D-Penicillamine was used as a bifunctional agent and compared to DTPA in the labeling with 99mTc. Quality controls were established using thin layer radio chromatography (TLRC) and electrophoresis techniques. In addition, high performance liquid radio chromatography (HPLRC) was also performed for elimination of possible uncertainties. The radiolabeled complexes maintained their stabilities throughout the study. The results obtained showed that 99mTc-D-Penicillamine-somatostatin is a promising potential radiopharmaceutical and an alternative of 99mTc-DTPA-somatostatin for in vivo and in vitro applications. (author)

  8. Nanoscale intimacy in bifunctional catalysts for selective conversion of hydrocarbons

    Science.gov (United States)

    Zečević, Jovana; Vanbutsele, Gina; de Jong, Krijn P.; Martens, Johan A.

    2016-01-01

    The ability to precisely control nanoscale features is increasingly exploited to develop and improve monofunctional catalysts1–4. Striking effects might also be expected in the case of bifunctional catalysts, which play an important role in hydrocracking of fossil and renewable hydrocarbon sources to provide high-quality diesel fuel5–7. Such bifunctional hydrocracking catalysts contain metal sites and acid sites, and for more than 50 years the so-called ‘intimacy criterion’8 has dictated the maximum distance between the two site types beyond which catalytic activity decreases. The lack of synthesis and material characterization methods with nanometer precision has long prevented in-depth exploration of the criterion, which has often been interpreted simply as ‘the closer the better’ for positioning metal and acid sites8–11. Here we show for a bifunctional catalyst, comprised of an intimate mixture of zeolite Y and alumina binder and with platinum (Pt) metal controllably deposited20,21 on either the zeolite or the binder, that close proximity between metal and zeolite acid sites can be detrimental: the selectivity when cracking large hydrocarbon feedstock molecules for high-quality diesel production is optimized with the catalyst that contains Pt on the binder, i.e. with a larger distance between metal and acid sites. Cracking of the large and complex hydrocarbon molecules typically derived from alternative sources such as gas-to-liquid technology, vegetable oil or algal oil6–7 should thus benefit especially from bifunctional catalysts that avoid locating Pt on the zeolite as the traditionally assumed optimal location. More generally, we anticipate that the ability to spatially organize different active sites at the nanoscale demonstrated here will benefit the further development and optimization of the newly emerging generation of multifunctional catalysts12–15. PMID:26659185

  9. Nanoscale intimacy in bifunctional catalysts for selective conversion of hydrocarbons

    Science.gov (United States)

    Zecevic, Jovana; Vanbutsele, Gina; de Jong, Krijn P.; Martens, Johan A.

    2015-12-01

    The ability to control nanoscale features precisely is increasingly being exploited to develop and improve monofunctional catalysts. Striking effects might also be expected in the case of bifunctional catalysts, which are important in the hydrocracking of fossil and renewable hydrocarbon sources to provide high-quality diesel fuel. Such bifunctional hydrocracking catalysts contain metal sites and acid sites, and for more than 50 years the so-called intimacy criterion has dictated the maximum distance between the two types of site, beyond which catalytic activity decreases. A lack of synthesis and material-characterization methods with nanometre precision has long prevented in-depth exploration of the intimacy criterion, which has often been interpreted simply as ‘the closer the better’ for positioning metal and acid sites. Here we show for a bifunctional catalyst—comprising an intimate mixture of zeolite Y and alumina binder, and with platinum metal controllably deposited on either the zeolite or the binder—that closest proximity between metal and zeolite acid sites can be detrimental. Specifically, the selectivity when cracking large hydrocarbon feedstock molecules for high-quality diesel production is optimized with the catalyst that contains platinum on the binder, that is, with a nanoscale rather than closest intimacy of the metal and acid sites. Thus, cracking of the large and complex hydrocarbon molecules that are typically derived from alternative sources, such as gas-to-liquid technology, vegetable oil or algal oil, should benefit especially from bifunctional catalysts that avoid locating platinum on the zeolite (the traditionally assumed optimal location). More generally, we anticipate that the ability demonstrated here to spatially organize different active sites at the nanoscale will benefit the further development and optimization of the emerging generation of multifunctional catalysts.

  10. [Bifunctional chelates of Rh-105, Au-199, and other metallic radionuclides as potential radiotherapeutic agents

    International Nuclear Information System (INIS)

    Progress during this period is reported under the following headings: Diethylenetriamine based and related bifunctional chelating agents and their complexation with Rh-105, Au-198, Pd-109, cu-67, In-111, and Co-57; studies of Pd-109, Rh-105 and Tc-99m with bifunctional chelates based on phenylenediamine; establishment of an appropriate protein assay method for conjugated proteins; studies of new bifunctional Bi, Tri and tetradentate amine oxime ligands with Rh-105; IgG and antibody B72.3 conjugation studies by HPLC Techniques with bifunctional metal chelates; and progress on ligand systems for Au(III)

  11. [Bifunctional chelates of Rh-105, Au-199, and other metallic radionuclides as potential radiotherapeutic agents

    Energy Technology Data Exchange (ETDEWEB)

    1991-12-31

    Progress during this period is reported under the following headings: Diethylenetriamine based and related bifunctional chelating agents and their complexation with Rh-105, Au-198, Pd-109, cu-67, In-111, and Co-57; studies of Pd-109, Rh-105 and Tc-99m with bifunctional chelates based on phenylenediamine; establishment of an appropriate protein assay method for conjugated proteins; studies of new bifunctional Bi, Tri and tetradentate amine oxime ligands with Rh-105; IgG and antibody B72.3 conjugation studies by HPLC Techniques with bifunctional metal chelates; and progress on ligand systems for Au(III).

  12. (Bifunctional chelates of Rh-105, Au-199, and other metallic radionuclides as potential radiotherapeutic agents)

    Energy Technology Data Exchange (ETDEWEB)

    1991-01-01

    Progress during this period is reported under the following headings: Diethylenetriamine based and related bifunctional chelating agents and their complexation with Rh-105, Au-198, Pd-109, cu-67, In-111, and Co-57; studies of Pd-109, Rh-105 and Tc-99m with bifunctional chelates based on phenylenediamine; establishment of an appropriate protein assay method for conjugated proteins; studies of new bifunctional Bi, Tri and tetradentate amine oxime ligands with Rh-105; IgG and antibody B72.3 conjugation studies by HPLC Techniques with bifunctional metal chelates; and progress on ligand systems for Au(III).

  13. Single-strand nuclease action on heat-denatured spermiogenic chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Hunter, J.D.; Bodner, A.J.; Hatch, F.T.; Balhorn, R.L.; Mazrimas, J.A.; McQueen, A.P.; Gledhill, B.L.

    1976-01-01

    The sensitivity of chromatin was compared from representative cellular stages of spermiogenesis to a single-stranded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S/sub 1/ nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90/sup 0/C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60/sup 0/C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80/sup 0/C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of roundspermatids labeled with /sup 3/H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot trichloracetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgenstainable material occurred in these cells and may be due to depurination and elimination of Fuelgren

  14. Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters.

    Science.gov (United States)

    Wang, Lihui; Ma, Keke; Zhang, Yaodong

    2014-09-28

    S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uμl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uμl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis. PMID:25263815

  15. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    Science.gov (United States)

    Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

    2015-04-01

    The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

  16. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    Science.gov (United States)

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  17. AdnAB: a new DSB-resecting motor-nuclease from mycobacteria.

    Science.gov (United States)

    Sinha, Krishna Murari; Unciuleac, Mihaela-Carmen; Glickman, Michael S; Shuman, Stewart

    2009-06-15

    The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase-nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase-nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16-17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor-nuclease complexes. PMID:19470566

  18. Nuclease-mediated genome editing: At the front-line of functional genomics technology.

    Science.gov (United States)

    Sakuma, Tetsushi; Woltjen, Knut

    2014-01-01

    Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose. PMID:24387662

  19. TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES-AMAZING TOOL FOR GENOME EDITING

    Directory of Open Access Journals (Sweden)

    Avantika Singh

    2013-01-01

    Full Text Available Transcription Activator-Like Effector Nucleases (TALENs are used to cut specific target DNA sequence in order to knock out a gene or to change its sequence. They are formed by the fusion of TALE protein serving as DNA binding domain along with non-specific DNA cleaving nucleases. Its specificity towards DNA bases in the target sequence is derived from the structure of the DNA binding domain, consisting of variable pair of amino acids in middle of the tandem array of repeated segment. Earlier targeted genome editing was performed using Zinc Finger Nucleases (ZFN. Recently TALENS have rapidly emerged as alternative to Zinc Finger Nucleases for genome editing and introducing targeted Double Stranded Break. The TALEN approach improves on tools currently available for genome modification. This breakthrough could eventually make it possible to efficiently modify plant, animal and even human genomes. Targeted nucleases offer the potential to correct or disrupt the gene product or sequences that causes the disorder and thus motivates the strategies for treatment of wide range of genetic and other diseases. The road to practical use of TALENs could still contain potholes. But particularly to academics, the potential of TALENs seems limitless.

  20. Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases.

    Science.gov (United States)

    Hu, Johnny H; Davis, Kevin M; Liu, David R

    2016-01-21

    Programmable DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR-Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA sequence. In their canonical forms, these nucleases induce double-strand breaks at a DNA locus of interest that can trigger cellular DNA repair processes that disrupt or replace genes. The fusion of these programmable nucleases with a variety of other protein domains has led to a rapidly growing suite of tools for activating, repressing, visualizing, and modifying loci of interest. Maximizing the usefulness and therapeutic relevance of these tools, however, requires precisely controlling their activity and specificity to minimize potentially toxic side effects arising from off-target activities. This need has motivated the application of chemical biology principles and methods to genome-editing proteins, including the engineering of variants of these proteins with improved or altered specificities, and the development of genetic, chemical, optical, and protein delivery methods that control the activity of these agents in cells. Advancing the capabilities, safety, effectiveness, and therapeutic relevance of genome-engineering proteins will continue to rely on chemical biology strategies that manipulate their activity, specificity, and localization. PMID:26933736

  1. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    This paper describes the development of a 5' nuclease assay for detection of virulence factor genes responsible for colonization factors and toxins in Escherichia coil isolated from pigs. Colonization factors were F4, F5, F6, F18, F41 and the outer membrane protein intimin. Toxins were heat stable...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

  2. Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

    International Nuclear Information System (INIS)

    DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems

  3. Interaction of nuclease colicins with membranes: insertion depth correlates with bilayer perturbation.

    Directory of Open Access Journals (Sweden)

    Mireille Vankemmelbeke

    Full Text Available BACKGROUND: Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases or RNA (RNases hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7 and one ribosomal RNase domain (E3 using a biomembrane model system. PRINCIPAL RESULTS: We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed. CONCLUSIONS/SIGNIFICANCE: Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.

  4. Bifunctional chelating agents for targeted α-particle radiotherapy

    International Nuclear Information System (INIS)

    An α-emitting radionuclide is proposed as a better choice for application in radiotherapy of either leukemias or lymphomas due to very high cytotoxicity, short emission path length, and immediate energy deposition minimizing collateral cytotoxicity. Metallic α-emitters that have been studied are 212Bi and 213Bi. Bifunctional derivatives of diethylenetriamine pentaacetic acid (DTPA) were found to form Bi(III) complexes that were labile in vivo. Pre-clinical experiments confirmed both the stability of the CHX-DTPA ligands for the Bi(III) isotopes and the therapeutic applicability of these α-emitting isotopes

  5. An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

    Science.gov (United States)

    Peng, Lan; Fan, Jialong; Tong, Chunyi; Xie, Zhenhua; Zhao, Chuan; Liu, Xuanming; Zhu, Yonghua; Liu, Bin

    2016-09-15

    Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo. PMID:27125839

  6. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

    Czech Academy of Sciences Publication Activity Database

    Stránský, J.; Koval, Tomáš; Podzimek, T.; Týcová, A.; Lipovová, P.; Matoušek, J.; Kolenko, Petr; Fejfarová, Karla; Dušková, J.; Skálová, T.; Hašek, J.; Dohnálek, Jan

    2015-01-01

    Roč. 71, č. 11 (2015), s. 1408-1415. ISSN 2053-230X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease Subject RIV: CE - Biochemistry

  7. Comprehensive analysis of the specificity of transcription activator-like effector nucleases

    DEFF Research Database (Denmark)

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien;

    2014-01-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their...... target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator...

  8. Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.

    OpenAIRE

    Antonino, L C; Kautz, R. A.; Nakano, T.; Fox, R. O.; Fink, A L

    1991-01-01

    Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28...

  9. Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

    OpenAIRE

    Kooistra, J; Venema, G

    1991-01-01

    The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA pro...

  10. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

    OpenAIRE

    Tsai, Shengdar Q.; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A.; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J.; Aryee, Martin J; Joung, J. Keith

    2014-01-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI Nucleases (RFNs) that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wi...

  11. Multispot array combined with S1 nuclease-mediated elimination of unpaired nucleotides

    DEFF Research Database (Denmark)

    Yoo, Seung Min; Kim, Dong Min; Lee, Sang Yup

    2015-01-01

    The accurate detection of mismatched base pairs is critical to many DNA hybridization-based applications in basic research and diagnostics. We herein demonstrate that mismatched DNAs on a multispot array can be accurately detected in a multiplexed way by employing the S1 nuclease-based mismatched...

  12. Programmable Site-Specific Nucleases for Targeted Genome Engineering in Higher Eukaryotes.

    Science.gov (United States)

    Govindan, Ganesan; Ramalingam, Sivaprakash

    2016-11-01

    Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site-specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site-specific genomic double-strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error-prone non-homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology-directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here, we summarize the role of three classes of nucleases; zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site-specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. J. Cell. Physiol. 231: 2380-2392, 2016. © 2016 Wiley Periodicals, Inc. PMID:26945523

  13. MegaTevs: single-chain dual nucleases for efficient gene disruption.

    Science.gov (United States)

    Wolfs, Jason M; DaSilva, Matthew; Meister, Sarah E; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R

    2014-07-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications. PMID:25013171

  14. Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects

    Czech Academy of Sciences Publication Activity Database

    Souček, J.; Škvor, J.; Poučková, P.; Matoušek, Jaroslav; Slavík, Tomáš; Matoušek, Josef

    2006-01-01

    Roč. 53, - (2006), s. 402-409. ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA523/04/0755 Keywords : mung bean * nuclease Subject RIV: FD - Oncology ; Hematology Impact factor: 1.247, year: 2006

  15. Antitumor and biological effects of black pine (Pinus nigra) pollen nuclease

    Czech Academy of Sciences Publication Activity Database

    Lipovová, P.; Podzimek, T.; Orctová, Lidmila; Matoušek, Jaroslav; Poučková, P.; Souček, J.; Matoušek, Josef

    2008-01-01

    Roč. 55, - (2008), s. 158-164. ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : pollen nuclease * Antitumor effect Subject RIV: FD - Oncology ; Hematology Impact factor: 1.179, year: 2008

  16. Application of a 5 ' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs

    DEFF Research Database (Denmark)

    Lindecrona, R. H.; Jensen, Tim Kåre; Andersen, P. H.; Møller, Kristian

    2002-01-01

    fecal samples derived from a herd known to be free from infection with L. intracellularis all tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1 L. intracellularis cell per PCR tube. In conclusion, the 5' nuclease assay that has...

  17. PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase.

    Science.gov (United States)

    Blanga-Kanfi, Shani; Amitsur, Michal; Azem, Abdussalam; Kaufmann, Gabriel

    2006-01-01

    The tRNA(Lys) anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129-143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNA(Lys) recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised. PMID:16790566

  18. Characterization, crystallization and preliminary X-ray diffraction studies of tomato nuclease I

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Dohnálek, Jan; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav

    2009-01-01

    Roč. 16, 1a (2009), b33. ISSN 1211-5894. [Discussions in Structural Molecular Biology /7./. 12.03.2009-14.03.2009, Nové Hrady] Institutional research plan: CEZ:AV0Z40500505 Keywords : X-ray diffraction * tomato nuclease Subject RIV: CD - Macromolecular Chemistry

  19. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, J.; Dušková, J.; Skálová, T.; Štěpánková, A.; Hašek, J.; Dohnálek, Jan

    2013-01-01

    Roč. 69, č. 2 (2013), s. 213-226. ISSN 0907-4449 Grant ostatní: AVČR(CZ) Praemium Academiae Institutional support: RVO:68378271 Keywords : TBN1 * nuclease * protein * x-ray structure Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 7.232, year: 2013

  20. Insights into how RNase R degrades structured RNA: analysis of the nuclease domain.

    Science.gov (United States)

    Vincent, Helen A; Deutscher, Murray P

    2009-04-01

    RNase R readily degrades highly structured RNA, whereas its paralogue, RNase II, is unable to do so. Furthermore, the nuclease domain of RNase R, devoid of all canonical RNA-binding domains, is sufficient for this activity. RNase R also binds RNA more tightly within its catalytic channel than does RNase II, which is thought to be important for its unique catalytic properties. To investigate this idea further, certain residues within the nuclease domain channel of RNase R were changed to those found in RNase II. Among the many examined, we identified one amino acid residue, R572, that has a significant role in the properties of RNase R. Conversion of this residue to lysine, as found in RNase II, results in weaker substrate binding within the nuclease domain channel, longer limit products, increased activity against a variety of substrates and a faster substrate on-rate. Most importantly, the mutant encounters difficulty in degrading structured RNA, pausing within a double-stranded region. Additional studies show that degradation of structured substrates is dependent upon temperature, suggesting a role for thermal breathing in the mechanism of action of RNase R. On the basis of these data, we propose a model in which tight binding within the nuclease domain allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNAs. PMID:19361424

  1. Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

    Science.gov (United States)

    Zhang, Hui; Zhao, Gang; Guo, Yusi; Menghwar, Harish; Chen, Yingyu; Chen, Huanchun; Guo, Aizhen

    2016-01-01

    This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca(2+) at 22-65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNase(Δ181-342) without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis. PMID:27136546

  2. Synthesis and Characterization of a New Bifunctional Dye Containing Spirobenzopyran and Cinnamoyl Moiety

    Institute of Scientific and Technical Information of China (English)

    申凯华; 崔东熏

    2005-01-01

    A novel bifunctional dye containing spirobenzopyran and cinnaznoyl moiety has been prepared and its photochromic behavior following irradiation at different wavelengths of monochrome UV light was investigated.The colourless bifunctional dye in film or solution exhibits unusual photochromism through structural and geometrical transformation from spirobenzopyran to merocyanine accompanying with photocrosslinking reaction in cinnamoyl moieties. Two kinds of photochemical reaction were achieved by irradiation at the different wavelengths of monochrome UV light (275 nm, 365 nm) selectively. The photochromic process of the bifunctional dye was discussed and the dynamic behaviors of the decolorization process were investigated.

  3. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    International Nuclear Information System (INIS)

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with 32Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of 32P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of 32P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the 32P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, [32P] HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14

  4. Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines*

    OpenAIRE

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2009-01-01

    Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (kcat 415 s−1), and the affinity of the motor for the ssDNA cofa...

  5. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

    DEFF Research Database (Denmark)

    Wendt, Toni; Holm, Preben Bach; Starker, Colby G;

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their...... broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation...

  6. Chemoselective Reactivity of Bifunctional Cyclooctynes on Si(001)

    CERN Document Server

    Reutzel, Marcel; Lipponer, Marcus A; Länger, Christian; Höfer, Ulrich; Koert, Ulrich; Dürr, Michael

    2016-01-01

    Controlled organic functionalization of silicon surfaces as integral part of semiconductor technology offers new perspectives for a wide range of applications. The high reactivity of the silicon dangling bonds, however, presents a major hindrance for the first basic reaction step of such a functionalization, i.e., the chemoselective attachment of bifunctional organic molecules on the pristine silicon surface. We overcome this problem by employing cyclooctyne as the major building block of our strategy. Functionalized cyclooctynes are shown to react on Si(001) selectively via the strained cyclooctyne triple bond while leaving the side groups intact. The achieved selectivity originates from the distinctly different adsorption dynamics of the separate functionalities: A direct adsorption pathway is demonstrated for cyclooctyne as opposed to the vast majority of other organic functional groups. The latter ones react on Si(001) via a metastable intermediate which makes them effectively unreactive in competition wi...

  7. Bifunctional chelates of RH-105 and AU199 as potential radiotherapeutic agents

    Energy Technology Data Exchange (ETDEWEB)

    Droege, P.

    1997-03-01

    Research is presented on new bifunctional chelating ligand systems with stability on the macroscopic and radiochemical levels. The synthesis of the following complexes are described: rhodium 105, palladium 109, and gold 198.

  8. Bifunctional chelating agent for the design and development of site specific radiopharmaceuticals and biomolecule conjugation strategy

    Science.gov (United States)

    Katti, Kattesh V.; Prabhu, Kandikere R.; Gali, Hariprasad; Pillarsetty, Nagavara Kishore; Volkert, Wynn A.

    2003-10-21

    There is provided a method of labeling a biomolecule with a transition metal or radiometal in a site specific manner to produce a diagnostic or therapeutic pharmaceutical compound by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radio metal or a transition metal, and covalently linking the resulting metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. Also provided is a method of synthesizing the --PR.sub.2 containing biomolecules by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radiometal or a transition metal, and covalently linking the resulting radio metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. There is provided a therapeutic or diagnostic agent comprising a --PR.sub.2 containing biomolecule.

  9. Novel 16-substituted bifunctional derivatives of huperzine B: multifunctional cholinesterase inhibitors

    OpenAIRE

    Shi, Yu-fang; Zhang, Hai-Yan; Wang, Wei; Fu, Yan; Xia, Yu; Tang, Xi-can; Bai, Dong-lu; He, Xu-chang

    2009-01-01

    Aim: To design novel bifunctional derivatives of huperzine B (HupB) based on the concept of dual binding site of acetylcholinesterase (AChE) and evaluate their pharmacological activities for seeking new drug candidates against Alzheimer's disease (AD). Methods: Novel 16-substituted bifunctional derivatives of HupB were synthesized through chemical reactions. The inhibitory activities of the derivatives toward AChE and butyrylcholinesterase (BuChE) were determined in vitro by modified Ellman's...

  10. Bifunctional Pt-Si Alloys for Small Organic Molecule Electro-oxidation

    DEFF Research Database (Denmark)

    Permyakova, Anastasia Aleksandrovna; Suntivich, Jin; Han, Binghong;

    bifunctional requirement, which demands both adsorption and water oxidation sites. In this contribution, we explore the possibility of using Pt-Si alloys to fulfill this bifunctional requirement. Silicon, a highly oxophillic element, is alloyed into Pt as a site for water oxidation, while Pt serves as a CO...... adsorption site. We will discuss the enhanced activity of Pt-Si alloys for small organic molecule oxidation, which can be attributed to the improved CO electro-oxidation kinetics on Pt-Si....

  11. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Genetically Modified Organisms (GMO

    2012-10-01

    Full Text Available

    The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3 which allows the integration of gene(s in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3 is used to describe the technique rather than ZFN-3 specifically. The EFSA GMO Panel considers that its guidance documents are applicable for the evaluation of food and feed products derived from plants developed using the SDN-3 technique and for performing an environmental risk assessment. However, on a case-by-case basis lesser amounts of event specific data may be needed for the risk assessment of plants developed using the SDN-3 technique. The EFSA GMO Panel compared the hazards associated with plants produced by the SDN-3 technique with those obtained by conventional plant breeding techniques and by currently used transgenesis. With respect to the genes introduced, the SDN-3 technique does not differ from transgenesis or from the other genetic modification techniques currently used, and can be used to introduce transgenes, intragenes or cisgenes. The main difference between the SDN-3 technique and transgenesis is that the insertion of DNA is targeted to a predefined region of the genome. Therefore, the SDN-3 technique can minimise hazards associated with the disruption of genes and/or regulatory elements in the recipient genome. Whilst the SDN-3 technique can induce off-target changes in the genome of the recipient plant these would be fewer than those occurring with most mutagenesis techniques. Furthermore, where such changes occur they would be of the same types as those produced by conventional breeding techniques.

  12. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

    OpenAIRE

    Vagner Laura L; Ziganshin Rustam; Staroverov Dmitry B; Menzorova Natalia I; Kozhemyako Valery B; Shagin Dmitry A; Rebrikov Denis V; Anisimova Veronika E; Rasskazov Valery A; Lukyanov Sergey A; Shcheglov Alex S

    2008-01-01

    Abstract Background Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properti...

  13. GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

    OpenAIRE

    Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith

    2014-01-01

    CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUID...

  14. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

    Czech Academy of Sciences Publication Activity Database

    Stránský, Jan; Koval, Tomáš; Podzimek, T.; Týcová, Anna; Lipovová, P.; Matoušek, Jaroslav; Kolenko, Petr; Fejfarová, Karla; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

    2015-01-01

    Roč. 71, č. 11 (2015), s. 1408-1415. ISSN 2053-230X R&D Projects: GA MŠk LG14009; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:60077344 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease * superhelix Subject RIV: EB - Genetics ; Molecular Biology

  15. Dynamics and denaturation of a protein. Simulations and neutron scattering on staphylococcus nuclease

    International Nuclear Information System (INIS)

    This research thesis reports simulations and experiments of inelastic scattering on the whole frequency spectrum to analyse the vibrations of the staphylococcus nuclease and its fragment, in order to study protein folding. Based on these experiments, information on eigenvectors which describe vibration modes can be directly obtained. Inelastic intensities are indeed fully determined by nuclear cross sections and the mean square displacement of each atom. Some experimentally noticed peaks are then explained by calculating a theoretical spectrum from an analysis of normal modes. The studied fragment is made of 136 c-terminal residues. The fragment structure obtained by molecular dynamics simulation is compared with available experimental data. Then, experiments of neutron scattering on the nuclease of staphylococcus and its fragment have been performed. Quasi elastic scattering spectra have been measured. The author then used simulations to try to reproduce the quasi-elastic spectrum. Experiments of inelastic scattering have then been performed

  16. Studies on a Novel Minor-groove Targeting Artificial Nuclease: Synthesis and DNA Binding Behavior

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleases play an important role in molecular biology, for example, in DNA sequencing. Synthetic polyamide conjugates can be considered as a novel tool for the selective inhibition of gene expressions and also as potential drugs in anticancer or antiviral chemotherapy. In this article, the synthesis of a novel minor-groove targeting artificial nuclease, an oligopyrrol-containing compound, has been reported. It was found that this novel compound can bind DNA in AT-rich minor groove with high affinity and site specificity. DNA binding behavior was determined by using UV-Vis and CD. It is indicated that compound 6 can enhance the Tm of DNA from 80. 4 C to 84. 4 ℃ and that it possesses a high binding constant value(Kb = 3.05×104 L/mol).

  17. Transcribed single nucleotide polymorphism: Ideal markers for detecting gene imprinting by 5' nuclease assay

    Institute of Scientific and Technical Information of China (English)

    ZHU Guan-shan; WAN Mo-bin; ZHU Zhong-zheng; ZHENG Rui-ying

    2002-01-01

    Objective:To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705 (C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lymphoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN,which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism.

  18. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA.

    Science.gov (United States)

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping

    2016-02-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction. PMID:26688865

  19. Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH)

    Institute of Scientific and Technical Information of China (English)

    ZHEN Yu; MI Tiezhu; YU Zhigang

    2008-01-01

    Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China. In this study, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was used to qualitatively and quantitatively detect P. globosa. Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields. The linear regression equation for P. globosa was obtained, and the lowest detection number of cells was 1.8×104 cells. Statistics showed that there was no distinct difference between the results of detecting the microalgae by NPA-SH and traditional microscopy. This technique has good reliability, accuracy, and can give a remarkably high sample processing rate. Sandwich hybridization integrated with nuclease protection assay will provide an efficient alternative to microscopic method for monitoring and investigating the bloom of P. globosa.

  20. Studies of interaction between a new synthesized minor-groove targeting artificial nuclease and DNA

    Science.gov (United States)

    Yin, Qiang; Zhang, Zhen; Zhao, Yu-Fen

    2007-04-01

    Nuclease plays an important role in molecular biology, such as DNA sequencing. Synthetic polyamide conjugates can be considered as new tool in the selective inhibition of gene expression and as potential drugs in anticancer or antiviral chemotherapy. In this paper, a new synthesized minor-groove targeting artificial nuclease, oligopyrrol-containing peptide, was reported. It was found that this new compound can bind DNA in AT-riched minor groove with high affinity and site specificity. DNA binding behavior was determined by UV-vis and circular dichroism (CD) methods. It was indicated that compound 6 can enhance the Tm of oligomer DNA from 51.8 to 63.5 °C and possesses large binding constant ( Kb = 8.83 × 10 4 L/mol).

  1. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    (STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

  2. Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

    Directory of Open Access Journals (Sweden)

    Zylicz-Stachula Agnieszka

    2009-05-01

    Full Text Available Abstract Background Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases, however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II. Results Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase and methyltransferase (MTase activities of wild type (wt TspGWI (either recombinant or isolated from Thermus sp. are dependent on the presence of divalent cations. Conclusion TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/EXK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module

  3. Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

    OpenAIRE

    Terazono Hideyuki; Anzai Yu; Soloviev Mikhail; Yasuda Kenji

    2010-01-01

    Abstract A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by ...

  4. Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

    OpenAIRE

    Onishi, Hiroshi; Mori, Tatsuro; Takeuchi, Setsuo; Tani, Keiko; KOBAYASHI, Takekazu; Kamekura, Masahiro

    1983-01-01

    A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular wei...

  5. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    OpenAIRE

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Ping WANG; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for ea...

  6. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    OpenAIRE

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells.

  7. Plant Ribonucleases and Nucleases as Antiproliferative Agens Targeting Human Tumors Growing in Mice

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Matoušek, Josef

    2010-01-01

    Roč. 4, č. 1 (2010), s. 29-39. ISSN 1872-2156 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : antiproliferative cytotoxic * effect human * plant nuclease Subject RIV: EB - Genetics ; Molecular Biology

  8. Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

    OpenAIRE

    Mueller, Niklaus H.; Gilden, Don; Cohrs, Randall J.

    2013-01-01

    Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic t...

  9. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  10. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping; Gjoka, Monika; Duda, Katarzyna; Taunton, Jack; Collingwood, Trevor N; Frödin, Morten; Davis, Gregory D

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mut...... mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions....

  11. Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases

    OpenAIRE

    Li XU; Zhao, Piming; Mariano, Andrew; Han, Renzhi

    2013-01-01

    Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin ge...

  12. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

    OpenAIRE

    Citorik, Robert J.; Mimee, Mark; Lu, Timothy K.

    2014-01-01

    Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes...

  13. Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

    Science.gov (United States)

    Butler, Nathaniel M.; Baltes, Nicholas J.; Voytas, Daniel F.; Douches, David S.

    2016-01-01

    Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

  14. Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.

    Science.gov (United States)

    Butler, Nathaniel M; Baltes, Nicholas J; Voytas, Daniel F; Douches, David S

    2016-01-01

    Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

  15. Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.

    Science.gov (United States)

    Crossan, Gerry P; van der Weyden, Louise; Rosado, Ivan V; Langevin, Frederic; Gaillard, Pierre-Henri L; McIntyre, Rebecca E; Gallagher, Ferdia; Kettunen, Mikko I; Lewis, David Y; Brindle, Kevin; Arends, Mark J; Adams, David J; Patel, Ketan J

    2011-02-01

    The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway. PMID:21240276

  16. Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.

    Science.gov (United States)

    Jo, Young-Il; Kim, Hyongbum; Ramakrishna, Suresh

    2015-10-01

    Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17 years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology. PMID:26089249

  17. Magnetic-plasmonic bifunctional CoO–Ag heterostructure nanoparticles

    International Nuclear Information System (INIS)

    We demonstrate the synthesis of CoO–Ag heterostructure nanoparticles by chemical reduction of AgNO3 in the presence of Co nanoparticles in oleylamine (OAm). OAm plays multiple roles as a surfactant, solvent, and reducing agent. The mechanism of surface-activated heterogeneous nucleation and growth on the preformed seeds has been proposed. At the same time, the Co nanoparticles are oxidized to form hollow CoO nanoparticles through the Kirkendall effect. The resulting CoO–Ag heterostructures display mushroom-like morphology, Ag nanoparticle as ‘cap’ attached on the ‘stem’ of hollow CoO nanoparticles. The size of Ag domains in the heterostructure nanoparticles can be tuned by controlling the volume of Co nanoparticles. The plasmonic absorption and the magnetization of the bifunctional nanoparticles were investigated. The combination of the hollow structure of the CoO and the surface plasmon resonances of the Ag domains may make them suitable for catalysis, drug delivery, therapy, and surface-enhanced Raman scattering. (papers)

  18. Gold(I) catalysts with bifunctional P, N ligands.

    Science.gov (United States)

    Wetzel, Corinna; Kunz, Peter C; Thiel, Indre; Spingler, Bernhard

    2011-08-15

    A series of phosphanes with imidazolyl substituents were prepared as hemilabile PN ligands. The corresponding gold(I) complexes were tested as bifunctional catalysts in the Markovnikov hydration of 1-octyne, as well as in the synthesis of propargylamines by the three component coupling reaction of piperidine, benzaldehyde, and phenylacetylene. While the activity in the hydration of 1-octyne was low, the complexes are potent catalysts for the three component coupling reaction. In homogeneous solution the conversions to the respective propargylamine were considerably higher than under aqueous biphasic conditions. The connectivity of the imidazolyl substituents to the phosphorus atom, their substitution pattern, as well as the number of heteroaromatic substituents have pronounced effects on the catalytic activity of the corresponding gold(I) complexes. Furthermore, formation of polymetallic species with Au(2), Au(3), and Au(4) units has been observed and the solid-state structures of the compounds [(5)(2)Au(3)Cl(2)]Cl and [(3c)(2)Au(4)Cl(2)]Cl(2) (3c = tris(2-isopropylimidazol-4(5)-yl phosphane, 5 = 2-tert-butylimidazol-4(5)-yldiphenyl phosphane) were determined. The gold(I) complexes of imidazol-2-yl phosphane ligands proved to be a novel source for bis(NHC)gold(I) complexes (NHC = N-heterocyclic carbene). PMID:21761834

  19. Gold-Copper Nanoparticles: Nanostructural Evolution and Bifunctional Catalytic Sites

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Jun; Shan, Shiyao; Yang, Lefu; Mott, Derrick; Malis, Oana; Petkov, Valeri; Cai, Fan; Ng, Mei; Luo, Jin; Chen, Bing H.; Engelhard, Mark H.; Zhong, Chuan-Jian

    2012-12-12

    Understanding of the atomic-scale structure is essential for exploiting the unique catalytic properties of any nanoalloy catalyst. This report describes novel findings of an investigation of the nanoscale alloying of gold-copper (AuCu) nanoparticles and its impact on the surface catalytic functions. Two pathways have been explored for the formation of AuCu nanoparticles of different compositons, including wet chemical synthesis from mixed Au- and Cu-precursor molecules, and nanoscale alloying via an evolution of mixed Au- and Cu-precursor nanoparticles near the nanoscale melting temperatures. For the evolution of mixed precursor nanoparticles, synchrotron x-ray based in-situ real time XRD was used to monitor the structural changes, revealing nanoscale alloying and reshaping towards an fcc-type nanoalloy (particle or cube) via a partial melting–resolidification mechanism. The nanoalloys supported on carbon or silica were characterized by in-situ high-energy XRD/PDFs, revealing an intriguing lattice "expanding-shrinking" phenomenon depending on whether the catalyst is thermochemically processed under oxidative or reductive atmosphere. This type of controllable structural changes is found to play an important role in determining the catalytic activity of the catalysts for carbon monoxide oxidation reaction. The tunable catalytic activities of the nanoalloys under thermochemically oxidative and reductive atmospheres are also discussed in terms of the bifunctional sites and the surface oxygenated metal species for carbon monoxide and oxygen activation.

  20. Novel Polyamide Proton Exchange Membranes with Bi-Functional Sulfonimide Bridges for Fuel Cell Applications

    International Nuclear Information System (INIS)

    Graphical abstract: A polymer proton conductor crosslinked with bi-functional sulfonamide bridges is synthesized for PEM fuel cell applications. The architecture simultaneously enhances mechanical strength and improves water retention of the PEMs. With an appropriate degree of crosslinking, the bi-functional PEM exhibits comparable performance to that of a commercial Nafion membrane tested in a direct methanol fuel cell. - Abstract: We design and successfully synthesize non-fluorinated polyamides with controlled crosslinking using sulfonimide as a bi-functional linker to interconnect polymer backbones and as a bridge for proton conduction. We show that the bi-functional linkers are highly beneficial not only for mechanical enforcement of the proton exchange membranes but also for enhancement of water retention capacity. With an appropriate degree of crosslinking, higher water retention capacity than that of commercial Nafion membranes can be obtained. The maximum proton conductivity of the membranes is found to be as high as 0.139 S cm−1 at 80 °C, almost the same as that of a Nafion 117 membrane. Excellent performance with the bi-functional polymer membranes in an air-breathing direct methanol fuel cell prototype, comparable to the performance of a Nafion 117 membrane, is demonstrated

  1. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille;

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha...

  2. Enhanced Bifunctional Oxygen Catalysis in Strained LaNiO3 Perovskites

    Energy Technology Data Exchange (ETDEWEB)

    Petrie, Jonathan R.; Cooper, Valentino R.; Freeland, John W.; Meyer, Tricia L.; Zhang, Zhiyong; Lutterman, Daniel A.; Lee, Ho Nyung

    2016-03-02

    Strain is known to greatly influence low temperature oxygen electro catalysis on noble metal films, leading to significant enhancements in bifunctional activity essential for fuel cells and Metal-air batteries. However, its catalytic impact on transition-metal oxide thin films, such as perovskites, is not widely understood. Here, we epitaxially strain the conducting perovskite LaNiO3 to systematically determine its influence on both the oxygen reduction and oxygen evolution reaction. Uniquely, we found that compressive strain could significantly enhance both reactions, yielding a bifunctional catalyst that surpasses the performance of noble metals' such as Pt. We attribute the improved bifunctionality to strain induced splitting of the e(g) Orbitals, which can customize orbital asymmetry at the surface. Analogous to strain induced shifts in the d-band center of noble metals relative to the Fermi level, :such splitting can dramatically affect catalytic activity in this perovskite and other potentially more active Oxides.

  3. Nickel sulfide microsphere film on Ni foam as an efficient bifunctional electrocatalyst for overall water splitting.

    Science.gov (United States)

    Zhu, Wenxin; Yue, Xiaoyue; Zhang, Wentao; Yu, Shaoxuan; Zhang, Yuhuan; Wang, Jing; Wang, Jianlong

    2016-01-25

    Developing low-cost, efficient, and bifunctional electrocatalysts for both the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) is an appealing yet challenging task. Herein, for the first time, a NiS microsphere film was grown in situ on Ni foam (NiS/Ni foam) via a sulfurization reaction as an efficient bifunctional electrocatalyst for overall water splitting with superior activity and good durability. This NiS/Ni foam electrode delivers 20 mA cm(-2) at an overpotential of 158 mV for the HER and 50 mA cm(-2) at an overpotential of 335 mV for the OER in 1.0 M KOH. This bifunctional electrode also enables a high-efficiency alkaline water electrolyzer with 10 mA cm(-2) at a cell voltage of only 1.64 V, which could be promising in water splitting devices for large-scale hydrogen production. PMID:26661579

  4. Polymer-Supported Reagents: The Role of Bifunctionality in the Design of Ion-Selective Complexants

    Energy Technology Data Exchange (ETDEWEB)

    Alexandratos, S. D.

    2001-06-01

    The importance of multi-functionality in the preparation of ion-selective polymers is evident from the structure of enzymes where specific metal ions are bound through cooperative interactions among different amino acids. In synthetic polymers, ionic selectivity is enhanced when a chemical reaction is superimposed on an ion-exchange process. The concept of reactive ion exchange has been extended through the synthesis of crosslinked polymers whose metal ion selectivity is a function of reduction, coordination or precipitation reactions as determined by various covalently bound ligands. Development of three classes of dual mechanism bifunctional polymers, a new series of bifunctional diphosphonate polymers, and novel bifunctional ion-selective polymers with enhanced ionic accessibility has been accomplished.

  5. Synthesis, characterization and catalytic activity of acid-base bifunctional materials through protection of amino groups

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Yanqiu [College of Chemistry, Jilin University, Changchun 130023 (China); College of Chemistry, Mudanjiang Normal University, Mudanjiang 157012 (China); Liu, Heng; Yu, Xiaofang [College of Chemistry, Jilin University, Changchun 130023 (China); Guan, Jingqi, E-mail: guanjq@jlu.edu.cn [College of Chemistry, Jilin University, Changchun 130023 (China); Kan, Qiubin, E-mail: qkan@mail.jlu.edu.cn [College of Chemistry, Jilin University, Changchun 130023 (China)

    2012-03-15

    Graphical abstract: Acid-base bifunctional mesoporous material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized under low acidic medium through protection of amino groups. Highlights: Black-Right-Pointing-Pointer The acid-base bifunctional material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized through protection of amino groups. Black-Right-Pointing-Pointer The obtained bifunctional material was tested for aldol condensation. Black-Right-Pointing-Pointer The SO{sub 3}H-SBA-15-NH{sub 2} catalyst containing amine and sulfonic acid groups exhibited excellent acid-basic properties. -- Abstract: Acid-base bifunctional mesoporous material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized under low acidic medium through protection of amino groups. X-ray diffraction (XRD), N{sub 2} adsorption-desorption, transmission electron micrographs (TEM), back titration, {sup 13}C magic-angle spinning (MAS) NMR and {sup 29}Si magic-angle spinning (MAS) NMR were employed to characterize the synthesized materials. The obtained bifunctional material was tested for aldol condensation reaction between acetone and 4-nitrobenzaldehyde. Compared with monofunctional catalysts of SO{sub 3}H-SBA-15 and SBA-15-NH{sub 2}, the bifunctional sample of SO{sub 3}H-SBA-15-NH{sub 2} containing amine and sulfonic acid groups exhibited excellent acid-basic properties, which make it possess high activity for the aldol condensation.

  6. The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs.

    Science.gov (United States)

    Kleinstiver, Benjamin P; Wang, Li; Wolfs, Jason M; Kolaczyk, Tomasz; McDowell, Brendon; Wang, Xu; Schild-Poulter, Caroline; Bogdanove, Adam J; Edgell, David R

    2014-06-01

    Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5'-CNNNG-3'. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints. PMID:24739648

  7. ZFN-Site searches genomes for zinc finger nuclease target sites and off-target sites

    Directory of Open Access Journals (Sweden)

    Iseli Christian

    2011-05-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence. Results ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases. Conclusions ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.

  8. Physical properties of bifunctional BST/LSMO nanocomposites

    International Nuclear Information System (INIS)

    We report the fabrication of bifunctional nanocomposites consisting of ferroelectric Ba0.7Sr0.3TiO3 (BST) and ferromagnetic La0.67Sr0.33MnO3 (LSMO) at different concentrations via a high-temperature solid state route. The structural, dielectric, electrical, magnetodielectric (MD), magnetoelectric (ME) and magnetic properties of BST/LSMO nanocomposites were systematically investigated over a wide range of temperatures and frequencies. The X-Ray Diffraction analyses reveal the nanocrystalline nature of the heterostructures, wherein both perovskite phases co-exist. No parasitic phases were observed. The study of the dielectric properties shows that the nanocomposites exhibit relaxor ferroelectric character, with ferroelectric-paraelectric phase transition temperatures around 287–292 K that do not follow the Curie-Weiss law. The electrical measurements indicate that ac conductivities of the nanocomposites follow the Jonscher's universal power law, with activation energies of 0.42–0.63 eV based on Arrhenius-type behavior at high temperatures. The nanocomposites exhibit well-defined ferromagnetic hysteresis loops at room temperature (RT). The MD and ME measurements at RT indicate that BST/LSMO exhibits a nonlinear ME effect at low frequencies, with a threshold near 0.5 T. The magnetocapacitance (MCp) measurements evidence a quadratic dependence on magnetic field, further confirming the multiferroic nature of BST/LSMO. The order of MCp was found to be ∼7% per Tesla. The analysis of the MCp measurements indicates that one of the BST/LSMO compositions studied can be considered as a new multiferroic compound

  9. Physical properties of bifunctional BST/LSMO nanocomposites

    Energy Technology Data Exchange (ETDEWEB)

    Beltran-Huarac, Juan, E-mail: baristary26@gmail.com; Morell, Gerardo [Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, Puerto Rico 00931 (United States); Department of Physics, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico 00936 (United States); Martinez, Ricardo [Department of Mathematics and Physics, University of Puerto Rico, Cayey Campus, Cayey, Puerto Rico 00737 (United States)

    2014-02-28

    We report the fabrication of bifunctional nanocomposites consisting of ferroelectric Ba{sub 0.7}Sr{sub 0.3}TiO{sub 3} (BST) and ferromagnetic La{sub 0.67}Sr{sub 0.33}MnO{sub 3} (LSMO) at different concentrations via a high-temperature solid state route. The structural, dielectric, electrical, magnetodielectric (MD), magnetoelectric (ME) and magnetic properties of BST/LSMO nanocomposites were systematically investigated over a wide range of temperatures and frequencies. The X-Ray Diffraction analyses reveal the nanocrystalline nature of the heterostructures, wherein both perovskite phases co-exist. No parasitic phases were observed. The study of the dielectric properties shows that the nanocomposites exhibit relaxor ferroelectric character, with ferroelectric-paraelectric phase transition temperatures around 287–292 K that do not follow the Curie-Weiss law. The electrical measurements indicate that ac conductivities of the nanocomposites follow the Jonscher's universal power law, with activation energies of 0.42–0.63 eV based on Arrhenius-type behavior at high temperatures. The nanocomposites exhibit well-defined ferromagnetic hysteresis loops at room temperature (RT). The MD and ME measurements at RT indicate that BST/LSMO exhibits a nonlinear ME effect at low frequencies, with a threshold near 0.5 T. The magnetocapacitance (MC{sub p}) measurements evidence a quadratic dependence on magnetic field, further confirming the multiferroic nature of BST/LSMO. The order of MC{sub p} was found to be ∼7% per Tesla. The analysis of the MC{sub p} measurements indicates that one of the BST/LSMO compositions studied can be considered as a new multiferroic compound.

  10. Oxidations of amines with molecular oxygen using bifunctional gold–titania catalysts

    DEFF Research Database (Denmark)

    Klitgaard, Søren Kegnæs; Egeblad, Kresten; Mentzel, Uffe Vie;

    2008-01-01

    Over the past decades it has become clear that supported gold nanoparticles are surprisingly active and selective catalysts for several green oxidation reactions of oxygen-containing hydrocarbons using molecular oxygen as the stoichiometric oxidant. We here report that bifunctional gold–titania c......Over the past decades it has become clear that supported gold nanoparticles are surprisingly active and selective catalysts for several green oxidation reactions of oxygen-containing hydrocarbons using molecular oxygen as the stoichiometric oxidant. We here report that bifunctional gold...

  11. Structural basis for bifunctional peptide recognition at human δ-opioid receptor.

    Science.gov (United States)

    Fenalti, Gustavo; Zatsepin, Nadia A; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C H; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Gati, Cornelius; Yefanov, Oleksandr M; White, Thomas A; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W; Roth, Bryan L; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C; Cherezov, Vadim

    2015-03-01

    Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics. PMID:25686086

  12. A nanostructured bifunctional Pd/C gas-diffusion electrode for metal-air batteries

    OpenAIRE

    McKerracher, R.D.; Alegre, C.; Baglio, V.; Aricò, A.S.; Ponce de León, C.; Mornaghini, F.; Rodlert, M.; Walsh, F. C.

    2015-01-01

    Designing a bifunctional air electrode which catalyses both the oxygen reduction and oxygen evolution reactions is an essential part of progress towards fully rechargeable metal-air batteries, such as the iron-air battery which is environmentally friendly, low cost, and does not suffer risk of thermal runaway like lithium-ion batteries. This paper reports the development of a lightweight carbon-based bifunctional air electrode, catalysed by a small (0.5 mg cm?2) loading of 30 wt.% palladium o...

  13. Direct catalytic transformation of carbohydrates into 5-ethoxymethylfurfural with acid–base bifunctional hybrid nanospheres

    DEFF Research Database (Denmark)

    Li, Hu; Khokarale, Santosh Govind; Kotni, Ramakrishna;

    2014-01-01

    A series of acid–base bifunctional hybrid nanospheres prepared from the self-assembly of basic amino acids and phosphotungstic acid (HPA) with different molar ratios were employed as efficient and recyclable catalysts for synthesis of liquid biofuel 5-ethoxymethylfurfural (EMF) from various...... carbohydrates. A high EMF yield of 76.6%, 58.5%, 42.4%, and 36.5% could be achieved, when fructose, inulin, sorbose, and sucrose were used as starting materials, respectively. Although, the acid–base bifunctional nanocatalysts were inert for synthesis of EMF from glucose based carbohydrates, ethyl...

  14. Janus nanobelts: fabrication, structure and enhanced magnetic-fluorescent bifunctional performance

    Science.gov (United States)

    Ma, Qianli; Yu, Wensheng; Dong, Xiangting; Wang, Jinxian; Liu, Guixia

    2014-02-01

    A new nanostructure of magnetic-fluorescent bifunctional Janus nanobelts with Fe3O4/PMMA as one half and Tb(BA)3phen/PMMA as the other half has been successfully fabricated by a specially designed parallel spinneret electrospinning technology. The morphology and properties of the final products were investigated in detail by X-ray diffractometry (XRD), scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), biological microscopy (BM), vibrating sample magnetometry (VSM) and fluorescence spectroscopy. The results revealed that the [Fe3O4/PMMA]//[Tb(BA)3phen/PMMA] magnetic-fluorescent bifunctional Janus nanobelts possess superior magnetic and fluorescent properties due to their special nanostructure. Compared with Fe3O4/Tb(BA)3phen/PMMA composite nanobelts, the magnetic-fluorescent bifunctional Janus nanobelts provided better performance. The new magnetic-fluorescent bifunctional Janus nanobelts have potential applications in novel nano-bio-label materials, drug target delivery materials and future nanodevices due to their excellent magnetic-fluorescent properties, flexibility and insolubility. Moreover, the construction technique for the Janus nanobelts is of universal significance for the fabrication of other multifunctional Janus nanobelts.A new nanostructure of magnetic-fluorescent bifunctional Janus nanobelts with Fe3O4/PMMA as one half and Tb(BA)3phen/PMMA as the other half has been successfully fabricated by a specially designed parallel spinneret electrospinning technology. The morphology and properties of the final products were investigated in detail by X-ray diffractometry (XRD), scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), biological microscopy (BM), vibrating sample magnetometry (VSM) and fluorescence spectroscopy. The results revealed that the [Fe3O4/PMMA]//[Tb(BA)3phen/PMMA] magnetic-fluorescent bifunctional Janus nanobelts possess superior magnetic and fluorescent properties due to their special

  15. Energy Storage in Bifunctional TiO2 Composite Materials under UV and Visible Light

    Directory of Open Access Journals (Sweden)

    Jialin Li

    2009-11-01

    Full Text Available This paper provides an overview of recent studies on energy storage in bifunctional TiO2 composite materials under UV and visible light. The working mechanism, property improvements and applications of these bifunctional TiO2 composite systems are introduced, respectively. The latest results obtained in our laboratory, especially a new process for photoelectric conversion and energy storage in TiO2/Cu2O bilayer films under visible light, are also presented. Hopefully this review will stimulate more fundamental and applied research on this subject in the future.

  16. The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

    OpenAIRE

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire; Thibessard, Annabelle; Leblond, Pierre

    2014-01-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only pu...

  17. A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

    OpenAIRE

    Meineke, Birthe; Kast, Alene; Schwer, Beate; Meinhardt, Friedhelm; Shuman, Stewart; Klassen, Roland

    2012-01-01

    The PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases encoded by cytoplasmic DNA plasmids. Toxicity can be recapitulated conveniently by induced intracellular expression of PaOrf2 or γ-toxin in Saccharomyces cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, the authors report that PaOrf2 residues Glu9 and His287 (putative count...

  18. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    2013-01-01

    Roč. 69, č. 2 (2013), s. 213-226. ISSN 0907-4449 R&D Projects: GA MŠk EE2.3.30.0029; GA ČR GAP302/11/0855; GA ČR GA310/09/1407; GA ČR GA521/09/1214 Grant ostatní: AV ČR(CZ) Praemium Academiae Institutional support: RVO:61389013 ; RVO:60077344 Keywords : plant nucleases * catalytic zinc cluster * glycoproteins Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (BC-A) Impact factor: 7.232, year: 2013

  19. Coarse-grained modelling of pressure-related effects in staphylococcal nuclease and ubiquitin

    Energy Technology Data Exchange (ETDEWEB)

    Wojciechowski, Michal; Cieplak, Marek [Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, 02-668 Warsaw (Poland)

    2007-07-18

    Folding and unfolding kinetics of proteins are affected by the application of high hydrostatic pressure. In order to study the pressure-related effects within geometry-based coarse-grained models, we propose adding a pressure-controlled hump to the contact potentials. This approach qualitatively reproduces the experimental findings on conformational changes in staphylococcal nuclease and predicts a non-monotonic dependence of the unfolding time with pressure. The pressure-induced unfolding is shown to be distinct from unfolding induced by a high temperature.

  20. Ultrafast solvation dynamics at internal site of staphylococcal nuclease investigated by site-directed mutagenesis

    CERN Document Server

    Guang-yu, Gao; Wei, Wang; Shu-feng, Wang; Zhong, Dongping; Qi-huang, Gong

    2014-01-01

    Solvation is essential for protein activities. To study internal solvation of protein, site-directed mutagenesis is applied. Intrinsic fluorescent probe, tryptophan, is inserted into desired position inside protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics researches. We introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside caves of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at internal sites of the caves indicate clear characteristics of local environment. These solvation behaviors correlated to the enzyme activity directly.

  1. The High Dielectric Constant of Staphylococcal Nuclease is Encoded in its Structural Architecture

    OpenAIRE

    Goh, Garrett B.; Bertrand, García-Moreno E.; Brooks, Charles L.

    2011-01-01

    The pKa values of Lys-66, Glu-66 and Asp-66 buried in the interior of the staphylococcal nuclease Δ+PHS variant were reported to be shifted by as much as 5 pKa units from their normal values. Reproducing the pKa of these buried ionizable residues using continuum electrostatic calculations required the use of a high protein dielectric constant of 10 or higher. The apparent high dielectric constant has been rationalized as a consequence of a local structural reorganization or increased fluctuat...

  2. Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

    OpenAIRE

    Widmer, R. M.; Lezzi, M.; Koller, Th.

    1987-01-01

    We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of th...

  3. Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I gene.

    OpenAIRE

    Bacolla, A; Wu, F. Y.

    1991-01-01

    Mung bean nuclease, an enzyme specific for single-stranded DNA, was used to probe a non-B DNA structure present in the mouse metallothionein-I gene. The region sensitive to the enzyme was constituted by a 128 base-pair long polypurine.polypyrimidine sequence located at 1.2-kb from the start of transcription. A detailed analysis of the mung bean nuclease cleavage pattern revealed that: (i) under conditions of supercoiling and low pH a triplex structure was formed, (ii) the triplex was flanked ...

  4. The pH dependence of staphylococcal nuclease stability is incompatible with a three-state denaturation model

    OpenAIRE

    Spencer, Daniel; Bertrand García-Moreno, E.; Stites, Wesley E.

    2013-01-01

    Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.2. Within experimental error the values of ΔGH2O and mGuHCl for...

  5. (NZCH...O Contacts assist crystallization of a ParB-like nuclease

    Directory of Open Access Journals (Sweden)

    Rao Zihe

    2007-07-01

    Full Text Available Abstract Background The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization Results Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ CH...O contacts (3.2 – 3.7 Å by the addition of 2 methyl groups to the side chain amine nitrogen (NZ of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 Å resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. Conclusion In this study, introduction of novel cohesive (NZCH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZCH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.

  6. Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA

    Science.gov (United States)

    Stoddard, Thomas J.; Clasen, Benjamin M.; Baltes, Nicholas J.; Demorest, Zachary L.; Voytas, Daniel F.; Zhang, Feng; Luo, Song

    2016-01-01

    Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing. PMID:27176769

  7. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  8. Rh D blood group conversion using transcription activator-like effector nucleases.

    Science.gov (United States)

    Kim, Young-Hoon; Kim, Hyun O; Baek, Eun J; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-01-01

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine. PMID:26078220

  9. Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

    Science.gov (United States)

    Christensen, Laura A; Wang, Hong; Van Houten, Bennett; Vasquez, Karen M

    2008-12-01

    Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification. PMID:18996898

  10. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

    Science.gov (United States)

    Merlin, Christine; Beaver, Lauren E; Taylor, Orley R; Wolfe, Scot A; Reppert, Steven M

    2013-01-01

    The development of reverse-genetic tools in "nonmodel" insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into "one nucleus" stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and "nonmodel" insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

  11. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  12. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

    Science.gov (United States)

    Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J; Aryee, Martin J; Joung, J Keith

    2014-06-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing. PMID:24770325

  13. Comparison of bifunctional chelates for {sup 64}Cu antibody imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Cara L.; Crisp, Sarah; Bensimon, Corinne [MDS Nordion, Vancouver, BC (Canada); Yapp, Donald T.T.; Ng, Sylvia S.W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); University of British Columba, The Faculty of Pharmaceutical Sciences, Vancouver, BC (Canada); Sutherland, Brent W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); Gleave, Martin [Prostate Centre at Vancouver General Hospital, Vancouver, BC (Canada); Jurek, Paul; Kiefer, Garry E. [Macrocyclics Inc., Dallas, TX (United States)

    2010-11-15

    Improved bifunctional chelates (BFCs) are needed to facilitate efficient {sup 64}Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with {sup 64}Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. {sup 64}Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of {sup 64}Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the {sup 64}Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the {sup 64}Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior {sup 64}Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, {sup 64}Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than

  14. Bifunctional phage-based pretargeted imaging of human prostate carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Newton-Northup, Jessica R. [Department of Biochemistry, University of Missouri, Columbia, MO 65211 (United States)], E-mail: newtonj@missouri.edu; Figueroa, Said D. [Department of Radiology, University of Missouri, Columbia, MO 65211 (United States); Quinn, Thomas P.; Deutscher, Susan L. [Department of Biochemistry, University of Missouri, Columbia, MO 65211 (United States); Research Service, Harry S. Truman Veterans Memorial Hospital, Columbia, MO 65201 (United States)

    2009-10-15

    Introduction: Two-step and three-step pretargeting systems utilizing biotinylated prostate tumor-homing bacteriophage (phage) and {sup 111}In-radiolabeled streptavidin or biotin were developed for use in cancer radioimaging. The in vivo selected prostate carcinoma-specific phage (G1) displaying up to five copies of the peptide IAGLATPGWSHWLAL was the focus of the present study. Methods: The ability of G1 phage to extravasate and target prostate tumor cells was investigated using immunohistochemistry. G1 phages were biotinylated, streptavidin was conjugated to diethylenetriaminepentaacetic acid (DTPA) and biotin was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Biodistribution studies and single-photon emission computed tomography (SPECT)/CT imaging of xenografted PC-3 tumors via two-step pretargeted {sup 111}In-labeled streptavidin and three-step pretargeted {sup 111}In-labeled biotin were performed in SCID mice to determine the optimal pretargeting method. Results: The ability of G1 phage to extravasate the vasculature and bind directly to human PC-3 prostate carcinoma tumor cells in vivo was demonstrated via immunocytochemical analysis. Comparative biodistribution studies of the two-step and three-step pretargeting strategies indicated increased PC-3 human prostate carcinoma tumor uptake in SCID mice of 4.34{+-}0.26 %ID g{sup -1} at 0.5 h postinjection of {sup 111}In-radiolabeled biotin (utilized in a three-step protocol) compared to 0.67{+-}0.06 %ID g{sup -1} at 24 h postinjection of {sup 111}In radiolabeled streptavidin (employed in a two-step protocol). In vivo SPECT/CT imaging of xenografted PC-3 tumors in SCID mice with the three-step pretargeting method was superior to that of the two-step pretargeting method, and, importantly, blocking studies demonstrated specificity of tumor uptake of {sup 111}In-labeled biotin in the three-step pretargeting scheme. Conclusion: This study demonstrates the use of multivalent bifunctional

  15. Direct catalytic transformation of carbohydrates into 5-ethoxymethylfurfural with acid–base bifunctional hybrid nanospheres

    International Nuclear Information System (INIS)

    Graphical abstract: Catalytic conversion of carbohydrates into HMF and EMF in ethanol/DMSO with acid–base bifunctional hybrid nanospheres prepared from self-assembly of corresponding basic amino acids and HPA. - Highlights: • Acid–base bifunctional nanospheres were efficient for production of EMF from sugars. • Synthesis of EMF in a high yield of 76.6% was realized from fructose. • Fructose based biopolymers could also be converted into EMF with good yields. • Ethyl glucopyranoside was produced in good yields from glucose in ethanol. - Abstract: A series of acid–base bifunctional hybrid nanospheres prepared from the self-assembly of basic amino acids and phosphotungstic acid (HPA) with different molar ratios were employed as efficient and recyclable catalysts for synthesis of liquid biofuel 5-ethoxymethylfurfural (EMF) from various carbohydrates. A high EMF yield of 76.6%, 58.5%, 42.4%, and 36.5% could be achieved, when fructose, inulin, sorbose, and sucrose were used as starting materials, respectively. Although, the acid–base bifunctional nanocatalysts were inert for synthesis of EMF from glucose based carbohydrates, ethyl glucopyranoside in good yields could be obtained from glucose in ethanol. Moreover, the nanocatalyst functionalized with acid and basic sites was able to be reused several times with no significant loss in catalytic activity

  16. Synthesis, characterization and use of ATRP bifunctional initiator with trichloromethyl end-groups

    Czech Academy of Sciences Publication Activity Database

    Toman, Luděk; Janata, Miroslav; Spěváček, Jiří; Masař, Bohumil; Vlček, Petr; Látalová, Petra

    2002-01-01

    Roč. 43, č. 2 (2002), s. 18-19. ISSN 0032-3934 R&D Projects: GA ČR GA203/01/0513 Institutional research plan: CEZ:AV0Z4050913 Keywords : bifunctional initiator * ATRP polymerization * trichloromethyl end-groups Subject RIV: CD - Macromolecular Chemistry

  17. Structure and potential applications of amido lanthanide complexes chelated by bifunctional b-diketiminate ligand

    Czech Academy of Sciences Publication Activity Database

    Olejník, R.; Padělková, Z.; Fridrichová, A.; Horáček, Michal; Merna, J.; Růžička, A.

    2014-01-01

    Roč. 759, JUN 2014 (2014), s. 1-10. ISSN 0022-328X R&D Projects: GA ČR GAP106/10/0924 Institutional support: RVO:61388955 Keywords : Bifunctional b-diketiminates * lanthanides * hydroamination Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.173, year: 2014

  18. Asymmetric α-amination of β-keto esters using a guanidine–bisurea bifunctional organocatalyst

    Science.gov (United States)

    Yamamoto, Yoshiharu

    2016-01-01

    Summary An asymmetric α-amination of β-keto esters with azodicarboxylate in the presence of a guanidine–bisurea bifunctional organocatalyst was investigated. The α-amination products were obtained in up to 99% yield with up to 94% ee. PMID:26977179

  19. High surface area carbon for bifunctional air electrodes applied in zinc-air batteries

    Energy Technology Data Exchange (ETDEWEB)

    Arai, H. [on leave from NTT Laboratories (Japan); Mueller, S.; Haas, O. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1999-08-01

    Bifunctional air electrodes with high surface area carbon substrates showed low reduction overpotential, thus are promising for enhancing the energy efficiency and power capability of zinc-air batteries. The improved performance is attributed to lower overpotential due to diffusion of the reaction intermediate, namely the peroxide ion. (author) 1 fig., 2 refs.

  20. Bifunctional catalysts for the direct production of liquid fuels from syngas

    NARCIS (Netherlands)

    Sartipi, S.

    2014-01-01

    Design and development of catalyst formulations that maximize the direct production of liquid fuels by combining Fischer-Tropsch synthesis (FTS), hydrocarbon cracking, and isomerization into one single catalyst particle (bifunctional FTS catalyst) have been investigated in this thesis. To achieve th

  1. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

    International Nuclear Information System (INIS)

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C2221 with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution

  2. Preparation of recombinant nucleases with anticancerogenic potential, their molecular analysis and production in plants for medicinal utilization

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Podzimek, Tomáš; Orctová, Lidmila; Škopek, Josef; Matoušek, Josef

    Brussel: European Plant Science Organisation, 2008. s. 220. [EPSO Conference "Plants for Life" /4./. 22.06.2008-26.06.2008, Toulon] R&D Projects: GA ČR GA521/06/1149; GA AV ČR(CZ) 1QS500510558 Institutional research plan: CEZ:AV0Z50510513 Keywords : recombinant nucleases * medicinal utilization Subject RIV: EB - Genetics ; Molecular Biology

  3. The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    Science.gov (United States)

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2016-01-01

    The key event in the choice of repair pathways for DNA double-strand breaks (DSBs) is the initial processing of ends. Non-homologous end joining (NHEJ) involves limited processing, but homology-dependent repair (HDR) requires extensive resection of the 5′ strand. How cells decide if an end is channeled to resection or NHEJ is not well understood. We hypothesize that the structure of ends is a major determinant and tested this hypothesis with model DNA substrates in Xenopus egg extracts. While ends with normal nucleotides are efficiently channeled to NHEJ, ends with damaged nucleotides or bulky adducts are channeled to resection. Resection is dependent on Mre11, but its nuclease activity is critical only for ends with 5′ bulky adducts. CtIP is absolutely required for activating the nuclease-dependent mechanism of Mre11 but not the nuclease-independent mechanism. Together, these findings suggest that the structure of ends is a major determinant for the pathway choice of DSB repair and the Mre11 nuclease dependency of resection. PMID:27084932

  4. Development of Cell Lines Stably Expressing Staphylococcal Nuclease Fused to Dengue 2 Virus Capsid Protein for CTVI

    Institute of Scientific and Technical Information of China (English)

    Cheng-Feng QIN; E-De QIN

    2004-01-01

    To explore the potential application of capsid-targeted viral inactivation(CTVI)strategy in prophylactic model against dengue virus(DV)infection,here we fused a Ca2+-dependent nuclease,staphylococcal nuclease(SN),to the capsid protein of dengue 2 virus(D2C)at the carboxyl terminal,and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C.A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5 μg/ml blasticidin.The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays.The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay.It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained.The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells.Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

  5. Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

    OpenAIRE

    Kamekura, M; Hamakawa, T; Onishi, H.

    1982-01-01

    RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA.

  6. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.; Christensen, Hans Erik Mølager; Kožíšek, Milan; Nagata, Kyosuke; Czene, Aniko; Gyurcsik, Béla

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  7. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

    Directory of Open Access Journals (Sweden)

    Vagner Laura L

    2008-05-01

    Full Text Available Abstract Background Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking. Results Using degenerative primers and the rapid amplification of cDNA ends (RACE procedure, we cloned the Duplex-Specific Nuclease (DSN gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5 and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact. Conclusion We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate

  8. Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

    Science.gov (United States)

    Yacoub, Elhem; Ben Abdelmoumen Mardassi, Boutheina

    2016-01-01

    Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases. PMID:27010566

  9. Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

    Directory of Open Access Journals (Sweden)

    Wenwen Wang

    2012-08-01

    Full Text Available This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.

  10. DNA sensor's selectivity enhancement and protection from contaminating nucleases due to a hydrated ionic liquid.

    Science.gov (United States)

    Tateishi-Karimata, Hisae; Pramanik, Smritimoy; Sugimoto, Naoki

    2015-07-01

    The thermodynamic stability of certain mismatched base pairs has made the development of DNA sequence sensing systems challenging. Thus, the stability of fully matched and mismatched DNA oligonucleotides in the hydrated ionic liquid choline dihydrogen phosphate (choline dhp) was investigated. Mismatched base pairs were significantly destabilized in choline dhp relative to those in aqueous buffer. A molecular beacon that forms a triplex with a conserved HIV-1 sequence was then designed and tested in choline dhp. The molecular beacon specifically detected the target duplex via triplex formation at concentrations as low as 1 pmol per 10 μL with 10,000-fold sequence selectivity. Moreover, the molecular beacon was protected from a contaminating nuclease in choline dhp, and DNAs in aqueous solutions were not sufficiently stable for practical use. PMID:25919083

  11. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos.

    Science.gov (United States)

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-10-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos. PMID:27189642

  12. Functional identification of the non-specific nuclease from white spot syndrome virus

    International Nuclear Information System (INIS)

    The product encoded by the wsv191 gene from shrimp white spot syndrome virus (WSSV) is homologous with non-specific nucleases (NSN) of other organisms. To functionally identify the protein, the wsv191 gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with 6His-tag at C-terminal. The fusion protein (termed as rWSSV-NSN) was purified using Ni-NTA affinity chromatography under denatured conditions, renatured and characterized by three methods. The results showed that rWSSV-NSN could hydrolyze both DNA and RNA. 5'-RACE result revealed that the transcription initiation site of the wsv191 gene was located at nucleotide residue G of the predicted ATG triplet. Therefore, we concluded that the next ATG should be the genuine translation initiation codon of the wsv191 gene. Western blot analysis revealed that the molecular mass of natural WSSV-NSN was 37 kDa

  13. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  14. Potential application of FoldX force field based protein modeling in zinc finger nucleases design.

    Science.gov (United States)

    He, ZuYong; Mei, Gui; Zhao, ChunPeng; Chen, YaoSheng

    2011-05-01

    Engineered sequence-specific zinc finger nucleases (ZFNs) make the highly efficient modification of eukaryotic genomes possible. However, most current strategies for developing zinc finger nucleases with customized sequence specificities require the construction of numerous tandem arrays of zinc finger proteins (ZFPs), and subsequent largescale in vitro validation of their DNA binding affinities and specificities via bacterial selection. The labor and expertise required in this complex process limits the broad adoption of ZFN technology. An effective computational assisted design strategy will lower the complexity of the production of a pair of functional ZFNs. Here we used the FoldX force field to build 3D models of 420 ZFP-DNA complexes based on zinc finger arrays developed by the Zinc Finger Consortium using OPEN (oligomerized pool engineering). Using nonlinear and linear regression analysis, we found that the calculated protein-DNA binding energy in a modeled ZFP-DNA complex strongly correlates to the failure rate of the zinc finger array to show significant ZFN activity in human cells. In our models, less than 5% of the three-finger arrays with calculated protein-DNA binding energies lower than -13.132 kcal mol(-1) fail to form active ZFNs in human cells. By contrast, for arrays with calculated protein-DNA binding energies higher than -5 kcal mol(-1), as many as 40% lacked ZFN activity in human cells. Therefore, we suggest that the FoldX force field can be useful in reducing the failure rate and increasing efficiency in the design of ZFNs. PMID:21455692

  15. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  16. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  17. A 5' Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens.

    Science.gov (United States)

    Kristiansen, Gitte Qvist; Lisby, Jan Gorm; Schønning, Kristian

    2016-06-01

    Rapid and sensitive detection of macrolide resistance in Mycoplasma genitalium is required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene of M. genitalium result in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the present study was to develop a 5' nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene of Mycoplasma genitalium that are associated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5' nuclease genotyping assay was used as a referral test to be used on M. genitalium-positive samples and was validated on 259 positive samples, of which 253 (97.7%) were successfully sequenced. With the newly developed assay, 237/259 (91.5%) investigated M. genitalium-positive samples were genotyped. The positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistant M. genitalium in clinical specimens possessing A2058G, A2058C, A2058T, and A2059G mutations with a sensitivity of 94.4% (95% confidence interval [CI], 90.7% to 98.2%) and a specificity of 92.7% (95% CI, 87.8% to 97.6%) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment of M. genitalium infections. PMID:27053672

  18. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    Science.gov (United States)

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  19. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

    KAUST Repository

    Li, Lixin

    2012-01-22

    Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.

  20. Analysis of cigarette-smoke-induced DNA adducts by butanol extraction and nuclease P1-enhanced 32P-postlabeling in human lymphocytes and granulocytes.

    OpenAIRE

    Savela, K; Hemminki, K

    1993-01-01

    In an earlier study, we analyzed the aromatic DNA adducts separated from lymphocytes and granulocytes of smokers and nonsmokers using the nuclease P1-enhanced 32P-postlabeling assay. Here we compare the butanol extraction and nuclease P1-enhanced procedure on the same kind of samples. The DNA adducts of 42 per 10(8) nucleotides from smokers' lymphocytes were statistically higher (p < 0.05) than those of 11 from nonsmokers', when analyzed by the nuclease P1 treatment, but not by the 1-butanol ...

  1. Bifunctional Nb/Ti-MCM-41 catalyst in oxidative acidic reaction of cyclohexene to diol

    International Nuclear Information System (INIS)

    Bifunctional oxidative and acidic catalyst was prepared by incorporating titanium ion (Ti4+) and niobic acid in meso porous molecular sieves MCM-41 structure. The catalyst is active both in oxidation, and acid-catalyzed reaction of olefin to diol. Nb/ Ti-MCM-41 catalyst was prepared by first synthesizing Ti-MCM-41 by hydrothermal method, followed by subsequent impregnation of niobic acid (Nb) into Ti-MCM-41 at various % wt Nb loading. The framework structure of Ti-MCM-41 collapsed after incorporation of Nb but the tetrahedral form of Ti4+ still maintained with octahedral Nb species. Both Bronsted and Lewis acid sites are present in all Nb/ Ti-MCM-41 samples. The formation of cyclohexanediol in the epoxidation of cyclohexene proved the bifunctional oxidative and acidic catalyst through the formation of cyclohexane oxide. The yield increased with the increase amount of the Bronsted acid sites provided by niobium species. (author)

  2. Tethering metal ions to photocatalyst particulate surfaces by bifunctional molecular linkers for efficient hydrogen evolution

    KAUST Repository

    Yu, Weili

    2014-08-19

    A simple and versatile method for the preparation of photocatalyst particulates modified with effective cocatalysts is presented; the method involves the sequential soaking of photocatalyst particulates in solutions containing bifunctional organic linkers and metal ions. The modification of the particulate surfaces is a universal and reproducible method because the molecular linkers utilize strong covalent bonds, which in turn result in modified monolayer with a small but controlled quantity of metals. The photocatalysis results indicated that the CdS with likely photochemically reduced Pd and Ni, which were initially immobilized via ethanedithiol (EDT) as a linker, were highly efficient for photocatalytic hydrogen evolution from Na2S-Na2SO3-containing aqueous solutions. The method developed in this study opens a new synthesis route for the preparation of effective photocatalysts with various combinations of bifunctional linkers, metals, and photocatalyst particulate materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. First-Principles Study of Structure Property Relationships of Monolayer (Hydroxy)Oxide-Metal Bifunctional Electrocatalysts

    DEFF Research Database (Denmark)

    Zeng, Zhenhua; Kubal, Joseph; Greeley, Jeffrey Philip

    2015-01-01

    information that is inaccessible by purely experimental means, and these structures, in turn, strongly suggest that a bifunctional reaction mechanism for alkaline HER will be operative at the interface between the films, the metal substrates, and the surrounding aqueous medium. This bifunctionality produces...... under alkaline electrochemical conditions. We demonstrate that the structure and oxidation state of the films can be systematically tuned by changing the applied electrode potential and/or the nature of substrates. Structural features determined from the theoretical calculations provide a wealth of...... important changes in the calculated barriers of key elementary reaction steps, including water activation and dissociation, as compared to traditional monofunctional Pt surfaces. The successful identification of the structures of thin metal films and three-phase boundary catalysts is not only an important...

  4. Sorption of Pu(IV) from nitric acid by bifunctional anion-exchange resins

    International Nuclear Information System (INIS)

    Anion exchange is attractive for separating plutonium because the Pu(IV) nitrate complex is very strongly sorbed and few other metal ions form competing anionic nitrate complexes. The major disadvantage of this process has been the unusually slow rate at which the Pu(IV) nitrate complex is sorbed by the resin. The paper summarizes the concept of bifunctional anion-exchange resins, proposed mechanism for Pu(IV) sorption, synthesis of the alkylating agent, calculation of Kd values from Pu(IV) sorption results, and conclusions from the study of Pu(IV) sorption from 7M nitric acid by macroporous anion-exchange resins including level of crosslinking, level of alkylation, length of spacer, and bifunctional vs. monofunctional anion-exchange resins

  5. Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines.

    Science.gov (United States)

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-01-22

    Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme. PMID:19920138

  6. Radiation Induced Crosslinking of Polyethylene in the Presence of Bifunctional Vinyl Monomers

    DEFF Research Database (Denmark)

    Joshi, M. S.; Singer, Klaus Albert Julius; Silverman, J.

    Several reports have been published showing that the radiation induced grafting of bifunctional vinyl monomers to low density polyethylene results in a product with an unusually high density of crosslinks. The same grafting reactions are shown to reduce the incipient gel dose by more than a factor...... of fifty. This paper is concerned with the apparent crosslinking produced by the radiation grafting of two monomers to polyethylene: acrylic acid and acrylonitrile....

  7. Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa

    OpenAIRE

    Mauline, Léïla; Gressier, Marie; Roques, Christine; Hammer, Peter,; Ribeiro, Sidney J. L.; Caiut, José Maurício A.; Menu, Marie-Joëlle

    2013-01-01

    Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium (II) complex. The surface properties of the silica particles were designed by reaction with am...

  8. L-Proline Derived Bifunctional Organocatalysts: Enantioselective Michael Addition of Dithiomalonates to trans-β-Nitroolefins.

    Science.gov (United States)

    Jin, Hui; Kim, Seung Tae; Hwang, Geum-Sook; Ryu, Do Hyun

    2016-04-15

    A series of novel L-proline derived tertiary amine bifunctional organocatalysts 9 are reported, which were applied to the asymmetric Michael addition of dithiomalonates 2 to trans-β-nitroolefins 1. The reaction proceeded in high yields (up to 99%) with high enantioselectivities (up to 97% ee). The synthetic utility of this methodology was demonstrated in the short synthesis of (R)-phenibut in high yield. PMID:26989804

  9. GST-TAT-SOD: Cell Permeable Bifunctional Antioxidant Enzyme—A Potential Selective Radioprotector

    OpenAIRE

    Jianru Pan; Huocong He; Ying Su; Guangjin Zheng; Junxin Wu; Shutao Liu; Pingfan Rao

    2016-01-01

    Superoxide dismutase (SOD) fusion of TAT was proved to be radioprotective in our previous work. On that basis, a bifunctional recombinant protein which was the fusion of glutathione S-transferase (GST), SOD, and TAT was constructed and named GST-TAT-SOD. Herein we report the investigation of the cytotoxicity, cell-penetrating activity, and in vitro radioprotective effect of GST-TAT-SOD compared with wild SOD, single-function recombinant protein SOD-TAT, and amifostine. We demonstrated that wi...

  10. 3D Ordered Mesoporous Bifunctional Oxygen Catalyst for Electrically Rechargeable Zinc-Air Batteries.

    Science.gov (United States)

    Park, Moon Gyu; Lee, Dong Un; Seo, Min Ho; Cano, Zachary Paul; Chen, Zhongwei

    2016-05-01

    To enhance energy efficiency and durability, a highly active and durable 3D ordered mesoporous cobalt oxide framework has been developed for rechargeable zinc-air batteries. The bifunctional air electrode consisting of 3DOM Co3 O4 having high active surface area and robust structure, results in superior charge and discharge battery voltages, and durable performance for electrically rechargeable zinc-air batteries. PMID:27043451

  11. Synthesis of acid-base bifunctional mesoporous materials by oxidation and thermolysis

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Xiaofang [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023 (China); Zou, Yongcun [State Key Laboratory of Inoranic Synthesis and Preparative Chemistryg, College of Chemistry, Jilin University, Changchun 130012 (China); Wu, Shujie; Liu, Heng [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023 (China); Guan, Jingqi, E-mail: guanjq@jlu.edu.cn [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023 (China); Kan, Qiubin, E-mail: qkan@jlu.edu.cn [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023 (China)

    2011-06-15

    Graphical abstract: A novel and efficient method has been developed for the synthesis of acid-base bifunctional catalyst. The obtained sample of SO{sub 3}H-MCM-41-NH{sub 2} containing amine and sulfonic acids exhibits excellent catalytic activity in aldol condensation reaction. Research highlights: {yields} Synthesize acid-base bifunctional mesoporous materials SO{sub 3}H-MCM-41-NH{sub 2}. {yields} Oxidation and then thermolysis to generate acidic site and basic site. {yields} Exhibit good catalytic performance in aldol condensation reaction between acetone and various aldehydes. -- Abstract: A novel and efficient method has been developed for the synthesis of acid-base bifunctional catalyst SO{sub 3}H-MCM-41-NH{sub 2}. This method was achieved by co-condensation of tetraethylorthosilicate (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and (3-triethoxysilylpropyl) carbamicacid-1-methylcyclohexylester (3TAME) in the presence of cetyltrimethylammonium bromide (CTAB), followed by oxidation and then thermolysis to generate acidic site and basic site. X-ray diffraction (XRD) and transmission electron micrographs (TEM) show that the resultant materials keep mesoporous structure. Thermogravimetric analysis (TGA), X-ray photoelectron spectra (XPS), back titration, solid-state {sup 13}C CP/MAS NMR and solid-state {sup 29}Si MAS NMR confirm that the organosiloxanes were condensed as a part of the silica framework. The bifunctional sample (SO{sub 3}H-MCM-41-NH{sub 2}) containing amine and sulfonic acids exhibits excellent acid-basic properties, which make it possess high activity in aldol condensation reaction between acetone and various aldehydes.

  12. 67Ga(NODASA): a new potential bifunctional radioligand for coupling to peptides

    International Nuclear Information System (INIS)

    A new bifunctional chelator NODASA (1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid) has been synthesised and its Ga(III) complex was crystallographically characterized by X-ray diffraction. The complex showed to be stable in serum and in acidic conditions and its stability constant was determined using a competition method with an auxiliary ligand. The conjugation of Ga(NODASA) to a model aminoacidamide proved the feasibility of a prelabelling approach. (author)

  13. Bifunctional catalysts for the direct production of liquid fuels from syngas

    OpenAIRE

    Sartipi, S.

    2014-01-01

    Design and development of catalyst formulations that maximize the direct production of liquid fuels by combining Fischer-Tropsch synthesis (FTS), hydrocarbon cracking, and isomerization into one single catalyst particle (bifunctional FTS catalyst) have been investigated in this thesis. To achieve this aim, a second functionality (other than FTS) has to be added to the catalyst formulation to break the limitation of a classical Anderson-Schulz-Flory (ASF) distribution of FTS products. Since up...

  14. A new bifunctional hybrid nanostructure as an active platform for photothermal therapy and MR imaging

    OpenAIRE

    Mona Khafaji; Manouchehr Vossoughi; M. Reza Hormozi-Nezhad; Rassoul Dinarvand; Felix Börrnert; Azam Irajizad

    2016-01-01

    As a bi-functional cancer treatment agent, a new hybrid nanostructure is presented which can be used for photothermal therapy by exposure to one order of magnitude lower laser powers compared to similar nanostructures in addition to substantial enhancment in magnetic resonance imaging (MRI) contrast. This gold-iron oxide hybrid nanostructure (GIHN) is synthesized by a cost-effective and high yield water-based approach. The GIHN is sheilded by PEG. Therefore, it shows high hemo and biocompatib...

  15. Radiation Induced Crosslinking of Polyethylene in the Presence of Bifunctional Vinyl Monomers

    DEFF Research Database (Denmark)

    Joshi, M. S.; Singer, Klaus Albert Julius; Silverman, J.

    Several reports have been published showing that the radiation induced grafting of bifunctional vinyl monomers to low density polyethylene results in a product with an unusually high density of crosslinks. The same grafting reactions are shown to reduce the incipient gel dose by more than a facto...... of fifty. This paper is concerned with the apparent crosslinking produced by the radiation grafting of two monomers to polyethylene: acrylic acid and acrylonitrile....

  16. Bifunctional Metamaterials with Simultaneous and Independent Manipulation of Thermal and Electric Fields

    OpenAIRE

    Lan, Chuwen; Li, Bo; Zhou, Ji

    2015-01-01

    Metamaterials offer a powerful way to manipulate a variety of physical fields ranging from wave fields (electromagnetic field, acoustic field, elastic wave, etc.), static fields (static magnetic field, static electric field) to diffusive fields (thermal field, diffusive mass). However, the relevant reports and studies are usually conducted on a single physical field or functionality. In this study, we proposed and experimentally demonstrated a bifunctional metamaterial which can manipulate th...

  17. Bifunctional Catalysts for Upgrading of Biomass-Derived Oxygenates: A Review

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Allison M.; Hensley, Jesse E.; Medlin, J. Will

    2016-08-05

    Deoxygenation is an important reaction in the conversion of biomass-derived oxygenates to fuels and chemicals. A key route for biomass refining involves the production of pyrolysis oil through rapid heating of the raw biomass feedstock. Pyrolysis oil as produced is highly oxygenated, so the feasibility of this approach depends in large part on the ability to selectively deoxygenate pyrolysis oil components to create a stream of high-value finished products. Identification of catalytic materials that are active and selective for deoxygenation of pyrolysis oil components has therefore represented a major research area. One catalyst is rarely capable of performing the different types of elementary reaction steps required to deoxygenate biomass-derived compounds. For this reason, considerable attention has been placed on bifunctional catalysts, where two different active materials are used to provide catalytic sites for diverse reaction steps. Here, we review recent trends in the development of catalysts, with a focus on catalysts for which a bifunctional effect has been proposed. We summarize recent studies of hydrodeoxygenation (HDO) of pyrolysis oil and model compounds for a range of materials, including supported metal and bimetallic catalysts as well as transition-metal oxides, sulfides, carbides, nitrides, and phosphides. Particular emphasis is placed on how catalyst structure can be related to performance via molecular-level mechanisms. These studies demonstrate the importance of catalyst bifunctionality, with each class of materials requiring hydrogenation and C-O scission sites to perform HDO at reasonable rates.

  18. Comparison of Dynamical Behaviors Between Monofunctional and Bifunctional Two-Component Signaling Modules

    Science.gov (United States)

    Yang, Xiyan; Wu, Yahao; Yuan, Zhanjiang

    2015-06-01

    Two-component signaling modules exist extensively in bacteria and microbes. These modules can be, based on their distinct network structures, divided into two types: the monofunctional system (denoted by MFS) where the sensor kinase (SK) modulates only phosphorylation of the response regulator (RR), and the bifunctional system (denoted by BFS) where the SK catalyzes both phosphorylation and dephosphorylation of the RR. Here, we analyze dynamical behaviors of these two systems based on stability theory, focusing on differences between them. The analysis of the deterministic behavior indicates that there is no difference between the two modules, that is, each system has the unique stable steady state. However, there are significant differences in stochastic behavior between them. Specifically, if the mean phosphorylated SK level is kept the same for the two modules, then the variance and the Fano factor for the phosphorylated RR in the BFS are always no less than those in the MFS, indicating that bifunctionality always enhances fluctuations. The correlation between the phosphorylated SK and the phosphorylated RR in the BFS is always positive mainly due to competition between system components, but this correlation in the MFS may be positive, almost zero, or negative, depending on the ratio between two rate constants. Our overall analysis indicates that differences between dynamical behaviors of monofunctional and bifunctional signaling modules are mainly in the stochastic rather than deterministic aspect.

  19. Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Plastic Surgery and Burns, Shenzhen Second People' s Hospital, Shenzhen 518035 (China); Wang, Mingbo [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); She, Zhending [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shenzhen Lando Biomaterials Co., Ltd., Shenzhen 518057 (China); Fan, Kunwu; Xu, Cheng [Department of Plastic Surgery and Burns, Shenzhen Second People' s Hospital, Shenzhen 518035 (China); Chu, Bin; Chen, Changsheng [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shi, Shengjun, E-mail: shengjunshi@yahoo.com [The Burns Department of Zhujiang Hospital, Southern Medical University, Guangzhou 510280 (China); Tan, Rongwei, E-mail: tanrw@landobiom.com [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shenzhen Lando Biomaterials Co., Ltd., Shenzhen 518057 (China)

    2015-07-01

    Inspired from the sophisticated bilayer structures of natural dermis, here, we reported collagen/chitosan based two-compartment and bi-functional dermal scaffolds. Two functions refer to mediating rapid angiogenesis based on recombinant human vascular endothelial growth factor (rhVEGF) and antibacterial from gentamicin, which were encapsulated in PLGA microspheres. The gentamicin and rhVEGF encapsulated PLGA microspheres were further combined with collagen/chitosan mixtures in low (lower layer) and high (upper layer) concentrations, and molded to generate the two-compartment and bi-functional scaffolds. Based on morphology and pore structure analyses, it was found that the scaffold has a distinct double layered porous and connective structure with PLGA microspheres encapsulated. Statistical analysis indicated that the pores in the upper layer and in the lower layer have great variations in diameter, indicative of a two-compartment structure. The release profiles of gentamicin and rhVEGF exceeded 28 and 49 days, respectively. In vitro culture of mouse fibroblasts showed that the scaffold can facilitate cell adhesion and proliferation. Moreover, the scaffold can obviously inhibit proliferation of Staphylococcus aureus and Serratia marcescens, exhibiting its unique antibacterial effect. The two-compartment and bi-functional dermal scaffolds can be a promising candidate for skin regeneration. - Highlights: • The dermal scaffold is inspired from the bilayer structures of natural dermis. • The dermal scaffold has two-compartment structures. • The dermal scaffold containing VEGF and gentamicin encapsulated PLGA microspheres • The dermal scaffold can facilitate cell adhesion and proliferation.

  20. Bifunctional Perovskite Oxide Catalysts for Oxygen Reduction and Evolution in Alkaline Media.

    Science.gov (United States)

    Gupta, Shiva; Kellogg, William; Xu, Hui; Liu, Xien; Cho, Jaephil; Wu, Gang

    2016-01-01

    Oxygen electrocatalysis, namely of the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER), governs the performance of numerous electrochemical energy systems such as reversible fuel cells, metal-air batteries, and water electrolyzers. However, the sluggish kinetics of these two reactions and their dependency on expensive noble metal catalysts (e.g, Pt or Ir) prohibit the sustainable commercialization of these highly innovative and in-demand technologies. Bifunctional perovskite oxides have emerged as a new class of highly efficient non-precious metal catalysts (NPMC) for oxygen electrocatalysis in alkaline media. In this review, we discuss the state-of-the-art understanding of bifunctional properties of perovskites with regards to their OER/ORR activity in alkaline media and review the associated reaction mechanisms on the oxides surface and the related activity descriptors developed in the recent literature. We also summarize the present strategies to modify their electronic structure and to further improve their performance for the ORR/OER through highlighting the new concepts relating to the role of surface redox chemistry and oxygen deficiency of perovskite oxides for the ORR/OER activity. In addition, we provide a brief account of recently developed advanced perovskite-nanocarbon hybrid bifunctional catalysts with much improved performances. PMID:26247625

  1. Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration

    International Nuclear Information System (INIS)

    Inspired from the sophisticated bilayer structures of natural dermis, here, we reported collagen/chitosan based two-compartment and bi-functional dermal scaffolds. Two functions refer to mediating rapid angiogenesis based on recombinant human vascular endothelial growth factor (rhVEGF) and antibacterial from gentamicin, which were encapsulated in PLGA microspheres. The gentamicin and rhVEGF encapsulated PLGA microspheres were further combined with collagen/chitosan mixtures in low (lower layer) and high (upper layer) concentrations, and molded to generate the two-compartment and bi-functional scaffolds. Based on morphology and pore structure analyses, it was found that the scaffold has a distinct double layered porous and connective structure with PLGA microspheres encapsulated. Statistical analysis indicated that the pores in the upper layer and in the lower layer have great variations in diameter, indicative of a two-compartment structure. The release profiles of gentamicin and rhVEGF exceeded 28 and 49 days, respectively. In vitro culture of mouse fibroblasts showed that the scaffold can facilitate cell adhesion and proliferation. Moreover, the scaffold can obviously inhibit proliferation of Staphylococcus aureus and Serratia marcescens, exhibiting its unique antibacterial effect. The two-compartment and bi-functional dermal scaffolds can be a promising candidate for skin regeneration. - Highlights: • The dermal scaffold is inspired from the bilayer structures of natural dermis. • The dermal scaffold has two-compartment structures. • The dermal scaffold containing VEGF and gentamicin encapsulated PLGA microspheres • The dermal scaffold can facilitate cell adhesion and proliferation

  2. Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

    International Nuclear Information System (INIS)

    Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH 5.5 (2H2O) at 45 degree C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments spectrometer fields of 14.09 and 11.74 T. The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparellel β-sheet (dαα NOE's) were observed in the 1H(13C)-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of α-helix were observed in the 1H(15N)SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H(1H) 2D NMR experiments

  3. TT2014 meeting report on the 12th Transgenic Technology meeting in Edinburgh: new era of transgenic technologies with programmable nucleases in the foreground

    Czech Academy of Sciences Publication Activity Database

    Beck, Inken; Sedláček, Radislav

    2015-01-01

    Roč. 24, č. 1 (2015), s. 179-183. ISSN 0962-8819 Institutional support: RVO:68378050 Keywords : Transgenic * Nuclease * Gene Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.322, year: 2014

  4. Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

    OpenAIRE

    Eftink, M R; Ghiron, C A; Kautz, R. A.; Fox, R. O.

    1989-01-01

    Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of...

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

    OpenAIRE

    Horner, Stacy M.; DiMaio, Daniel

    2007-01-01

    Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA bind...

  6. Production of 5′ Nucleotide by Using Halophilic Nuclease H Preferentially Adsorbed on Flocculated Cells of the Halophile Micrococcus varians subsp. halophilus

    OpenAIRE

    Onishi, Hiroshi; Kamekura, Masahiro; Yokoi, Haruhiko; Kobayashi, Takekazu

    1988-01-01

    A bioreactor with a column of flocculated cells of the moderate halophile Micrococcus varians subsp. halophilus which adsorbed the halophilic nuclease H was designed to be used in the production of 5′ nucleotides from RNA. A remarkable characteristic of the flocculated cells was that they preferentially adsorbed much exogenous nuclease, excluding adsorbed 5′ nucleotidase. Furthermore, desalting treatment of the flocculated cells in the presence of 2% MgSO4 · 7H2O gave rise to selective inacti...

  7. CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

    Science.gov (United States)

    Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2016-01-01

    The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases. PMID:27557689

  8. Response surface optimization of carbon and nitrogen sources for nuclease P1 production by Penicillium citrinum F-5-5

    International Nuclear Information System (INIS)

    Penicillium citrinum F-5-5, a nuclease P1 high-producing strain with 978.6 U/ml in potato glucose medium, was derived from the original Penicillium citrinum CICC 4011 with 60Co γ-rays irradiation mutation and then protoplasts fusion treatment. Culture components were optimized for the nuclease P1 production, and response surface methodology was applied for the critical medium components(carbon and nitrogen sources) which were preselected by Plackett-Burman design approach. Glucose, soluble starch and corn steep powder showed significant effects on production of nuclease. Central composite design was used for the optimization levels by software Minitab 15, and it showed that, the optimal values for the concentration of glucose, soluble starch and corn steep powder were 30.89, 42.46 and 11.60 g/L, respectively. With this medium,an enzyme activity of 1687.16 U/ml could be obtained theoretically. Using this optimized medium, an experimental enzyme activity of 1672.6 U/ml was reached. (authors)

  9. GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

    Science.gov (United States)

    Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith

    2014-01-01

    CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. PMID:25513782

  10. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula.

    Science.gov (United States)

    Lomate, Purushottam R; Bonning, Bryony C

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

  11. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

    Directory of Open Access Journals (Sweden)

    Yanwei Bi

    2014-05-01

    Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

  12. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    Science.gov (United States)

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  13. Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events.

    Science.gov (United States)

    Johnson, Graham D; Jodar, Meritxell; Pique-Regi, Roger; Krawetz, Stephen A

    2016-01-01

    Nuclear remodeling to a condensed state is a hallmark of spermatogenesis. This is achieved by replacement of histones with protamines. Regions retaining nucleosomes may be of functional significance. To determine their potential roles, sperm from wild type and transgenic mice harboring a single copy insert of the human protamine cluster were subjected to Micrococcal Nuclease-seq. CENTIPEDE, a hierarchical Bayesian model, was used to identify multiple spatial patterns, "footprints", of MNase-seq reads along the sperm genome. Regions predicted by CENTIPEDE analysis to be bound by a regulatory factor in sperm were correlated with genomic landmarks and higher order chromatin structure datasets to identify potential roles for these factors in regulating either prior or post spermatogenic, i.e., early embryonic events. This approach linked robust endogenous protamine transcription and transgene suppression to its chromatin environment within topologically associated domains. Of the candidate enhancer-bound regulatory proteins, Ctcf, was associated with chromatin domain boundaries in testes and embryonic stem cells. The continuity of Ctcf binding through the murine germline may permit rapid reconstitution of chromatin organization following fertilization. This likely reflects its preparation for early zygotic genome activation and comparatively accelerated preimplantation embryonic development program observed in mouse as compared to human and bull. PMID:27184706

  14. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5 ' nuclease PCR assay

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    included assays for the detection of verocytotoxin genes (vtx1, vtx2), pO157-associated genes (ehxA, katP, espP, and etpD), a recently identified adhesin (saa), intimin (eae, all variants), seven subtypes of eae, four subtypes of tir, and three subtypes of espD. A number of reference strains (VTEC and......In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization...... of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5' nuclease PCR assay (real-time PCR) that targets vtx1 and vtx2. VTEC isolates were obtained by colony replication by use of...

  15. Structural characterization of the virulence factor Sda1 nuclease from Streptococcus pyogenes.

    Science.gov (United States)

    Moon, Andrea F; Krahn, Juno M; Lu, Xun; Cuneo, Matthew J; Pedersen, Lars C

    2016-05-01

    Infection by Group A Streptococcus pyogenes (GAS) is a leading cause of severe invasive disease in humans, including streptococcal toxic shock syndrome and necrotizing fasciitis. GAS infections lead to nearly 163,000 annual deaths worldwide. Hypervirulent strains of S. pyogenes have evolved a plethora of virulence factors that aid in disease-by promoting bacterial adhesion to host cells, subsequent invasion of deeper tissues and blocking the immune system's attempts to eradicate the infection. Expression and secretion of the extracellular nuclease Sda1 is advantageous for promoting bacterial dissemination throughout the host organism, and evasion of the host's innate immune response. Here we present two crystal structures of Sda1, as well as biochemical studies to address key structural features and surface residues involved in DNA binding and catalysis. In the active site, Asn211 is observed to directly chelate a hydrated divalent metal ion and Arg124, on the putative substrate binding loop, likely stabilizes the transition state during phosphodiester bond cleavage. These structures provide a foundation for rational drug design of small molecule inhibitors to be used in prevention of invasive streptococcal disease. PMID:26969731

  16. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    Science.gov (United States)

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  17. Mutational and Biochemical Analysis of the DNA-entry Nuclease EndA from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    M Midon; P Schafer; A Pingoud; M Ghosh; A Moon; M Cuneo; R London; G Meiss

    2011-12-31

    EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays.

  18. Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease

    International Nuclear Information System (INIS)

    A recent paper from our laboratories Biochemistry 26, 6278] described the generation of site-directed substitutions for the putative general base Glu-43 in the active site of Staphylococcal nuclease (SNase) and the use of 1H NMR spectroscopy to characterize the effect of the substitutions on the conformations of the mutant proteins. The replacements for Glu-43 (Asp, Gln, Asn, Ser, and Ala) both decreased the catalytic efficiency and changed the one- and two-dimensional NMR spectral properties of the mutant enzymes. We have prepared and studied the NMR spectral properties of several samples of deuteriated wild-type SNase that allow sequence-specific resonance assignments for several aromatic and aliphatic amino acid side chains that experience changes both in normal one-dimensional spectra and in two-dimensional NOESY spectra. Due to severe spectral congestion of resonances in the one- and two-dimensional spectra of protiated SNase, the assignments would have been difficult, if not impossible, to obtain without deuteriation of selected amino acids. The spectra we have obtained demonstrate that changes in NOE intensities involve a valine residue that is spatially adjacent to two phenylalanine residues; given the X-ray structure for SNase, these residues must be Val-74, Phe-34, and Phe-76. In addition, a leucine residue experiencing changes in NOE intensities spatially adjacent to Val-74 and Phe-34 can be assigned to Leu-25

  19. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

    Science.gov (United States)

    Sauer, Noel J; Narváez-Vásquez, Javier; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Woodward, Melody J; Mihiret, Yohannes A; Lincoln, Tracey A; Segami, Rosa E; Sanders, Steven L; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-04-01

    Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants. PMID:26864017

  20. Oxidative stress-mediated protein conformation changes: ESR study of spin-labelled staphylococcal nuclease

    International Nuclear Information System (INIS)

    We report on the electron spin resonance (ESR) study of the photo-oxidative stress-mediated protein conformation changes in the spin-labelled protein staphylococcal nuclease (SNase). The photo-oxidative stress was brought on by photosensitization of singlet oxygen (1Δg) in the presence of a novel photosensitizer, water-soluble fullerol C60(OH)19(ONa)17, and resulted in partial protein denaturation. This process was monitored via ESR measurements performed for a spin-labelled SNase Thr-62-Cys mutant, with MTSSL spin label (SL) attached to the cysteine 62 residue (SNase T62C-SL). Prior to ESR measurements of the oxidative stress-induced alterations in protein conformations, the efficiency of C60(OH)19(ONa)17 for 1Δg-generation was confirmed by three different techniques: (i) selective reactive scavenging of 1Δg and ESR detection of the resulting paramagnetic product (ii) 1Δg-mediated photo-oxidative loss of tryptophan and (iii) by measuring the characteristic near-infrared phosphorescence of singlet oxygen at 1270 nm. The observed evolution of the ESR spectra of SNase T62C-SL as a function of exposure to the photo-oxidative stress points to marked changes in protein conformation due to the deleterious action of 1Δg

  1. Preparation and characteristics of Fe3O4-YVO4:Eu3+ bifunctional magnetic-luminescent nanocomposites

    International Nuclear Information System (INIS)

    Graphical abstract: Highlights: → Bifunctional magnetic-luminescent nanocomposites with Fe3O4 nanoparticles as the core and YVO4:Eu3+ as the shell. → A cubic spinel structrue of Fe3O4 core and a tetragonal phase of YVO4 shell were obtained. → The nanocomposites displayed a strong red emission and superparamagnetic behavior at room temperature. - Abstract: A facile direct precipitation method has been developed for the synthesis of bifunctional magnetic-luminescent nanocomposites with Fe3O4 nanoparticles as the core and YVO4:Eu3+ as the shell. Transmission electron microscopy (TEM) images revealed that the obtained bifunctional nanocomposites had a core-shell structure and a spherical morphology. The average size was ∼150 nm, and the thickness of the shell was ∼15 nm. The X-ray diffraction (XRD) patterns showed that a cubic spinel structure of Fe3O4 core and a tetragonal phase of YVO4 shell were obtained. Fourier transform infrared (FT-IR) spectra confirmed that the YVO4:Eu3+ had been successfully deposited on the surface of Fe3O4 nanoparticles. Photoluminescence (PL) spectra indicated that the nanocomposites displayed a strong red characteristic emission of Eu3+. Magnetic measurements showed that the obtained bifunctional nanocomposites exhibited superparamagnetic behavior at room temperature. Therefore, the bifunctional nanocomposites are expected to develop many potential applications in biomedical fields.

  2. Nanostructured Perovskite LaCo1-xMnxO3 as Bifunctional Catalysts for Rechargeable Metal-Air Batteries

    Science.gov (United States)

    Ge, Xiaoming; Li, Bing; Wuu, Delvin; Sumboja, Afriyanti; An, Tao; Hor, T. S. Andy; Zong, Yun; Liu, Zhaolin

    2015-09-01

    Bifunctional catalyst that is active for both oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) is one of the most important components of rechargeable metal-air batteries. Nanostructured perovskite bifunctional catalysts comprising La, Co and Mn(LaCo1-xMnxO3, LCMO) are synthesized by hydrothermal methods. The morphology, structure and electrochemical activity of the perovskite bifunctional catalysts are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and rotating disk electrode (RDE) techniques. Nanorod, nanodisc and nanoparticle are typical morphologies of LCMO. The electrocatalytic activity of LCMO is significantly improved by the addition of conductive materials such as carbon nanotube. To demonstrate the practical utilization, LCMO in the composition of LaCo0.8Mn0.2O3(LCMO82) is used as air cathode catalysts for rechargeable zinc-air batteries. The battery prototype can sustain 470 h or 40 discharge-charge cycles equivalent.

  3. Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage

    International Nuclear Information System (INIS)

    Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage

  4. Mechanical characterization of a bifunctional Tetronic hydrogel adhesive for soft tissues.

    Science.gov (United States)

    Sanders, Lindsey; Stone, Roland; Webb, Kenneth; Mefford, Thompson; Nagatomi, Jiro

    2015-03-01

    Although a number of tissue adhesives and sealants for surgical use are currently available, attaining a useful balance in high strength, high compliance, and low swelling has proven difficult. Recent studies have demonstrated that a four-arm poly(propylene oxide)-poly(ethylene oxide) block copolymer, Tetronic, can be chemically modified to form a hydrogel tissue adhesive (Cho et al., Acta Biomater 2012;8:2223-2232; Barrett et al., Adv Health Mater 2012;1-11; Balakrishnan, Evaluating mechanical performance of hydrogel-based adhesives for soft tissue applications. Clemson University, All Theses, Paper 1574: Tiger Prints; 2013). Building on the success of these studies, this study explored bifunctionalization of Tetronic with acrylates for chemical crosslinking of the hydrogel and N-hydroxysuccinimide (NHS) for reaction with tissue amines. The adhesive bond strengths of various uni and bifunctional Tetronic blends (T1107 ACR: T1107 ACR/NHS) determined by lap shear testing ranged between 8 and 74 kPa, with the 75:25 (T1107 ACR: T1107 ACR/NHS) blend displaying the highest value. These results indicated that addition of NHS led to improvement of tissue bond strength over acrylation alone. Furthermore, ex vivo pressure tests using the rat bladder demonstrated that the bifunctional Tetronic adhesive exhibited high compliance and maintained pressures under hundreds of filling and emptying cycles. Together, the results of this study provided evidence that the bifunctional Tetronic adhesive with a proper blend ratio may be used to achieve an accurate balance in bulk and tissue bond strengths, as well as the compliance and durability for soft tissue such as the bladder. PMID:25111445

  5. Bifunctional phase-transfer catalysis in the asymmetric synthesis of biologically active isoindolinones

    Science.gov (United States)

    Di Mola, Antonia; Tiffner, Maximilian; Scorzelli, Francesco; Palombi, Laura; Filosa, Rosanna; De Caprariis, Paolo

    2015-01-01

    Summary New bifunctional chiral ammonium salts were investigated in an asymmetric cascade synthesis of a key building block for a variety of biologically relevant isoindolinones. With this chiral compound in hand, the development of further transformations allowed for the synthesis of diverse derivatives of high pharmaceutical value, such as the Belliotti (S)-PD172938 and arylated analogues with hypnotic sedative activity, obtained in good overall total yield (50%) and high enantiomeric purity (95% ee). The synthetic routes developed herein are particularly convenient in comparison with the current methods available in literature and are particularly promising for large scale applications. PMID:26734105

  6. Tunable catalytic properties of bi-functional mixed oxides in ethanol conversion to high value compounds

    Energy Technology Data Exchange (ETDEWEB)

    Ramasamy, Karthikeyan K.; Gray, Michel J.; Job, Heather M.; Smith, Colin D.; Wang, Yong

    2016-04-10

    tA highly versatile ethanol conversion process to selectively generate high value compounds is pre-sented here. By changing the reaction temperature, ethanol can be selectively converted to >C2alcohols/oxygenates or phenolic compounds over hydrotalcite derived bi-functional MgO–Al2O3cata-lyst via complex cascade mechanism. Reaction temperature plays a role in whether aldol condensationor the acetone formation is the path taken in changing the product composition. This article containsthe catalytic activity comparison between the mono-functional and physical mixture counterpart to thehydrotalcite derived mixed oxides and the detailed discussion on the reaction mechanisms.

  7. Bifunctional Organic Polymeric Catalysts with a Tunable Acid-Base Distance and Framework Flexibility

    Science.gov (United States)

    Chen, Huanhui; Wang, Yanan; Wang, Qunlong; Li, Junhui; Yang, Shiqi; Zhu, Zhirong

    2014-09-01

    Acid-base bifunctional organic polymeric catalysts were synthesized with tunable structures. we demonstrated two synthesis approaches for structural fine-tune. In the first case, the framework flexibility was tuned by changing the ratio of rigid blocks to flexible blocks within the polymer framework. In the second case, we precisely adjusted the acid-base distance by distributing basic monomers to be adjacent to acidic monomers, and by changing the chain length of acidic monomers. In a standard test reaction for the aldol condensation of 4-nitrobenzaldehyde with acetone, the catalysts showed good reusability upon recycling and maintained relatively high conversion percentage.

  8. Bifunctional Brønsted Base Catalyzes Direct Asymmetric Aldol Reaction of α-Keto Amides.

    Science.gov (United States)

    Echave, Haizea; López, Rosa; Palomo, Claudio

    2016-03-01

    The first enantioselective direct cross-aldol reaction of α-keto amides with aldehydes, mediated by a bifunctional ureidopeptide-based Brønsted base catalyst, is described. The appropriate combination of a tertiary amine base and an aminal, and urea hydrogen-bond donor groups in the catalyst structure promoted the exclusive generation of the α-keto amide enolate which reacted with either non-enolizable or enolizable aldehydes to produce highly enantioenriched polyoxygenated aldol adducts without side-products resulting from dehydration, α-keto amide self-condensation, aldehyde enolization, and isotetronic acid formation. PMID:26835655

  9. The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

    DEFF Research Database (Denmark)

    Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio;

    2014-01-01

    are specifically presumed to deliver the diverse array of nucleotide sugars found in plants. This study has developed a novel approach that enabled functional characterization of six bifunctional UDP-rhamnose (Rha)/UDP-galactose (Gal) transporters from Arabidopsis. An analysis of loss-of-function and...... overexpression lines for two of these transporters identified biochemical alterations supporting their roles in the biosynthesis of Rha- and Gal-containing polysaccharides. Thus, cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is likely also regulated by substrate transport mechanisms....

  10. Bifunctional phase-transfer catalysis in the asymmetric synthesis of biologically active isoindolinones.

    Science.gov (United States)

    Di Mola, Antonia; Tiffner, Maximilian; Scorzelli, Francesco; Palombi, Laura; Filosa, Rosanna; De Caprariis, Paolo; Waser, Mario; Massa, Antonio

    2015-01-01

    New bifunctional chiral ammonium salts were investigated in an asymmetric cascade synthesis of a key building block for a variety of biologically relevant isoindolinones. With this chiral compound in hand, the development of further transformations allowed for the synthesis of diverse derivatives of high pharmaceutical value, such as the Belliotti (S)-PD172938 and arylated analogues with hypnotic sedative activity, obtained in good overall total yield (50%) and high enantiomeric purity (95% ee). The synthetic routes developed herein are particularly convenient in comparison with the current methods available in literature and are particularly promising for large scale applications. PMID:26734105

  11. An efficient bifunctional electrocatalyst for water splitting based on cobalt phosphide

    Science.gov (United States)

    Yang, Libin; Qi, Honglan; Zhang, Chengxiao; Sun, Xuping

    2016-06-01

    The development of highly efficient electrocatalysts for water splitting is critical for various renewable-energy technologies. In this letter, we demonstrate a cobalt phosphide nanowire array grown on a Ti mesh (CoP/TM) behaving as a bifunctional electrocatalyst for water splitting. The CoP/TM electrode delivers 10 mA cm‑2 at an overpotential of 72 mV for the hydrogen evolution reaction (HER) and 310 mV for the oxygen evolution reaction (OER) in 1.0 M KOH. Furthermore, its corresponding two-electrode alkaline electrolyzer displays 10 mA cm‑2 at 1.64 V.

  12. Neurodegeneration in D-bifunctional protein deficiency: diagnostic clues and natural history using serial magnetic resonance imaging

    International Nuclear Information System (INIS)

    We report serial neurodegenerative changes on neuroimaging in a rare peroxisomal disease called D-bifunctional protein deficiency. The pattern of posterior to anterior demyelination with white matter disease resembles X-linked adrenoleukodystrophy. We feel this case is important to (1) highlight that D-bifunctional protein deficiency should be considered in cases where the neuroimaging resembles X-linked adrenoleukodystrophy, (2) to show different stages of progression to help identify this disease using neuroimaging in children, and (3) to show that neuroimaging suggesting a leukodystrophy can warrant peroxisomal beta-oxidation studies in skin fibroblasts even when plasma very long chain fatty acids are normal. (orig.)

  13. Cobalt nanoparticles embedded in N-doped carbon as an efficient bifunctional electrocatalyst for oxygen reduction and evolution reactions

    Science.gov (United States)

    Su, Yunhe; Zhu, Yihua; Jiang, Hongliang; Shen, Jianhua; Yang, Xiaoling; Zou, Wenjian; Chen, Jianding; Li, Chunzhong

    2014-11-01

    Cobalt based catalysts are promising bifunctional electrocatalysts for both oxygen reduction and oxygen evolution reactions (ORR and OER) in unitized regenerative fuel cells (URFCs) operating with alkaline electrolytes. Here we report a hybrid composite of cobalt nanoparticles embedded in nitrogen-doped carbon (Co/N-C) via a solvothermal carbonization strategy. With the synergistic effect arising from the N-doped carbon and cobalt nanoparticles in the composite, the Co/N-C hybrid catalyst exhibits highly efficient bifunctional catalytic activity and excellent stability toward both ORR and OER. The ΔE (oxygen electrode activity parameter for judging the overall electrocatalytic activity of a bifunctional electrocatalyst) value for Co/N-C is 0.859 V, which is smaller than those of Pt/C and most of the non-precious metal catalysts in previous studies. Furthermore, the Co/N-C composite also shows better bifunctional catalytic activity than its oxidative counterparts, which could be attributed to the high specific surface area and the efficient charge transfer ability of the composite, as well as the good synergistic effect between N-doped carbon and the Co nanoparticles in the Co/N-C composite.Cobalt based catalysts are promising bifunctional electrocatalysts for both oxygen reduction and oxygen evolution reactions (ORR and OER) in unitized regenerative fuel cells (URFCs) operating with alkaline electrolytes. Here we report a hybrid composite of cobalt nanoparticles embedded in nitrogen-doped carbon (Co/N-C) via a solvothermal carbonization strategy. With the synergistic effect arising from the N-doped carbon and cobalt nanoparticles in the composite, the Co/N-C hybrid catalyst exhibits highly efficient bifunctional catalytic activity and excellent stability toward both ORR and OER. The ΔE (oxygen electrode activity parameter for judging the overall electrocatalytic activity of a bifunctional electrocatalyst) value for Co/N-C is 0.859 V, which is smaller than those

  14. Preparation of Pt/Irx(IrO2)10 - x bifunctional oxygen catalyst for unitized regenerative fuel cell

    Science.gov (United States)

    Kong, Fan-Dong; Zhang, Sheng; Yin, Ge-Ping; Zhang, Na; Wang, Zhen-Bo; Du, Chun-Yu

    2012-07-01

    Bifunctional Pt/Irx(IrO2)10 - x (x oxygen reduction reaction (ORR) (21.71 mA mg-1 at 0.85 V). Meanwhile, considerably high activity towards oxygen evolution reaction (OER) (42.35 mA mg-1 at 1.55 V) is also observed for Pt/Ir3(IrO2)7 catalyst. Kinetic analyses indicate that ORR on Pt/Ir3(IrO2)7 catalyst follows four-electron mechanism. This work opens a new way to fabricate efficient bifunctional oxygen catalyst for URFC.

  15. A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids.

    OpenAIRE

    El-Azaz, Jorge; Cánovas, Francisco M.; de la Torre, Fernando; Ávila, Concepción

    2014-01-01

    A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids. Fernando de la Torre, Jorge El-Azaz, Concepción Ávila, Francisco M. Cánovas Departamento de Biología Molecular y Bioquímica. Universidad de Málaga. Bifunctional aspartate/prephenate aminotransferases (AAT/PAT) are plastid-located enzymes encoded by a single locus in all reported plants, which develop two different enzymatic activities: aspartate aminotransferas...

  16. Neurodegeneration in D-bifunctional protein deficiency: diagnostic clues and natural history using serial magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Aneal [University of Calgary, Department of Medical Genetics and Pediatrics, Alberta Children' s Hospital, Calgary, AB (Canada); Wei, Xing-Chang [University of Calgary, Department of Radiology, Alberta Children' s Hospital, Calgary, AB (Canada); Snyder, Floyd F. [Alberta Children' s Hospital, Biochemical Genetics Laboratory, Calgary, AB (Canada); Mah, Jean K. [University of Calgary, Division of Neurology, Department of Pediatrics, Calgary, AB (Canada); Waterham, Hans; Wanders, Ronald J.A. [University of Amsterdam, Academic Medical Center, Lab Genetic Metabolic Diseases, Amsterdam (Netherlands)

    2010-12-15

    We report serial neurodegenerative changes on neuroimaging in a rare peroxisomal disease called D-bifunctional protein deficiency. The pattern of posterior to anterior demyelination with white matter disease resembles X-linked adrenoleukodystrophy. We feel this case is important to (1) highlight that D-bifunctional protein deficiency should be considered in cases where the neuroimaging resembles X-linked adrenoleukodystrophy, (2) to show different stages of progression to help identify this disease using neuroimaging in children, and (3) to show that neuroimaging suggesting a leukodystrophy can warrant peroxisomal beta-oxidation studies in skin fibroblasts even when plasma very long chain fatty acids are normal. (orig.)

  17. Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

    Directory of Open Access Journals (Sweden)

    Zschemisch Nils-Holger

    2012-11-01

    Full Text Available Abstract Background Engineered zinc-finger nucleases (ZFN represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB and initiated non-homologous end joining (NHEJ after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(DJ recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. Results After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. Conclusion The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

  18. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  19. Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks by Artemis Nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Povrik, Lawrence F.; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J.; Yannone, Steven M.

    2005-10-01

    The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double-strand breaks. To assess the possibility that Artemis functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.

  20. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Directory of Open Access Journals (Sweden)

    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  1. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Science.gov (United States)

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  2. Structural Plasticity of Staphylococcal Nuclease Probed by Perturbation with Pressure and pH

    Science.gov (United States)

    Kitahara, Ryo; Hata, Kazumi; Maeno, Akihiro; Akasaka, Kazuyuki; Chimenti, Michael; Bertrand Garcia-Moreno, E; Schroer, Martin A.; Jeworrek, Christoph; Tolan, Metin; Winter, Roland; Roche, Julien; Roumestand, Christian; de Guillen, Karine Montet; Royer, Catherine A.

    2012-01-01

    The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. SAXS and NMR spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (< 2 kbar) at pH values where Lys-66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant non-linear changes in chemical shifts in the helix where Lys-66 is located. Above 2 kbar pressure at these pH values the protein with Lys-66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys-66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys-66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure. PMID:21254234

  3. Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

    Directory of Open Access Journals (Sweden)

    Lingyan Xing

    Full Text Available foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd coding exon: a 17 base-pair (bp deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

  4. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    Science.gov (United States)

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  5. Large-scale Generation of Patterned Bubble Arrays on Printed Bi-functional Boiling Surfaces

    Science.gov (United States)

    Choi, Chang-Ho; David, Michele; Gao, Zhongwei; Chang, Alvin; Allen, Marshall; Wang, Hailei; Chang, Chih-Hung

    2016-04-01

    Bubble nucleation control, growth and departure dynamics is important in understanding boiling phenomena and enhancing nucleate boiling heat transfer performance. We report a novel bi-functional heterogeneous surface structure that is capable of tuning bubble nucleation, growth and departure dynamics. For the fabrication of the surface, hydrophobic polymer dot arrays are first printed on a substrate, followed by hydrophilic ZnO nanostructure deposition via microreactor-assisted nanomaterial deposition (MAND) processing. Wettability contrast between the hydrophobic polymer dot arrays and aqueous ZnO solution allows for the fabrication of heterogeneous surfaces with distinct wettability regions. Heterogeneous surfaces with various configurations were fabricated and their bubble dynamics were examined at elevated heat flux, revealing various nucleate boiling phenomena. In particular, aligned and patterned bubbles with a tunable departure frequency and diameter were demonstrated in a boiling experiment for the first time. Taking advantage of our fabrication method, a 6 inch wafer size heterogeneous surface was prepared. Pool boiling experiments were also performed to demonstrate a heat flux enhancement up to 3X at the same surface superheat using bi-functional surfaces, compared to a bare stainless steel surface.

  6. Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase.

    Science.gov (United States)

    Fushinobu, Shinya; Nishimasu, Hiroshi; Hattori, Daiki; Song, Hyun-Jin; Wakagi, Takayoshi

    2011-10-27

    Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-Å resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction. PMID:21983966

  7. Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase.

    Science.gov (United States)

    Du, Juan; Say, Rafael F; Lü, Wei; Fuchs, Georg; Einsle, Oliver

    2011-10-27

    Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3 Å. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality. PMID:21983965

  8. Subnanometer Cobalt-Hydroxide-Anchored N-Doped Carbon Nanotube Forest for Bifunctional Oxygen Catalyst.

    Science.gov (United States)

    Kim, Ji Eun; Lim, Joonwon; Lee, Gil Yong; Choi, Sun Hee; Maiti, Uday Narayan; Lee, Won Jun; Lee, Ho Jin; Kim, Sang Ouk

    2016-01-27

    Electrochemical oxygen redox reactions are the crucial elements for energy conversion and storage including fuel cells and metal air batteries. Despite tremendous research efforts, developing high-efficient, low-cost, and durable bifunctional oxygen catalysts remains a major challenge. We report a new class of hybrid material consisting of subnanometer thick amorphous cobalt hydroxide anchored on NCNT as a durable ORR/OER bifunctional catalyst. Although amorphous cobalt species-based catalysts are known as good OER catalysts, hybridizing with NCNT successfully enhanced ORR activity by promoting a 4e reduction pathway. Abundant charge carriers in amorphous cobalt hydroxide are found to trigger the superior OER activity with high current density and low Tafel slope as low as 36 mV/decade. A remarkably high OER turnover frequency (TOF) of 2.3 s(-1) at an overpotential of 300 mV was obtained, one of the highest values reported so far. Moreover, the catalytic activity was maintained over 120 h of cycling. The unique subnanometer scale morphology of amorphous hydroxide cobalt species along with intimate cobalt species-NCNT interaction minimizes the deactivation of catalyst during prolonged repeated cycles. PMID:26766495

  9. Highly Efficient and Robust Nickel Phosphides as Bifunctional Electrocatalysts for Overall Water-Splitting.

    Science.gov (United States)

    Li, Jiayuan; Li, Jing; Zhou, Xuemei; Xia, Zhaoming; Gao, Wei; Ma, Yuanyuan; Qu, Yongquan

    2016-05-01

    To search for the efficient non-noble metal based and/or earth-abundant electrocatalysts for overall water-splitting is critical to promote the clean-energy technologies for hydrogen economy. Herein, we report nickel phosphide (NixPy) catalysts with the controllable phases as the efficient bifunctional catalysts for water electrolysis. The phases of NixPy were determined by the temperatures of the solid-phase reaction between the ultrathin Ni(OH)2 plates and NaH2PO2·H2O. The NixPy with the richest Ni5P4 phase synthesized at 325 °C (NixPy-325) delivered efficient and robust catalytic performance for hydrogen evolution reaction (HER) in the electrolytes with a wide pH range. The NixPy-325 catalysts also exhibited a remarkable performance for oxygen evolution reaction (OER) in a strong alkaline electrolyte (1.0 M KOH) due to the formation of surface NiOOH species. Furthermore, the bifunctional NixPy-325 catalysts enabled a highly performed overall water-splitting with ∼100% Faradaic efficiency in 1.0 M KOH electrolyte, in which a low applied external potential of 1.57 V led to a stabilized catalytic current density of 10 mA/cm(2) over 60 h. PMID:27064172

  10. Introducing new 99m-Tc-bifunctional radiopharmaceutical containing dithiosemicarbazone chelate group

    International Nuclear Information System (INIS)

    In our attempt to develop 99m-Tc-Bifunctional Radiopharmaceuticals (BR) of biomolecules, our interest has been focussed on the di-thiosemicarbazone chelating group (DTS); its coordination moiety allows a 1:1 complex with a tetravalent TcO2+, generating a neutral chelate of great stability and compactness. For ligand containing S N coordination, satisfactory labeling has been achieved at pH 5-6, in the presence of Sn-Resin. Based on the above mentioned, the use of DTS is tested in two different modalities by selecting glucose and albumin as the biomolecules of interest. The synthesis of a glucosone-1,2-bis (thiosemicarbazone) (Glu-DTS) and upon its labelling, the 99m-Tc-Glu-DTS was tested in animals. Scintigraphic studies revealed high activity in the brain and heart. This result offered strong support for the use of DTS as a bifunctional chelating agent (BCA). DTS-COOH and DTS-NH2 were synthesized for the coupling with biomolecules. As a first trial, they were coupled with albumin and their functionality proven. The results gathered provided conclusive evidence for the remarkable characteristic of DTS as a basic structure for the development of 99m-Tc-RP

  11. Construction of a bifunctional enzyme fusion for the combined determination of biogenic amines in foods.

    Science.gov (United States)

    Lee, Jae-Ick; Jang, Jun-Hyuck; Yu, Mi-Ji; Kim, Young-Wan

    2013-09-25

    Biogenic amines (BAs) are a group of low-molecular-mass organic bases derived from free amino acids. Due to the undesirable effects of BAs on human health, amine oxidase-based detection methods for BAs in foods have been developed. Here, we developed a bifunctional enzyme fusion (MAPO) using a Cu(2+)-containing monoamine oxidase (AMAO2) and a flavin adenine dinucleotide-containing putrescine oxidase (APUO) from Arthrobacter aurescens. It was necessary to activate MAPO with supplementary Cu(2+) ions, leading to a 6- to 12-fold improvement in catalytic efficiency (kcat/KM) for monoamines. The optimal temperatures of Cu(2+)-activated MAPO (cMAPO) for both tyramine and putrescine were 50 °C, and the optimal pH values for tyramine and putrescine were pH 7.0 and pH 8.0, respectively, consistent with those of AMAO2 and APUO, respectively. The cMAPO showed relative specific activities of 100, 99, 32, and 32 for 2-phenylethylamine, tyramine, histamine, and putrescine, respectively. The tyramine-equivalent BA contents of fermented soybean pastes by cMAPO were more than 90% of the total BA determined by HPLC. In conclusion, cMAPO is fully bifunctional toward biogenic monoamines and putrescine, allowing the combined determination of multiple BAs in foods. This colorimetric determination method could be useful for point-of-care testing to screen safety-guaranteed products prior to instrumental analyses. PMID:24001036

  12. Bifunctional composite microspheres of silica/lanthanide-polyoxometalates/Au: Study on luminescence and catalytic properties

    International Nuclear Information System (INIS)

    In this paper, the synthesis and properties of composite silica microspheres grafted with gold nanoparticles and lanthanide-polyoxometalates are described. This synthesis employs polyethyleneimine as the crosslink polymer to immobilize the Au nanoparticles and lanthanide-polyoxometalates on silica spheres, which results in the formation of bifunctional composite microspheres of silica/lanthanide-polyoxometalates/Au. The composite material was found to be catalytically active in the oxidation of styrene, and benzaldehyde and styrene oxide were the main products. Catalyzed oxidation of styrene demonstrates the size-dependent activity of catalysts and the smaller catalyst shows the higher selectivity. Moreover, the composite particles show bright red luminescence under UV light, which could be seen by naked eyes. The luminescence properties of composite material and the effect of Au nanoparticles on the luminescence of Eu ion were investigated, and energy could be more effectively transferred from ligand to lanthanide ion when Au nanoparticles were grafted on silica spheres. The integration of luminescent components and Au particles makes it possible to label catalyst and monitor the catalyzed reactions. - Highlights: • The bifunctional composite microspheres were fabricated. • Both polyoxometalates and Au nanoparticles could be grafted on silica spheres. • The composite particles exhibit the excellent luminescence and catalytic activity. • The Au nanoparticles affect the luminescence properties of Eu3+ ions

  13. Determining the Origin of Half-bandgap-voltage Electroluminescence in Bifunctional Rubrene/C60 Devices

    Science.gov (United States)

    Chen, Qiusong; Jia, Weiyao; Chen, Lixiang; Yuan, De; Zou, Yue; Xiong, Zuhong

    2016-01-01

    Lowering the driving voltage of organic light-emitting diodes (OLEDs) is an important approach to reduce their energy consumption. We have fabricated a series of bifunctional devices (OLEDs and photovoltaics) using rubrene and fullerene (C60) as the active layer, in which the electroluminescence threshold voltage(~1.1 V) was half the value of the bandgap of rubrene. Magneto-electroluminescence (MEL) response of planner heterojunction diodes exhibited a small increase in response to a low magnetic field strength (20 mT). When a hole-transport layer with a low mobility was included in these devices, the MEL response reversed in shape, and simultaneously, the EL threshold voltage became larger than the bandgap voltage. When bulk heterojunction device was examined, the amplitude of MEL curves presented an anomalous voltage-dependence. Following an analysis of the MEL responses of these devices, we proposed that the EL of half-bandgap-voltage device originated from bimolecular triplet-triplet annihilation in the rubrene film, rather than from singlet excitons that formed via an interface auger recombination. This work provides critical insight into the mechanisms of OLED emission and will help advance the applications of bifunctional devices. PMID:27142285

  14. Determining the Origin of Half-bandgap-voltage Electroluminescence in Bifunctional Rubrene/C60 Devices.

    Science.gov (United States)

    Chen, Qiusong; Jia, Weiyao; Chen, Lixiang; Yuan, De; Zou, Yue; Xiong, Zuhong

    2016-01-01

    Lowering the driving voltage of organic light-emitting diodes (OLEDs) is an important approach to reduce their energy consumption. We have fabricated a series of bifunctional devices (OLEDs and photovoltaics) using rubrene and fullerene (C60) as the active layer, in which the electroluminescence threshold voltage(~1.1 V) was half the value of the bandgap of rubrene. Magneto-electroluminescence (MEL) response of planner heterojunction diodes exhibited a small increase in response to a low magnetic field strength (20 mT). When a hole-transport layer with a low mobility was included in these devices, the MEL response reversed in shape, and simultaneously, the EL threshold voltage became larger than the bandgap voltage. When bulk heterojunction device was examined, the amplitude of MEL curves presented an anomalous voltage-dependence. Following an analysis of the MEL responses of these devices, we proposed that the EL of half-bandgap-voltage device originated from bimolecular triplet-triplet annihilation in the rubrene film, rather than from singlet excitons that formed via an interface auger recombination. This work provides critical insight into the mechanisms of OLED emission and will help advance the applications of bifunctional devices. PMID:27142285

  15. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification

    OpenAIRE

    Guilinger, John P.; Thompson, David B.; Liu, David R.

    2014-01-01

    Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ~15 or 25 base pairs apart. In human cells, fCas9 modified target DNA ...

  16. Recognition and repair of the CC-1065-(N3-Adenine)-DNA adduct by the UVRABC nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Tang, M.; Lee, C.S.; Doisy, R.; Ross, L.; Needham-VanDevanter, D.R.; Hurley, L.H.

    1988-02-09

    The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, the authors have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, they have found that UVRABC nuclease incises at the eight phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure.

  17. Recognition and repair of the CC-1065-(N3-Adenine)-DNA adduct by the UVRABC nuclease

    International Nuclear Information System (INIS)

    The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, the authors have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, they have found that UVRABC nuclease incises at the eight phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure

  18. Conformational consequences of ionization of Lys, Asp and Glu buried at position 66 in staphylococcal nuclease

    OpenAIRE

    Karp, Daniel A.; Stahley, Mary R.; Bertrand, García-Moreno E.

    2010-01-01

    The pKa values measured previously for the internal Lys-66, Asp-66 and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pKa units relative to normal pKa values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of t...

  19. Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases

    OpenAIRE

    Karginov, Fedor V.; Cheloufi, Sihem; Chong, Mark M.W.; Stark, Alexander; Smith, Andrew D; Hannon, Gregory J.

    2010-01-01

    The lifespan of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here, we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5′-phosphate groups in m...

  20. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone

    Indian Academy of Sciences (India)

    KARREDDULA RAJA; AKKILI SUSEELAMMA; KATREDDI HUSSAIN REDDY

    2016-08-01

    Lanthanide(III) complexes of general formula [La(BPAH)₂(NO₃)₃] and [Ce(BPAH)₂(NO₃)(H₂O)₂] 2NO₃.H₂O (where, BPAH = 2-benzoylpyridine acetyl hydrazone), were synthesized and characterized by elemental analysis, molar conductance, IR spectroscopy and single crystal X-ray diffraction and Hirschfeld studies. The central metal ion is 12-coordinate in lanthanum complex and 10-coordinated in the cerium complex. The coordination polyhedra around the lanthanum and cerium were found to have distorted icosahedron and distorted bicapped square antiprism respectively. DNA binding and nuclease activity of these complexes were also investigated in the present work.

  1. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.

    Science.gov (United States)

    Wyvekens, Nicolas; Topkar, Ved V; Khayter, Cyd; Joung, J Keith; Tsai, Shengdar Q

    2015-07-01

    Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. PMID:26068112

  2. Pd(0)@UiO-68-AP: chelation-directed bifunctional heterogeneous catalyst for stepwise organic transformations.

    Science.gov (United States)

    Li, Yan-An; Yang, Song; Liu, Qi-Kui; Chen, Gong-Jun; Ma, Jian-Ping; Dong, Yu-Bin

    2016-05-01

    A bifunctional heterogeneous catalyst Pd(0)@UiO-68-AP based on a chelation-directed post-synthetic approach is reported. It exhibits typical heterogeneous catalytic behaviour and can promote benzyl alcohol oxidiation-Knoevenagel condensation in a stepwise way. PMID:27035589

  3. Complexation and biodistribution study of 111In complexes of bifunctional phosphinic acid analogues of H4DOTA

    Czech Academy of Sciences Publication Activity Database

    Forsterová, Michaela; Zimová, Jana; Petrík, M.; Lázníček, M.; Lázníčková, A.; Hermann, P.; Melichar, František

    2007-01-01

    Roč. 2, č. 337 (2007), s. 34-34. ISSN 1619-7070 R&D Projects: GA AV ČR 1QS100480501 Institutional research plan: CEZ:AV0Z10480505 Keywords : bifunctional H4DOTA ligands * phosphinic acid analogues, * complexation of 111In Subject RIV: FR - Pharmacology ; Medidal Chemistry

  4. Bifunctional, Carbon-Free Nickel/Cobalt-Oxide Cathodes for Lithium-Air Batteries with an Aqueous Alkaline Electrolyte

    International Nuclear Information System (INIS)

    Highlights: • High activity bi-functional catalyst combination for ORR and OER . • An optimum ratio of high active bi-functional catalysts was found. • Novel electrodes without carbon to avoid carbon corrosion during OER mode. • EIS model for OER describes influence of a growing oxide layers. • Long-term test exhibited an excellent long-term stability over 1200 cycles. - Abstract: Lithium-air batteries with an aqueous alkaline electrolyte promise a very high practical energy density and capacity. These batteries are mainly limited by high overpotentials on the bifunctional cathode during charge and discharge. To reduce overpotentials the bifunctional cathode of such batteries must be improved significantly. Nickel is relatively inexpensive and has a good catalytic activity in alkaline media. Co3O4 was found to be a promising metal oxide catalyst for oxygen evolution in alkaline media but it has a low electronic conductivity. On the other hand since nickel has a good electronic conductivity Co3O4 can be added to pure nickel electrodes to enhance performance due to a synergetic effect. Due to the poor stability of carbon materials at high anodic potentials, gas diffusion electrodes were prepared without carbon to improve especially long-term stability. Gas diffusion electrodes were electrochemically investigated in a half cell. In addition, cyclic voltammogrametry (CV) and electrochemical impedance spectroscopy (EIS) were carried out. SEM was used for the physical and morphological investigations. Investigations showed that electrodes containing 20 wt.% Co3O4 exhibited the highest performance

  5. Scalable Fabrication of Nanoporous Carbon Fiber Films as Bifunctional Catalytic Electrodes for Flexible Zn-Air Batteries.

    Science.gov (United States)

    Liu, Qin; Wang, Yaobing; Dai, Liming; Yao, Jiannian

    2016-04-01

    A flexible nanoporous carbon-fiber film for wearable electronics is prepared by a facile and scalable method through pyrolysis of electrospun polyimide. It exhibits excellent bifunctional electrocatalytic activities for oxygen reduction and oxygen evolution. Flexible rechargeable zinc-air batteries based on the carbon-fiber film show high round-trip efficiency and mechanical stability. PMID:26914270

  6. ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

    Directory of Open Access Journals (Sweden)

    Voytas Daniel F

    2011-01-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms. Description ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s. Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence. Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the

  7. A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

    Science.gov (United States)

    Meineke, Birthe; Kast, Alene; Schwer, Beate; Meinhardt, Friedhelm; Shuman, Stewart; Klassen, Roland

    2012-09-01

    PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases. PMID:22836353

  8. Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.

    Science.gov (United States)

    McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J; Lew, Ala E

    2006-03-01

    A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport. PMID:16517880

  9. Comparison of Culture and a Novel 5′ Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

    Science.gov (United States)

    McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J.; Lew, Ala E.

    2006-01-01

    A Campylobacter fetus subsp. venerealis-specific 5′ Taq nuclease PCR assay using a 3′ minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5′ Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5′ Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5′ Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5′ Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5′ Taq nuclease assay demonstrates a statistically significant association with culture (χ2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport. PMID:16517880

  10. Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)*

    OpenAIRE

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-01-01

    Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevent...

  11. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

    DEFF Research Database (Denmark)

    Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael;

    2014-01-01

    Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant...

  12. Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair

    OpenAIRE

    Hodskinson, Michael R.G.; Silhan, Jan; Crossan, Gerry P.; Garaycoechea, Juan I.; Mukherjee, Shivam; Johnson, Christopher M.; Schärer, Orlando D.; Patel, Ketan J.

    2014-01-01

    Summary SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal ...

  13. Locked nucleic acid couples with Fok I nucleases to target and cleave hepatitis B virus's gene in vitro.

    Science.gov (United States)

    Li, Ma; Hongyan, Chen; Huaxing, Zhu; Wei, Li; Daru, Lu

    2016-04-01

    Hepatitis B virus (HBV) is a dented double-stranded DNA virus. After infecting human hepatic cells, it forms cccDNA that replicates persistently and integrates randomly into the host’s genome during the process of reserve transcription. On average, in each cell with chronic HBV infection, there are about 33 copies of cccDNA with a half of 35-57 days, which can be difficult to eradicate. A new strategy is to inhibit HBV transcription by using locked nucleic acid (LNA). Besides, cleaving HBV genome by targeted genome editing technologies could potentially cure patients. In this study, we explored new genome editing tools for HBV treatment. Based on LNA’s ability to form triple helix by binding to duplex DNA, its stability towards nuclease and polymerase, and its sensitivity to single base mismatches, we designed LNA-modified oligonucleotides as DNA binding domain to effectively increase the specificity of target gene recognition. Meanwhile, by utilizing the small molecular weight and dimerization dependent activity of nuclease Fok I, we used Fok I recombinant dimer protein as DNA cleavage domain. Here, we established a method by chemical coupling of LNA-oligonucleotide with Fok I cleavage domain, and also validated the targeted cleavage of HBV genes with our new tools in vitro. These results provide new possibilities for future in vivo anti-virus gene therapy with high specificity and no integration risk. PMID:27103458

  14. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  15. Expression of the mouse metallothionein-I gene alters the nuclease hypersensitivity of its 5' regulatory region

    International Nuclear Information System (INIS)

    We have been using the mouse metallothionein-I (MT-I) gene as a model system for studies of gene regulation and hormone action at the molecular level. Expression of the MT-I gene is induced at the transcriptional level both by heavy metals and by glucocorticoid hormones in animals and cultured cells. Transfection experiments reveal that the sequences required for cadmium-regulated expression lie within a 221-bp region (-148 to +63) overlapping the 5' end (+1) of the gene; similarly, only sequences between -95 and +63 are required in mouse oocyte injection experiments. We find that in a variety of tissues and cell lines that can express the MT-I gene, there is a region of DNA 250 to 300 bp long near the 5' end of the MT-I gene that is hypersensitive to both DNase I and staphylococcal nuclease. After induction with cadmium, we observe significantly increased nuclease hypersensitivity within a region between -30 and +60 in a variety of cell types. Thus, all of the sequences primarily responsible for transcriptional regulation of this gene may lie within the hypersensitive region. We suggest that various features of this chromatin structure, established during gene commitment, may be important for the efficient interaction of RNA polymerase and various regulatory proteins with specific DNA sequences

  16. Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases.

    Science.gov (United States)

    Karginov, Fedor V; Cheloufi, Sihem; Chong, Mark M W; Stark, Alexander; Smith, Andrew D; Hannon, Gregory J

    2010-06-25

    The life span of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5' phosphate groups in mouse embryonic stem cells (mESCs). We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mESCs, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals. PMID:20620951

  17. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Arildo Nerys-Junior

    2014-01-01

    Full Text Available Engineered nucleases such as zinc finger nucleases (ZFN and transcription activator-like effector nucleases (TALEN are one of the most promising tools for modifying genomes. These site-specific enzymes cause double- strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  18. A nanostructured bifunctional Pd/C gas-diffusion electrode for metal-air batteries

    International Nuclear Information System (INIS)

    Graphical Abstract: Images showing (a) a scanning electrode microscope cross section of the Pd/C air electrode, (b) a TEM micrograph of the Pd/C catalyst, and (c) charge–discharge cycling of the air electrode in 6 mol dm−3 KOH at 20 mA cm−2 under 100 ml min−1 oxygen flow. - Highlights: • The Pd/C air electrode shows consistent good performance at modest current densities (20–80 mA cm−2), and is stable for 1000 cycles at room temperature. • The Pd/C air electrode has a potential difference of 0.53 V between oxygen evolution and oxygen reduction. • The air electrode makes efficient use of a small precious metal loading (0.5 mg cm−2), and is mainly made from lightweight carbon materials. • The air electrode has better stability than a commercial 2 mg cm−2 Pt/C electrode on repeated charge–discharge cycling, despite having a lower (and therefore less expensive) loading of catalyst. - Abstract: Designing a bifunctional air electrode which catalyses both the oxygen reduction and oxygen evolution reactions is an essential part of progress towards fully rechargeable metal-air batteries, such as the iron-air battery which is environmentally friendly, low cost, and does not suffer risk of thermal runaway like lithium-ion batteries. This paper reports the development of a lightweight carbon-based bifunctional air electrode, catalysed by a small (0.5 mg cm−2) loading of 30 wt.% palladium on carbon. The Pd-catalysed air electrode showed good bifunctional activity, with 0.53 V potential difference between oxygen reduction and evolution. The Pd/C air electrode showed improved catalytic activity at high current densities (≥ 50 mA cm−2) and enhanced durability compared with two commercial Pt/C air electrodes produced by Gaskatel GmbH and Johnson Matthey. A stable oxygen evolution potential was maintained over 1,000 charge-discharge cycles

  19. Oxygen electrode bifunctional electrocatalyst NiCo2O4 spinel

    Science.gov (United States)

    Fielder, William L.; Singer, Joseph

    1988-01-01

    A significant increase in energy density may be possible if a two-unit alkaline regenerative H2-O2 fuel cell is replaced with a single-unit system that uses passive means for H2O transfer and thermal control. For this single-unit system, new electrocatalysts for the O2 electrode will be required which are not only bifunctionally active but also chemically and electrochemically stable between the voltage range of about 0.7 and 1.5 V. NiCo2O4 spinel is reported to have certain characteristics that make it useful for a study of electrode fabrication techniques. High surface area NiCo2O4 powder was fabricated into unsupported, bifunctional, PTFE-bonded, porous gas fuel cell electrodes by commercial sources using varying PTFE contents and sintering temperatures. The object of this study is to measure the bifunctional activities of these electrodes and to observe what performance differences might result from different commercial electrode fabricators. O2 evolution and O2 reduction data were obtained at 80 C (31 percent KOH). An irreversible reaction (i.e., aging) occurred during O2 evolution at potentials greater than about 1.5 V. Anodic Tafel slopes of 0.06 and 0.12 V/decade were obtained for the aged electrodes. Within the range of 15 to 25 percent, the PTFE content was not a critical parameter for optimizing the electrode for O2 evolution activity. Sintering temperatures between 300 and 340 C may be adequate but heating at 275 C may not be sufficient to properly sinter the PTFE-NiCo2O4 mixture. Electrode disintegration was observed during O2 reduction. Transport of O2 to the NiCo2O4 surface became prohibitive at greater than about -0.02 A/sq cm. Cathodic Tafel slopes of -0.6 and -0.12 V/decade were assumed for the O2 reduction process. A PTFE content of 25 percent (or greater) appears to be preferable for sintering the PTFE-NiCo2O4 mixture.

  20. A Cascade-Reaction Nanoreactor Composed of a Bifunctional Molecularly Imprinted Polymer that Contains Pt Nanoparticles.

    Science.gov (United States)

    Wang, Jiao; Zhu, Maiyong; Shen, Xiaojuan; Li, Songjun

    2015-05-11

    This study was aimed at addressing the present challenge of cascade reactions, namely, how to furnish the catalysts with desired and hierarchical catalytic ability. This issue was addressed by constructing a cascade-reaction nanoreactor made of a bifunctional molecularly imprinted polymer containing acidic catalytic sites and Pt nanoparticles. The acidic catalytic sites within the imprinted polymer allowed one specified reaction, whereas the encapsulated Pt nanoparticles were responsible for another coupled reaction. To that end, the unique imprinted polymer was fabricated by using two well-coupled templates, that is, 4-nitrophenyl acetate and 4-nitrophenol. The catalytic hydrolysis of the former compound at the acidic catalytic sites led to the formation of the latter compound, which was further reduced by the encapsulated Pt nanoparticles to 4-aminophenol. Therefore, this nanoreactor demonstrated a catalytic-cascade ability. This protocol opens up the opportunity to develop functional catalysts for complicated chemical processes. PMID:25846700

  1. Bifunctional Nanoparticle-SILP Catalysts (NPs@SILP) for the Selective Deoxygenation of Biomass Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Luska, Kylie L. [RWTH Aachen Univ. (Germany); Julis, Jennifer [RWTH Aachen Univ. (Germany); Evonik Industries AG, Marl (Germany); Stavitski, Eli [Brookhaven National Lab. (BNL), Upton, NY (United States); Zakharov, Dmitri N. [Brookhaven National Lab. (BNL), Upton, NY (United States); Adams, Alina [RWTH Aachen Univ. (Germany); Leitner, Walter [RWTH Aachen Univ. (Germany); Max Planck Inst. for Coal Research, Ruhr (Germany)

    2014-08-27

    We immobilized ruthenium nanoparticles onto an acidic supported ionic liquid phase (RuNPs@SILP) in the development of bifunctional catalysts for the selective deoxygenation of biomass substrates. RuNPs@SILPs possessed high catalytic activities, selectivities and recyclabilities in the hydrogenolytic deoxygenation and ring opening of C8- and C9-substrates derived from furfural or 5-hydroxymethylfurfural and acetone. When we tailor the acidity of the SILP through the ionic liquid loading provided a molecular parameter by which the catalytic activity and selectivity of the RuNPs@SILPs were controlled to provide a flexible catalyst system toward the formation of different classes of value-added products: cyclic ethers, primary alcohols or aliphatic ethers.

  2. Bifunctional hairy silica nanoparticles as high-performance additives for lubricant

    Science.gov (United States)

    Sui, Tianyi; Song, Baoyu; Wen, Yu-Ho; Zhang, Feng

    2016-03-01

    Bifunctional hairy silica nanoparticles (BHSNs), which are silica nanoparticles covered with alkyl and amino organic chains, were prepared as high-performance additives for lubricants. Compared with hairy silica nanoparticles covered by a single type of organic chain, binary hairy silica nanoparticles exhibit the advantages of both types of organic chains, which exhibit excellent compatibility with lubricants and adsorbability to metal surfaces. Nanoparticles with different ratios of amino and alkyl ligands were investigated. In comparison to an untreated lubricant, BHSNs reduce the friction coefficient and wear scar diameter by 40% and 60%, respectively. The wear mechanism of BHSNs was investigated, and the protective and filling effect of the nanoparticles improved because of collaboration of amino and alkyl ligands.

  3. Dyes as bifunctional markers of DNA hybridization on surfaces and mutation detection.

    Science.gov (United States)

    García-Mendiola, Tania; Cerro, María Ramos; López-Moreno, José María; Pariente, Félix; Lorenzo, Encarnación

    2016-10-01

    The interaction of small molecules with DNA has found diagnostic and therapeutic applications. In this work, we propose the use of two different dyes, in particular Azure A and Safranine, as bifunctional markers of on-surface DNA hybridization and potent tools for screening of specific gene mutations directly in real DNA PCR amplicons extracted from blood cells. By combining spectroscopic and electrochemical methods we demonstrate that both dyes can interact with single and double stranded DNA to a different extent, allowing reliable hybridization detection. From these data, we have also elucidated the nature of the interaction. We conclude that the binding mode is fundamentally intercalative with an electrostatic component. The dye fluorescence allows their use as nucleic acid stains for the detection of on-surfaces DNA hybridization. Its redox activity is exploited in the development of selective electrochemical DNA biosensors. PMID:27317997

  4. Synthesis and application of glycolic esters in methanol-gasoline as bifunctional additives

    International Nuclear Information System (INIS)

    To explore new and multifunctional additives for methanol-gasoline, glycolic esters were synthesized and screened as phase stabilizer and saturation vapor pressure depressor. The effect of the esters structure on the efficiency was discussed. It was found that the stability of the blends depend on the length of the glycolic esters' alkoxy group, and hexyl glycolic and octyl glycolic were found to be the most effective in various gasoline-methanol blends. Additionally, the glycolic esters can depress the saturation vapor pressure of methanol-gasoline effectively as well, and decyl glycolic is the most effective one. With these data, it can be concluded that the glycolic esters have the great potential to be used as bifunctional gasoline-methanol additives. (author)

  5. Novel 3-nitrotriazole-based amides and carbinols as bifunctional antichagasic agents.

    Science.gov (United States)

    Papadopoulou, Maria V; Bloomer, William D; Lepesheva, Galina I; Rosenzweig, Howard S; Kaiser, Marcel; Aguilera-Venegas, Benjamín; Wilkinson, Shane R; Chatelain, Eric; Ioset, Jean-Robert

    2015-02-12

    3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogues were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed. PMID:25580906

  6. A Bifunctional Electrocatalyst for Oxygen Evolution and Oxygen Reduction Reactions in Water

    Science.gov (United States)

    Faschinger, Felix; Chattopadhyay, Samir; Bhakta, Snehadri; Mondal, Biswajit; Elemans, Johannes A. A. W.; Müllegger, Stefan; Tebi, Stefano; Koch, Reinhold; Klappenberger, Florian; Paszkiewicz, Mateusz; Barth, Johannes V.; Rauls, Eva; Aldahhak, Hazem; Schmidt, Wolf Gero

    2016-01-01

    Abstract Oxygen reduction and water oxidation are two key processes in fuel cell applications. The oxidation of water to dioxygen is a 4 H+/4 e− process, while oxygen can be fully reduced to water by a 4 e−/4 H+ process or partially reduced by fewer electrons to reactive oxygen species such as H2O2 and O2 −. We demonstrate that a novel manganese corrole complex behaves as a bifunctional catalyst for both the electrocatalytic generation of dioxygen as well as the reduction of dioxygen in aqueous media. Furthermore, our combined kinetic, spectroscopic, and electrochemical study of manganese corroles adsorbed on different electrode materials (down to a submolecular level) reveals mechanistic details of the oxygen evolution and reduction processes.

  7. Cage-like bifunctional chelators, copper-64 radiopharmaceuticals and PET imaging using the same

    Energy Technology Data Exchange (ETDEWEB)

    Conti, Peter S.; Cai, Hancheng; Li, Zibo; Liu, Shuanglong

    2016-08-02

    Disclosed is a class of versatile Sarcophagine based bifunctional chelators (BFCs) containing a hexa-aza cage for labeling with metals having either imaging, therapeutic or contrast applications radiolabeling and one or more linkers (A) and (B). The compounds have the general formula ##STR00001## where A is a functional group selected from group consisting of an amine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and an alkene, and B is a functional group selected from the group consisting of hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, a thiol, an azide and an alkene. Also disclosed are conjugate of the BFC and a targeting moiety, which may be a peptide or antibody. Also disclosed are metal complexes of the BFC/targeting moiety conjugates that are useful as radiopharmaceuticals, imaging agents or contrast agents.

  8. Mono- and bi-functional arenethiols as surfactants for gold nanoparticles: synthesis and characterization

    Directory of Open Access Journals (Sweden)

    Fratoddi Ilaria

    2011-01-01

    Full Text Available Abstract Stable gold nanoparticles stabilized by different mono and bi-functional arenethiols, namely, benzylthiol and 1,4-benzenedimethanethiol, have been prepared by using a modified Brust's two-phase synthesis. The size, shape, and crystalline structure of the gold nanoparticles have been determined by high-resolution electron microscopy and full-pattern X-ray powder diffraction analyses. Nanocrystals diameters have been tuned in the range 2 ÷ 9 nm by a proper variation of Au/S molar ratio. The chemical composition of gold nanoparticles and their interaction with thiols have been investigated by X-ray photoelectron spectroscopy. In particular, the formation of networks has been observed with interconnected gold nanoparticles containing 1,4-benzenedimethanethiol as ligand.

  9. Bi-functional biobased packing of the cassava starch, glycerol, licuri nanocellulose and red propolis.

    Directory of Open Access Journals (Sweden)

    Samantha Serra Costa

    Full Text Available The aim of this study was to characterize and determine the bi-functional efficacy of active packaging films produced with starch (4% and glycerol (1.0%, reinforced with cellulose nanocrystals (0-1% and activated with alcoholic extracts of red propolis (0.4 to 1.0%. The cellulose nanocrystals used in this study were extracted from licuri leaves. The films were characterized using moisture, water-activity analyses and water vapor-permeability tests and were tested regarding their total phenolic compounds and mechanical properties. The antimicrobial and antioxidant efficacy of the films were evaluated by monitoring the use of the active films for packaging cheese curds and butter, respectively. The cellulose nanocrystals increased the mechanical strength of the films and reduced the water permeability and water activity. The active film had an antimicrobial effect on coagulase-positive staphylococci in cheese curds and reduced the oxidation of butter during storage.

  10. Bi-Functional Biobased Packing of the Cassava Starch, Glycerol, Licuri Nanocellulose and Red Propolis

    Science.gov (United States)

    Costa, Samantha Serra; Druzian, Janice Izabel; Machado, Bruna Aparecida Souza; de Souza, Carolina Oliveira; Guimarães, Alaíse Gil

    2014-01-01

    The aim of this study was to characterize and determine the bi-functional efficacy of active packaging films produced with starch (4%) and glycerol (1.0%), reinforced with cellulose nanocrystals (0–1%) and activated with alcoholic extracts of red propolis (0.4 to 1.0%). The cellulose nanocrystals used in this study were extracted from licuri leaves. The films were characterized using moisture, water-activity analyses and water vapor-permeability tests and were tested regarding their total phenolic compounds and mechanical properties. The antimicrobial and antioxidant efficacy of the films were evaluated by monitoring the use of the active films for packaging cheese curds and butter, respectively. The cellulose nanocrystals increased the mechanical strength of the films and reduced the water permeability and water activity. The active film had an antimicrobial effect on coagulase-positive staphylococci in cheese curds and reduced the oxidation of butter during storage. PMID:25383783

  11. Experimental verifications on chemical carcinogenesis, a bifunctional alkylation between DNA interstrands

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is evidenced by the filter elution method that two carcinogenic aromatic hydrocarbons, benzo[a]pyrene and dibenzo[a,h]anthracene, two carcinogenic metal salts, beryllium chloride and cadmium chloride, four carcinogenic aromatic amines, 2-aminofluorene, β-naphthylamine, 4-aminobiphenyl and benzidine, can all induce DNA interstrand and DNA-protein cross-link in L1210 culture. However, under the same condition, the corresponding non-carcinogenic compounds, including benzo[k]fluorancene, anthracene, magnesium chloride, zinc chloride, a -naphthylamine, 2-aminobiphenyl and m-toluidine, cannot produce any cross-link adducts. All these results are consistent with the di-region theory that carcinogens are bio-bifunctional alkylation agents. This method can also be used to discriminate carcinogens and non-carcinogens.

  12. On the molecular basis of D-bifunctional protein deficiency type III.

    Directory of Open Access Journals (Sweden)

    Maija L Mehtälä

    Full Text Available Molecular basis of D-bifunctional protein (D-BP deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2 protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m, V(max and k(cat of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.

  13. RNA Secondary Structure Modulates FMRP's Bi-Functional Role in the MicroRNA Pathway.

    Science.gov (United States)

    Kenny, Phillip; Ceman, Stephanie

    2016-01-01

    MicroRNAs act by post-transcriptionally regulating the gene expression of 30%-60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP) and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP's interaction with the RNA helicase Moloney leukemia virus 10 (MOV10), which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex. PMID:27338369

  14. A sustainable method inspired by nature for the production of original bi-functional nutraceuticals containing prebiotic fibers and highly bio-available forms of calcium and magnesium

    OpenAIRE

    Goffin, Dorothée; Blecker, Christophe; Paquot, Michel

    2012-01-01

    A sustainable method inspired by nature is proposed leading to an original bifunctional product active on gut health, metabolism regulation, immunity and mineral fortification, fighting against topical chronic diseases (type2 diabetes, osteoporosis, colon cancer…). Peer reviewed

  15. Chemical modification of multiwalled carbon nanotube with a bifunctional caged ligand for radioactive labelling

    International Nuclear Information System (INIS)

    Graphical abstract: The findings of this work describe a new method that can be utilised to radioactively label multiwalled carbon nanotube (MWCNT) powders with the use of a bi-functional cage ligand. -- Abstract: Carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs) have been successfully radiolabelled with cobalt-57 (57Co) (T1/2 = 270 days) via the attachment of the bifunctional caged ligand MeAMN3S3sar. In this study MeAMN3S3sar has been synthesized and coupled to MWCNTs to form the conjugate MWCNT–MeAMN3S3sar. Synthesis was confirmed with nuclear magnetic resonance. X-ray photoelectron spectroscopy (XPS) confirmed the conjugation. Non-radioactive labelling of this conjugate was completed with Cu(II) ions to confirm the stability of the MeAMN3S3sar after coupling with the MWCNTs. The complexation of the Cu(II) was also confirmed with XPS. Transmission electron microscopy was used to demonstrate that the coupling reaction had a negligible effect on the size and shape of the MWCNTs. Radiolabelling of the MWCNT–MeAMN3S3sar conjugate and pristine (untreated) MWCNTs (non-specific) with the gamma-emitting radioactive isotope 57Co were compared. The radiolabelling efficiency of the MWCNT–MeAMN3S3sar conjugate was significantly higher (95% vs. 0.1%) (P ⩽ 0.001) than for the unconjugated pristine MWCNTs. This will allow for the potential tracking of nanoparticle movement in vitro and in vivo

  16. Cobalt Selenide Nanostructures: An Efficient Bifunctional Catalyst with High Current Density at Low Coverage.

    Science.gov (United States)

    Masud, Jahangir; Swesi, Abdurazag T; Liyanage, Wipula P R; Nath, Manashi

    2016-07-13

    Electrodeposited Co7Se8 nanostructures exhibiting flake-like morphology show bifunctional catalytic activity for oxygen evolution and hydrogen evolution reaction (OER and HER, respectively) in alkaline medium with long-term durability (>12 h) and high Faradaic efficiency (99.62%). In addition to low Tafel slope (32.6 mV per decade), the Co7Se8 OER electrocatalyst also exhibited very low overpotential to achieve 10 mA cm(-2) (0.26 V) which is lower than other transition metal chalcogenide based OER electrocatalysts reported in the literature and significantly lower than the state-of-the-art precious metal oxides. A low Tafel slope (59.1 mV per decade) was also obtained for the HER catalytic activity in alkaline electrolyte. The OER catalytic activity could be further improved by creating arrays of 3-dimensional rod-like and tubular structures of Co7Se8 through confined electrodeposition on lithographically patterned nanoelectrodes. Such arrays of patterned nanostructures produced exceptionally high mass activity and gravimetric current density (∼68 000 A g(-1)) compared to the planar thin films (∼220 A g(-1)). Such high mass activity of the catalysts underlines reduction in usage of the active material without compromising efficiency and their practical applicability. The catalyst layer could be electrodeposited on different substrates, and an effect of the substrate surface on the catalytic activity was also investigated. The Co7Se8 bifunctional catalyst enabled water electrolysis in alkaline solution at a cell voltage of 1.6 V. The electrodeposition works with exceptional reproducibility on any conducting substrate and shows unprecedented catalytic performance especially with the patterned growth of catalyst rods and tubes. PMID:27309595

  17. Gently reduced graphene oxide incorporated into cobalt oxalate rods as bifunctional oxygen electrocatalyst

    International Nuclear Information System (INIS)

    Graphical abstract: - Abstract: Water-oxygen electrochemistry is at the heart of key renewable energy technologies (fuel cells, electrolyzers, and metal-air batteries) due to the sluggish kinetics of oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). Although much effort has been devoted to the development of improved bifunctional electrocatalysts, an inexpensive, highly active oxygen electrocatalyst, however, remains to be a challenge. In this paper, we present a facile and robust method to create gently reduced graphene oxide incorporated into cobalt oxalate microstructures (CoC2O4/gRGO) and demonstrate its excellent and stable electrocatalytic activity in both OER and ORR, arising from the inherent properties of the components and their physicochemical interaction. Our synthesis technique also explores a single pot method to partially reduce graphene oxide and form CoC2O4 structures while maintaining the solution processability of reduced graphene oxide. While the OER activity of CoC2O4/gRGO is exclusively due to CoC2O4, which transformed into OER-active Co species, the combination with gRGO significantly improves OER stability. On the other hand, CoC2O4/gRGO exhibits synergistic effect towards ORR, via a quasi-four-electron pathway, leading to a slightly higher ORR limiting current than Pt/C. Remarkably, gRGO offers dual functionality, contributing to ORR activity via the N-functional groups and also enhancing OER stability through the gRGO coating around CoC2O4 structures. Our results suggest a new class of metal-carbon composite that has the potential to be alternative bifunctional catalysts for regenerative fuel cells and metal-air batteries

  18. Evaluation of copper-labeled bifunctional chelate-albumin conjugates for blood pool imaging

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.J.; Rocque, P.A.; Welch, M.J. (Washington Univ., St. Louis, MO (United States). Edward Mallinckrodt Inst. of Radiology); Weinheimer, C.J. (Washington Univ., St. Louis, MO (United States). School of Medicine)

    1993-05-01

    [sup 62]Cu is a generator-produced positron-emitting radionuclide with a half-life amenable to blood-pool imaging with PET. Three bifunctional chelates [cyclic anhydride of diethylenetriamine-pentaacetic acid (cDTPAA), 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N'', N''' tetraacetic acid (BAT), and p-carboxyethylphenylglyoxal-bis-([sup 4])N-methyl-thiosemicarbazone (CE-DTS)] were conjugated to HSA and labeled with [sup 67]Cu. Blood clearance and biodistribution of these three [sup 67]Cu-labeled conjugates were determined in rats. Of the three [sup 67]Cu-labeled bifunctional chelate-HSA conjugates, [sup 67]Cu-benzyl-TETA-HSA remained in the blood pool the longest, achieving stable blood levels at times longer than 24 h post-injection. The [sup 67]Cu radioactivity cleared the blood within 60 min post-injection of [sup 67]Cu-DTS-HSA, and within 10 min after administration of [sup 67]Cu-DTPA-HSA, indicating the dissociation of Cu[sup 2+] from these conjugates. Copper-labeled DTS-HSA achieved stable blood concentrations for at least 30 min post-injection and was therefore evaluated as a vascular imaging agent. DTS-HSA and benzy-TETA-HSA were labeled with [sup 62]Cu and administered to a dog for blood-pool imaging using PET. Because of the high labeling efficiency, DTS-HSA can be labeled with [sup 62]Cu without purification, making it more practical than [sup 62]Cu-benzyl-TETA-HSA as a blood-pool imaging agent. Generator-produced [sup 62]Cu-DTS-HSA should be a viable alternative blood pool agent to cyclotron-produced C[sup 15]O for PET facilities without cyclotrons. (author).

  19. Dimerization and Bifunctionality Confer Robustness to the Isocitrate Dehydrogenase Regulatory System in Escherichia coli*

    Science.gov (United States)

    Dexter, Joseph P.; Gunawardena, Jeremy

    2013-01-01

    An important goal of systems biology is to develop quantitative models that explain how specific molecular features give rise to systems-level properties. Metabolic and regulatory pathways that contain multifunctional proteins are especially interesting to study from this perspective because they have frequently been observed to exhibit robustness: the ability for a system to perform its proper function even as levels of its components change. In this study, we use extensive biochemical data and algebraic modeling to develop and analyze a model that shows how robust behavior arises in the isocitrate dehydrogenase (IDH) regulatory system of Escherichia coli, which was shown in 1985 to experimentally exhibit robustness. E. coli IDH is regulated by reversible phosphorylation catalyzed by the bifunctional isocitrate dehydrogenase kinase/phosphatase (IDHKP), and the level of IDH activity determines whether carbon flux is directed through the glyoxylate bypass (for growth on two-carbon substrates) or the full tricarboxylic acid cycle. Our model, which incorporates recent structural data on IDHKP, identifies several specific biochemical features of the system (including homodimerization of IDH and bifunctionality of IDHKP) that provide a potential explanation for robustness. Using algebraic techniques, we derive an invariant that summarizes the steady-state relationship between the phospho-forms of IDH. We use the invariant in combination with kinetic data on IDHKP to calculate IDH activity at a range of total IDH levels and find that our model predicts robustness. Our work unifies much of the known biochemistry of the IDH regulatory system into a single quantitative framework and highlights the importance of constructing biochemically realistic models in systems biology. PMID:23192354

  20. Bifunctional Effect of Human IFN-γon Cultured Human Fibroblasts from Tenon's Capsule

    Institute of Scientific and Technical Information of China (English)

    Yan Guo; Jian Ge; Haiquan Liu; Yanyan Li; Jianliang Zheng; Xiangkun Huang; Yuqing Lan

    2000-01-01

    Purpose: To study the effect of human IFN-γ on in vitro cultured human fibroblasts from Tenon's capsuleMaterials and methods: The effect of different concentrations of human IFN-γ and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MIT [3-(4, 5-dimethylthiazo-2-yl)] -2,5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0version. The difference was considered to be significant if P < 0. 05.Results: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-γ on the growth of HTCF were both negative (102 ~ l04 units/ml in two experiments) and positive (106, 105, 10 units/ml in two experiments) . The inhibition rate of MMC ranged from 5.73% to 46. 9%, which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92% ( P= 0. 351) . The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu ( P < 0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition) on proliferation of cultured HTCF. The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu. Further study has to be carried out to document the inhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation. Eye Science 2000; 16: 43~ 47.

  1. Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents

    International Nuclear Information System (INIS)

    DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity. Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Although the initial number of cross-links was similar for the three cell lines, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes

  2. Bifunctional Effect of Human IFN-γ on Cultured Human Fibroblasts from Tenon‘s Capsule

    Institute of Scientific and Technical Information of China (English)

    YanGuo; JianGe; 等

    2002-01-01

    Purpose:To study the effect of human IFN-γ on in ivtro cultured human fibroblasts from Tenon's capsule.Materials and methods:The effect of different concentrations of human IFN-γand mitomycin-C (MMC),5-fluorouracil(5-Fu) on cultured human Tenon's capsule fibroblasts(HTCF) was measured using a MTT[3-(4,5-dimethylthiazo-2-yI)]-2,5-diphenylterazolium bromide;Thiazolyl blue) colorimetric assay.The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0 version.The difference was considered to be significant if P<0.05.Results:The effects of MMC and 5-Fu on the growth of HTCF were negative,while the effects of IFN-γon the growth of HTCF were both negative(102-104 units/ml in two experiments)and positive(106,105,10 units /ml in two experiments).The inhibition rate of MMC ranged from 5.73% to 46.9% ,which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92%(P=0.351).The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu (P<0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition)on proliferation of cultured HTCF.The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu.Further study has to be carried out to document theinhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation.Eye Science 2000;16:43-47.

  3. A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

    DEFF Research Database (Denmark)

    Czene, Aniko; Tóth, Eszter; Németh, Eszter;

    2014-01-01

    The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant ΔN4-NColE7-C* - where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag - is catalytically inactive...

  4. Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.

    Science.gov (United States)

    Hodskinson, Michael R G; Silhan, Jan; Crossan, Gerry P; Garaycoechea, Juan I; Mukherjee, Shivam; Johnson, Christopher M; Schärer, Orlando D; Patel, Ketan J

    2014-05-01

    SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repair. PMID:24726326

  5. DIFFERENCES IN DETECTION OF DNA ADDUCTS IN THE 32P-POSTLABELING ASSAY AFTER EITHER 1-BUTANOL EXTRACTION OR NUCLEASE P1 TREATMENT

    Science.gov (United States)

    The use of nuclease Pl treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabe1ling assay for DNA adducts have been compared. lthough similar results were obtained with the two methods for standard adducts formed with benzo(a)pyrene diol epoxide I, nucl...

  6. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    Science.gov (United States)

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

  7. Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism.

    Science.gov (United States)

    Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y; Varnado, Brittany; Beutler, John A; Murelli, Ryan P; Le Grice, Stuart F J; Tang, Liang

    2015-12-15

    Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg(2+). A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg(2+) from Ca(2+). Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

  8. Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

    Science.gov (United States)

    Zhu, Jing; Wang, Lei; Xu, Xiaowen; Wei, Haiping; Jiang, Wei

    2016-04-01

    Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery. PMID:26943244

  9. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    Science.gov (United States)

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system. PMID:26950513

  10. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

    Energy Technology Data Exchange (ETDEWEB)

    Beloglazova, Natalia; Petit, Pierre; Flick, Robert; Brown, Greg; Savchenko, Alexei; Yakunin, Alexander F. (Toronto)

    2012-03-15

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

  11. Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

    Directory of Open Access Journals (Sweden)

    Garvin Alex M

    2012-12-01

    Full Text Available Abstract Background Profiling sperm DNA present on vaginal swabs taken from rape victims often contributes to identifying and incarcerating rapists. Large amounts of the victim’s epithelial cells contaminate the sperm present on swabs, however, and complicate this process. The standard method for obtaining relatively pure sperm DNA from a vaginal swab is to digest the epithelial cells with Proteinase K in order to solubilize the victim’s DNA, and to then physically separate the soluble DNA from the intact sperm by pelleting the sperm, removing the victim’s fraction, and repeatedly washing the sperm pellet. An alternative approach that does not require washing steps is to digest with Proteinase K, pellet the sperm, remove the victim’s fraction, and then digest the residual victim’s DNA with a nuclease. Methods The nuclease approach has been commercialized in a product, the Erase Sperm Isolation Kit (PTC Labs, Columbia, MO, USA, and five crime laboratories have tested it on semen-spiked female buccal swabs in a direct comparison with their standard methods. Comparisons have also been performed on timed post-coital vaginal swabs and evidence collected from sexual assault cases. Results For the semen-spiked buccal swabs, Erase outperformed the standard methods in all five laboratories and in most cases was able to provide a clean male profile from buccal swabs spiked with only 1,500 sperm. The vaginal swabs taken after consensual sex and the evidence collected from rape victims showed a similar pattern of Erase providing superior profiles. Conclusions In all samples tested, STR profiles of the male DNA fractions obtained with Erase were as good as or better than those obtained using the standard methods.

  12. Cloning, Purification and Initial Characterization of E. coli McrA, a Putative 5-methylcytosine-specific Nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Mulligan,E.; Dunn, J.

    2008-01-01

    Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m5C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 C. rMcrA protein, which is predicted to contain a Cys4-Zn2+ finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high a-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm5CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in 'restricting' m5C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m5C residues.

  13. Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease.

    Science.gov (United States)

    Mulligan, Elizabeth A; Dunn, John J

    2008-11-01

    Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues. PMID:18662788

  14. Functionalization of nanoparticle titanium dioxide with different bifunctional organic molecules and trimers of transition compounds for obtaining new materials

    International Nuclear Information System (INIS)

    Functionalization of titanium dioxide in nanoporous anatase phase is investigated for obtaining new nanomaterials. Functionalizations were performed using two heating methods: the conventional of refluxing heating method and microwave irradiation with bifunctional organic molecules is used to study how to anchor molecules and the change in the wettability of the material. Besides, reactions with organic molecules were performed as the derived from nanoproxene. The growth layer by layer is performed using the bifunctional molecules previous for the immobilization of cobalt trimers. Functionalized molecules were characterized by infrared spectroscopy, X-ray diffraction, contact angle, scanning electron microscopy, x-ray elemental analysis, plasma atomic emission spectroscopy coupled inductively, x-ray photoelectron spectroscopy and thermogravimetric analysis. This type of functionalizations on nanoporous titanium dioxide could potentially improve optical sensitivity and activity of this nanomaterial in the visible region. (author)

  15. Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa

    International Nuclear Information System (INIS)

    The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported. Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P21212, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%

  16. Dual-Doped Molybdenum Trioxide Nanowires: A Bifunctional Anode for Fiber-Shaped Asymmetric Supercapacitors and Microbial Fuel Cells.

    Science.gov (United States)

    Yu, Minghao; Cheng, Xinyu; Zeng, Yinxiang; Wang, Zilong; Tong, Yexiang; Lu, Xihong; Yang, Shihe

    2016-06-01

    A novel in situ N and low-valence-state Mo dual doping strategy was employed to significantly improve the conductivity, active-site accessibility, and electrochemical stability of MoO3 , drastically boosting its electrochemical properties. Consequently, our optimized N-MoO3-x nanowires exhibited exceptional performances as a bifunctional anode material for both fiber-shaped asymmetric supercapacitors (ASCs) and microbial fuel cells (MFCs). The flexible fiber-shaped ASC and MFC device based on the N-MoO3-x anode could deliver an unprecedentedly high energy density of 2.29 mWh cm(-3) and a remarkable power density of 0.76 μW cm(-1) , respectively. Such a bifunctional fiber-shaped N-MoO3-x electrode opens the way to integrate the electricity generation and storage for self-powered sources. PMID:27097987

  17. Self-organization of Au–CdSe hybrid nanoflowers at different length scales via bi-functional diamine linkers

    International Nuclear Information System (INIS)

    This work introduces a series of molecular bridging bi-functional linkers to produce laterally self-assembled nanostructures of the Au–CdSe nanoflowers on different length scales ranging from 10 nm to 100 microns. Assembly of Au nanocrystals within amorphous CdSe rods is found in the early stages of the growth of the Au–CdSe nanoflowers. The Au–CdSe nanoflowers are formed through a one-pot low temperature (150 °C) process where CdSe clusters are adsorbed on the surface of the Au cores, and they then start to form multiple arms and branches resulting in flower-shaped hybrid nanostructures. More complex assembly at a micron length scale can be achieved by means of bi-functional capping agents with appropriate alkyl chain lengths, such as 1,12-diaminododecane.

  18. Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe.

    Directory of Open Access Journals (Sweden)

    Roni F Rayes

    Full Text Available The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

  19. Synthesis and Characterization of Bifunctional Organic-Glasses Based on Diphenylhydrazone and Barbituric Acid Derivative for Photorefractive Application

    International Nuclear Information System (INIS)

    A series of amorphous molecules that possess both photoconductive and electro-optic properties was synthesized in order to investigate photorefractive properties of bifunctional organic-glasses. Diethylaminobenzaldehyde- diphenylhydrazone was covalently attached to 5-(4-diethylamino-benzylidene)-1,3-dimethylpyrimidine- 2,4,6-trione through a flexible alkyl chain (3, 4, 5, 6 and 10 carbons) containing two ether linkages. The longer linkage not only lowered the glass transition temperature (Tg) of the molecules, but also allowed faster orientation of the chromophore. To examine the photorefractive properties, a 50 μm-thick film was prepared from the mixture of a bifunctional molecule, butyl benzyl phthalate, and C60. The photoconductivity of this composite was as high as 8.01 x 10-12 S/cm at 60 V/μm, and the maximum diffraction efficiency (ηmax) of 50 μm-thick film was about 5% at 80 V/μm

  20. Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum

    International Nuclear Information System (INIS)

    The bifunctional enzyme catalase-phenol oxidase from S. thermophilum was crystallized by the hanging-drop vapour-diffusion method in space group P21 and diffraction data were collected to 2.8 Å resolution. Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit

  1. Self-organization of Au–CdSe hybrid nanoflowers at different length scales via bi-functional diamine linkers

    Energy Technology Data Exchange (ETDEWEB)

    AbouZeid, Khaled Mohamed [Virginia Commonwealth University, Department of Chemistry (United States); Mohamed, Mona Bakr [Cairo University, National Institute of Laser Enhanced Science (NILES) (Egypt); El-Shall, M. Samy, E-mail: mselshal@vcu.edu [Virginia Commonwealth University, Department of Chemistry (United States)

    2016-01-15

    This work introduces a series of molecular bridging bi-functional linkers to produce laterally self-assembled nanostructures of the Au–CdSe nanoflowers on different length scales ranging from 10 nm to 100 microns. Assembly of Au nanocrystals within amorphous CdSe rods is found in the early stages of the growth of the Au–CdSe nanoflowers. The Au–CdSe nanoflowers are formed through a one-pot low temperature (150 °C) process where CdSe clusters are adsorbed on the surface of the Au cores, and they then start to form multiple arms and branches resulting in flower-shaped hybrid nanostructures. More complex assembly at a micron length scale can be achieved by means of bi-functional capping agents with appropriate alkyl chain lengths, such as 1,12-diaminododecane.

  2. N,P-Codoped Carbon Networks as Efficient Metal-free Bifunctional Catalysts for Oxygen Reduction and Hydrogen Evolution Reactions.

    Science.gov (United States)

    Zhang, Jintao; Qu, Liangti; Shi, Gaoquan; Liu, Jiangyong; Chen, Jianfeng; Dai, Liming

    2016-02-01

    The high cost and scarcity of noble metal catalysts, such as Pt, have hindered the hydrogen production from electrochemical water splitting, the oxygen reduction in fuel cells and batteries. Herein, we developed a simple template-free approach to three-dimensional porous carbon networks codoped with nitrogen and phosphorus by pyrolysis of a supermolecular aggregate of self-assembled melamine, phytic acid, and graphene oxide (MPSA/GO). The pyrolyzed MPSA/GO acted as the first metal-free bifunctional catalyst with high activities for both oxygen reduction and hydrogen evolution. Zn-air batteries with the pyrolyzed MPSA/GO air electrode showed a high peak power density (310 W g(-1) ) and an excellent durability. Thus, the pyrolyzed MPSA/GO is a promising bifunctional catalyst for renewable energy technologies, particularly regenerative fuel cells. PMID:26709954

  3. Hydrocarbon fuel synthesis from sorbitol over bifunctional catalysts: Association of tungstated titania with platinum, palladium or iridium

    OpenAIRE

    Vilcocq, Léa; Cabiac, Amandine; Especel, Catherine; Lacombe, Sylvie; Duprez, Daniel

    2015-01-01

    To selectively transform sorbitol into hydrocarbons in aqueous phase, new bifunctional catalytic systems are designed by associating a hydrogenating metallic catalyst (M/ZrO2, M = Pt, Ir or Pd) and a dehydratingacidic catalyst (TiO2–WOx) in a mechanical mixture. Zirconium oxide was chosen as support because ithas virtually no dehydration activity and gives a good stability to the metal in water. This stability isevaluated through several characterizations before and after contact with the aqu...

  4. The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

    OpenAIRE

    Tiedtke Arno; Bockau Ulrike; Herrmann Lutz; Hartmann Marcus WW; Weide Thomas

    2006-01-01

    Abstract Background Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. Results Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to dest...

  5. Vanadium nanobelts coated nickel foam 3D bifunctional electrode with excellent catalytic activity and stability for water electrolysis

    Science.gov (United States)

    Yu, Yu; Li, Pei; Wang, Xiaofang; Gao, Wenyu; Shen, Zongxu; Zhu, Yanan; Yang, Shuliang; Song, Weiguo; Ding, Kejian

    2016-05-01

    Pursuit of highly active, stable and low-cost electrocatalysts for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) is the key point for large-scale water splitting. A vanadium nanobelts coating on a nickel foam (V/NF) is proposed as an excellent 3D bifunctional electrode for water electrolysis here, which exhibits high activities with overpotentials of 292 and 176 mV at 10 mA cm-2 for OER and HER, respectively. When employed as a bifunctional electrocatalyst in an alkaline water electrolyzer, a cell voltage of 1.80 V was required to achieve 20 mA cm-2 with a slight increase during a 24 h durability test. The existence of the appropriate amount of nitrogen and oxygen elements in the surface region of vanadium nanobelts is regarded to be responsible for the electrocatalytic activity.Pursuit of highly active, stable and low-cost electrocatalysts for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) is the key point for large-scale water splitting. A vanadium nanobelts coating on a nickel foam (V/NF) is proposed as an excellent 3D bifunctional electrode for water electrolysis here, which exhibits high activities with overpotentials of 292 and 176 mV at 10 mA cm-2 for OER and HER, respectively. When employed as a bifunctional electrocatalyst in an alkaline water electrolyzer, a cell voltage of 1.80 V was required to achieve 20 mA cm-2 with a slight increase during a 24 h durability test. The existence of the appropriate amount of nitrogen and oxygen elements in the surface region of vanadium nanobelts is regarded to be responsible for the electrocatalytic activity. Electronic supplementary information (ESI) available: More SEM, TEM images, XRD patterns, LSV curves, XPS spectra. See DOI: 10.1039/c6nr02395a

  6. Enhanced bifunctional activity of LaNiO3-based gas diffusion electrodes for regenerative fuel cells

    OpenAIRE

    Silva, R A; Soares, C. O.; Carvalho, M. D.; C. M. Rangel; Pereira, M. I. da Silva

    2013-01-01

    Perovskites are of great interest when searching replacements for precious metals as catalyst for bifunctional oxygen electrodes involving the oxygen evolution(OER) and oxygen reduction reaction (ORR) as is the case of regenerative fuel cells. In this work a full electrochemical study on the electrochemical properties of gas diffusion electrodes (GDEs) using LaNiO3-based catalysts, conducted in alkaline media, led to a study of cyclability and durability. The incorporation of GDEs in a low po...

  7. Morphology-controlled growth of magnetic iron oxide components on gold nanoparticles as bi-functional agents

    OpenAIRE

    Li, L.; Leung, CW; Ruotolo, A.; Jiang, C; Pong, PWT

    2015-01-01

    Summary form only given. Hybrid nanostructure can inherit the physiochemical properties of its individual components to realize its multi-functionality. The coupling of plasmonic effect of gold nanoparticles with magnetic properties of iron oxide nanoparticles has shown great promise as bi-functional agents allowing simultaneous magnetic resonance imaging (MRI)/computed tomography (CT) imaging and magnetic/photonic thermal therapy. However, since gold and iron oxide are two dissimilar materia...

  8. The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

    OpenAIRE

    Arthur Sarrade-Loucheur; Shuang-yong Xu; Siu-Hong Chan

    2013-01-01

    Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavag...

  9. L-Threonine-derived novel bifunctional phosphine-sulfonamide catalyst-promoted enantioselective aza-morita-Baylis-Hillman reaction

    KAUST Repository

    Zhong, Fangrui

    2011-03-18

    A series of novel bifunctional phosphine-sulfonamide organic catalysts were designed and readily prepared from natural amino acids, and they were utilized to promote enantioselective aza-Morita-Baylis-Hillman (MBH) reactions. l-Threonine-derived phosphine-sulfonamide 9b was found to be the most efficient catalyst, affording the desired aza-MBH adducts in high yields and with excellent enantioselectivities. © 2011 American Chemical Society.

  10. Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

    Institute of Scientific and Technical Information of China (English)

    Florian Graepler; Ulrike A Lauer; Reinhard Vonthein; Michael Gregor; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer; Marie-Luise Lemken; Wolfgang A Wybranietz; Ulrike Schmidt; Irina Smirnow; Christine D Groβ; Martin Spiegel; Andrea Schenk; Hansj(o)rg Graf

    2005-01-01

    AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model.METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene.RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH ceils stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin(MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application,whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>>YCD > > BCD > > > negative control) was defi ned as a result of a directin vivo comparison of all three suicide genes.CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model,thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

  11. Gold nanoparticle immobilization on ZnO nanorods via bi-functional monolayers: A facile method to tune interface properties

    Science.gov (United States)

    Jayaraman, Sundaramurthy; Suresh Kumar, P.; Mangalaraj, D.; Dharmarajan, Rajarathnam; Ramakrishna, Seeram; P Srinivasan, M.

    2015-11-01

    We demonstrated the functionalization of one dimensional (1-D) zinc oxide nanorods (ZnO NRs) using bi-functional organic molecules to create hybrid structures with surface functionalities and tuneable organic/inorganic interface. Bi-functional molecules with carboxylic acid, thiol and silane end groups and amine termination had been employed to functionalize the NRs by forming carboxylate, thiolate and hydroxylation bonds, respectively, with ZnO. The surface textures of NRs were preserved even after functionalization. The functionalized NRs were decorated with gold nanoparticles (AuNPs) and the hybrid structures exhibited a quenched blue shift ultraviolet emission which depended on the distance between the ZnO surface and the AuNPs. The NR functionalization with bi-functional molecules and decoration of NPs, and surface morphologies were analyzed using x-ray photoelectron spectroscopy, field emission scanning electron microscopy and transmission electron spectroscopy. These hybrid structures can play a vital role in tuning the interface properties and have potential applications in future photovoltaics, chemical sensors, biomarkers, and wavelength based biosensors.

  12. Construction and evaluation of a novel bifunctional phenylalanine-formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration.

    Science.gov (United States)

    Jiang, Wei; Fang, Bai-Shan

    2016-05-01

    Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of L-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of L-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH-FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5-9.0). The bifunctional enzyme can produce 153.9 mM L-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to L-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH-FDH with coenzyme regeneration for phenylpyruvic acid analysis and L-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field. PMID:26819086

  13. The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

    Directory of Open Access Journals (Sweden)

    Santos Diógenes S

    2008-04-01

    Full Text Available Abstract Background The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB. The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS, molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and

  14. Hexadentate bispidine derivatives as versatile bifunctional chelate agents for copper(II) radioisotopes.

    Science.gov (United States)

    Juran, Stefanie; Walther, Martin; Stephan, Holger; Bergmann, Ralf; Steinbach, Jörg; Kraus, Werner; Emmerling, Franziska; Comba, Peter

    2009-02-01

    The preparation and use of bispidine derivatives (3,7-diazabicyclo[3.3.1]nonane) as chelate ligands for radioactive copper isotopes for diagnosis (64Cu) or therapy (67Cu) are reported. Starting from the hexadentate bispidine-based bis(amine)tetrakis(pyridine) ligand 1 with a keto and two ester substituents, the corresponding mono-ol 2 and two dicarboxylic acid derivatives 3 and 5 have been synthesized. A range of techniques, including single-crystal X-ray structure analysis, UV/vis spectroscopy, cyclic voltammetry, thin-layer- (TLC), and high-performance liquid chromatography (HPLC), have been used to characterize the structure and stability of the copper(II)-bispidine complexes. A rapid formation (within 1 min) of stable copper(II)-bispidine complexes under mild conditions (ambient temperature, aqueous solution) has been observed. Challenge experiments of these complexes in the presence of a high excess of competing ligands, such as glutathione, cyclam, or superoxide dismutase (SOD), as well as in rat plasma, gave no evidence of demetalation or transchelation. The bifunctional bispidine derivative 5 can be readily functionalized with biologically active molecules at the pendant carboxylate groups. The coupling of a bombesin analogue betahomo-Glu-betaAla-betaAla-[Cha(13),Nle(14)]BBN(7-14), by condensation of a carboxylate of the bispidine backbone with the N-terminus of the peptide produced the bifunctional ligand 6. The radiocopper(II) complex of this bombesin-bispidine conjugate has a considerable hydrophilicity (log D(o/w) < -2.4), and this leads to a very fast blood clearance (blood: 0.28 +/- 0.02 SUV, 1 h p.i.), low liver tissue accumulation (liver: 1.20 +/- 0.27 SUV, 1 h p.i.), and rapid renal-urinary excretion (kidneys: 6.06 +/- 2.96 SUV, 1 h p.i.) as shown by biodistribution studies of 64Cu-6 in Wistar rats. Preliminary in vivo studies of 64Cu-6 in NMRI nu/nu mice, bearing the human prostate tumor PC-3 showed an accumulation of the conjugate in the tumor (2

  15. NHS-MAS3: a bifunctional chelator alternative to NHS-MAG3

    International Nuclear Information System (INIS)

    This laboratory uses an N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS-MAG3) to conjugate amines for subsequent labeling with 99mTc. However, the synthesis from triglycerine is general and not restricted to this tripeptide. We had earlier selected a small number of alternative tripeptides and synthesized the corresponding NHS derivatives. Each was then evaluated in a search for bifunctional chelators with properties superior to NHS-MAG3, such as lower serum protein binding or improved stability to cysteine challenge. Based on these preliminary results, NHS-S-acetylmercaptoacetyltriserine (NHS-MAS3) was selected for further investigation. We have now conjugated this bifunctional chelator to biocytin and to an amine-derivatized peptide nucleic acid (PNA). Both carriers were also conjugated with NHS-MAG3 under identical conditions and all were labeled with 99mTc at neutral pH and at boiling temperature while the conjugated PNAs were radiolabelled at neutral pH and at room temperature. Regardless of the chelator, reverse phase HPLC radiochromatograms of the labeled biotins and PNAs after purification showed a single peak. However, by size exclusion HPLC, the radiochromatograms always showed several peaks even after purification, but the MAS3 radiochromatograms were less complicated. For biotin and PNA both, radiolabeling via MAS3 showed improved 99mTc stability in 37 deg. C serum and in cysteine solution. The four preparations were administered to mice implanted in one thigh with avidin beads (biotins) or complementary PNA beads (PNAs). At 5 h post-administration, no significant differences were observed in the targeting of PNA beads between the two chelators, however the target thigh/normal thigh ratio was significantly higher for MAS3-biotin compared to MAG3-biotin. We conclude that labeling biocytin and amine-derivatized PNA with NHS-MAS3 compared to NHS-MAG3 provides simpler radiochromatographic profiles, improved stability of the label in

  16. Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa.

    Science.gov (United States)

    Mauline, L; Gressier, M; Roques, C; Hammer, P; Ribeiro, S J L; Caiut, J M A; Menu, M-J

    2013-01-01

    Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, (13)C and (29)Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68 nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix. PMID:23805884

  17. Antimutagenic activities of two suspected anticarcinogenic bifunctional organoiron seleno-terephthalate derivatives.

    Science.gov (United States)

    Maslat, Ahmed O; Jibril, Ibrahim; Mizyed, Shehdeh

    2010-07-01

    Two newly bifunctional organoiron seleno-terephthalate derivatives (S1 and S2) were synthesized as potential anticarcinogenic compounds. In a previous study, they were found to have antibacterial and/or antifungal activity, while they did not show any mutagenic action. Such compounds were investigated in the present study for their antimutagenic activity. Sodium azide, hydrogen peroxide, and 4-nitro-o-phenylenediamine, as known mutagens for strains TA100, TA102, and TA98 of Salmonella typhimurium, respectively, were used. Both (S1 and S2) compounds showed a strong antimutagenic action of >98% against sodium azide, >70% against hydrogen peroxide, and >65% activity against 4-nitro-o-phenylenediamine. Bearing in mind the strong correlation between mutagenicity and carcinogenicity, the above compounds can be considered as potentially promising anticarcinogens. Therefore, the present results are very encouraging to investigate the above compounds for other biological activities, including their evaluation as anticarcinogens. A suggested mechanism for the antimutagenicity of the tested compounds is presented. PMID:20462347

  18. Composite Films of Poly(vinyl alcohol) and Bifunctional Cross-linking Cellulose Nanocrystals.

    Science.gov (United States)

    Sirviö, Juho Antti; Honkaniemi, Samuli; Visanko, Miikka; Liimatainen, Henrikki

    2015-09-01

    Long and flexible cellulose nanofibrils or stiff and short cellulose nanocrystals (CNCs) are both promising lightweight materials with high strength and the potential to serve as reinforcing agents in many polymeric materials. In this study, bifunctional reactive cellulose nanocrystals (RCNCs) with carboxyl and aldehyde functionalities were used as reinforcements to prepare acetal-bonding cross-linked poly(vinyl alcohol) (PVA) films. Two RCNCs were obtained through the mechanical homogenization of partially carboxylated dialdehyde cellulose (DAC) with a residual aldehyde content of 0.55 and 1.93 mmol/g and a carboxyl content of 1.65 and 1.93 mmol/g, respectively. The mechanical, thermal, and barrier properties of PVA-RCNC films with a variable mass ratio of RCNCs (0.5-10%) were determined. Reference CNCs without reactive aldehydes were obtained through the reduction of aldehyde functionalities to primary hydroxide groups, and their reinforcing effect was compared to RCNCs. With the addition of 10% acetal-bonding RCNCs with respect to PVA weight, the tensile strength and Young's modulus were up to 2-fold greater than those of pure PVA film. An addition of only 0.5% RCNCs improved the tensile strength of the PVA film by 66% and the modulus by 61%. In comparison, a significantly lower reinforcing effect (19% with CNC loading of 0.5%) was found using reference CNCs. PVA's effective oxygen barrier and thermal properties were preserved when RCNCs were introduced into the films. PMID:26280660

  19. Bifunctional nanoparticles for surface-enhanced Raman spectroscopy-based leukemia biomarker detection

    Science.gov (United States)

    Mehn, Dora; Morasso, Carlo; Vanna, Renzo; Schiumarini, Domitilla; Bedoni, Marzia; Ciceri, Fabio; Gramatica, Furio

    2014-03-01

    The Wilms tumor gene (WT1) is a biomarker overexpressed in more than 90% of acute myeloid leukemia patients. Fast and sensitive detection of the WT1 in blood samples would allow monitoring of the minimal residual disease during clinical remission and would permit early detection of a potential relapse in acute myeloid leukemia. In this work, Surface Enhanced Raman Spectroscopy (SERS) based detection of the WT1 sequence using bifunctional, magnetic core - gold shell nanoparticles is presented. The classical co-precipitation method was applied to generate magnetic nanoparticles which were coated with a gold shell after modification with aminopropyltriethoxy silane and subsequent deposition of gold nanoparticle seeds. Simple hydroquinone based reduction procedure was applied for the shell growing in water based reaction mixture at room temperature. Thiolated ssDNA probes of the WT1 sequence were immobilized as capture oligonucleotides on the gold surface. Malachite green was applied both for testing the amplification performance of the core-shell colloidal SERS substrate and also as label dye of the target DNA sequence. The SERS enhancer efficacy of the core-shell nanomaterial was compared with the efficacy of classical spherical gold particles produced using the conventional citrate reduction method. The core-shell particles were found not only to provide an opportunity for facile separation in a heterogeneous reaction system but also to be superior regarding robustness as SERS enhancers.

  20. Designing calcium phosphate-based bifunctional nanocapsules with bone-targeting properties

    Energy Technology Data Exchange (ETDEWEB)

    Khung, Yit-Lung; Bastari, Kelsen; Cho, Xing Ling; Yee, Wu Aik; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

    2012-06-15

    Using sodium dodecyl sulphate micelles as template, hollow-cored calcium phosphate nanocapsules were produced. The surfaces of the nanocapsule were subsequently silanised by a polyethylene glycol (PEG)-based silane with an N-hydroxysuccinimide ester end groups which permits for further attachment with bisphosphonates (BP). Characterisations of these nanocapsules were investigated using Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy, Fourier Transform Infra-Red Spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Dynamic Light Scattering. To further validate the bone-targeting potential, dentine discs were incubated with these functionalised nanocapsules. FESEM analysis showed that these surface-modified nanocapsules would bind strongly to dentine surfaces compared to non-functionalised nanocapsules. We envisage that respective components would give this construct a bifunctional attribute, whereby (1) the shell of the calcium phosphate nanocapsule would serve as biocompatible coating aiding in gradual osteoconduction, while (2) surface BP moieties, acting as targeting ligands, would provide the bone-targeting potential of these calcium phosphate nanocapsules.

  1. Directed assembly of bifunctional silica-iron oxide nanocomposite with open shell structure.

    Science.gov (United States)

    Che, Hui Xin; Yeap, Swee Pin; Osman, Mohamed Syazwan; Ahmad, Abdul Latif; Lim, JitKang

    2014-10-01

    The synthesis of nanocomposite with controlled surface morphology plays a key role for pollutant removal from aqueous environments. The influence of the molecular size of the polyelectrolyte in synthesizing silica-iron oxide core-shell nanocomposite with open shell structure was investigated by using dynamic light scattering, atomic force microscopy, and quartz crystal microbalance with dissipation (QCM-D). Here, poly(diallydimethylammonium chloride) (PDDA) was used to promote the attachment of iron oxide nanoparticles (IONPs) onto the silica surface to assemble a nanocomposite with magnetic and catalytic bifunctionality. High molecular weight PDDA tended to adsorb on silica colloid, forming a more extended conformation layer than low molecular weight PDDA. Subsequent attachment of IONPs onto this extended PDDA layer was more randomly distributed, forming isolated islands with open space between them. By taking amoxicillin, an antibiotic commonly found in pharmaceutical waste, as the model system, better removal was observed for silica-iron oxide nanocomposite with a more extended open shell structure. PMID:25198872

  2. The rigid bi-functional sail, new concept concerning the reduction of the drag of ships

    Science.gov (United States)

    Țicu, I.; Popa, I.; Ristea, M.

    2015-11-01

    The policy of the European Union in the energy field, for the period to follow until 2020, is based on three fundamental objectives: sustainability, competitiveness and safety in energy supply. The “Energy - Climate Changes” program sets out a number of objectives for the EU for the year 2020, known as the “20-20-20 objectives”, namely: the reduction of greenhouse gas emissions by at least 20% from the level of those of 1990, a 20% increase in the share of renewable energy sources out of the total energy consumption as well as a target of 10% biofuels in the transports energy consumption. In this context, in order to produce or save a part of the propulsive power produced by the main propulsion machinery, by burning fossil fuels, we suggest the equipping of vessels designed for maritime transport with a bi-functional rigid sail. We consider that this device may have both the role of trapping wind energy and the role of acting as a deflector for reducing the resistance of the vessel's proceeding through the water by conveniently using the bow air current, as a result of the vessel's heading through the water with significant advantage in reducing the energy consumption for propulsion insurance.

  3. Carboxylation and Decarboxylation of Aluminum Oxide Nanoparticles Using Bifunctional Carboxylic Acids and Octylamine

    Directory of Open Access Journals (Sweden)

    Shirin Alexander

    2016-01-01

    Full Text Available The carboxylation of alumina nanoparticles (NPs, with bifunctional carboxylic acids, provides molecular anchors that are used for building more complexed structures via either physisorption or chemisorption. Colloidal suspensions of the NPs may be prepared by covalently bonding a series of carboxylic acids with secondary functional groups (HO2C-R-X to the surface of the NPs: lysine (X = NH2, p-hydroxybenzoic acid (X = OH, fumaric acid (X = CO2H, and 4-formylbenzoic acid (X = C(OH. Subsequent reaction with octylamine at either 25°C or 70°C was investigated. Fourier transform IR-attenuated reflectance spectroscopy (FTIR-ATR, thermogravimetric analysis (TGA, and scanning electron microscopy (SEM along with energy dispersive X-ray (EDX analysis were used to characterize the bifunctionalized monolayers and/or multilayer corona surrounding the alumina NPs and investigate the reaction mechanism of octylamine with the functional groups (X of the NPs. Except for the fumaric functionalized NPs, addition of octylamine to the functionalized NPs leads to removal of excess carboxylic acid corona from the surface via an amide formation. The extent of the multilayer is dependent on the strength of the acid⋯acid interaction.

  4. Bifunctional Crosslinking Agents Enhance Anion Exchange Membrane Efficacy for Vanadium Redox Flow Batteries.

    Science.gov (United States)

    Wang, Wenpin; Xu, Min; Wang, Shubo; Xie, Xiaofeng; Lv, Yafei; Ramani, Vijay K

    2014-06-01

    A series of cross-linked fluorinated poly (aryl ether oxadiazole) membranes (FPAEOM) derivatized with imidazolium groups were prepared. Poly (N-vinylimidazole) (PVI) was used as the bifunctional cross-linking agent to: a) lower vanadium permeability, b) enhance dimensional stability, and c) concomitantly provide added ion exchange capacity in the resultant anion exchange membranes. At a molar ratio of PVI to FPAEOM of 1.5, the resultant membrane (FPAEOM-1.5 PVI) had an ion exchange capacity of 2.2 meq g-1, a vanadium permeability of 6.8×10-7 cm2 min-1, a water uptake of 68 wt.%, and an ionic conductivity of 22.0 mS cm-1, all at 25°C. Single cells prepared with the FPAEOM-1.5 PVI membrane exhibited a higher coulombic efficiency (> 92%) and energy efficiency (> 86%) after 40 test cycles in vanadium redox flow battery. The imidazolium cation showed high chemical stability in highly acidic and oxidizing vanadium solution as opposed to poor stability in alkaline solutions. Based on our DFT studies, this was attributed to the lower HOMO energy (-7.265 eV) of the HSO4- ion (compared to the OH- ion; -5.496 eV) and the larger HOMO-LUMO energy gap (6.394 eV) of dimethylimidazolium bisulfate ([DMIM] [HSO4]) as compared to [DMIM] [OH] (5.387 eV). PMID:24884171

  5. Mechanism of tetralin ring opening and contraction over bifunctional Ir/SiO₂-Al₂O₃ Catalysts.

    Science.gov (United States)

    Piccolo, Laurent; Nassreddine, Salim; Toussaint, Guy; Geantet, Christophe

    2012-09-01

    The development of cleaner fuels from conventional resources requires the finding of new hydrotreatment processes able to improve the combustion performances of fuels and limit undesirable emissions. In the context of gas oil upgrading by selective ring opening, we have investigated the hydroconversion of tetralin over iridium nanoparticles supported on amorphous silica-alumina. The conversion of tetralin leads to hydrogenation, ring-contraction, and ring-opening products. The selectivity to ring-opening/-contraction products (ROCPs) increases linearly with the acid-metal site ratio and can be tuned by modifying the metal loading, the metal nanoparticle size, or the support composition. From the combination of catalytic tests at variable conversion and the products identification by two-dimensional gas chromatography, a mechanistic reaction scheme has been established. Aromatic ROCPs are formed through purely acidic steps, whereas the formation of saturated ROCPs mostly involves bifunctional reaction steps. Iridium-catalyzed hydrogenolysis appears to be a minor pathway with respect to iridium-catalyzed hydrogenation and Brønsted acid catalyzed isomerization. PMID:22949005

  6. Fe3O4 and CdS based bifunctional core–shell nanostructure

    International Nuclear Information System (INIS)

    Highlights: ► First report on a room temperature aqueous process for growth of a hybrid core shell nanostructure containing a magnetic core and a semiconducting shell. ► Formation of distinct core shell nanostructure revealed by high resolution transmission electron microscopy. ► A bifunctional nature combining magnetic as well as photoresponce for the as synthesised core shell nanostructures demonstrated. ► A tendency towards self organisation of the core–shell nanostructure. ► Possible applications including purification and isolation of biological materials, drug delivery system, bio-labels, spintronics, etc. -- Abstract: A room temperature solution process for synthesis of Fe3O4 nanoparticles and their hybrid core shell nanostructures using CdS as the shell material has been described. The as grown particles have been characterised using XRD, Rietveld refinement, high resolution transmission electron microscopy, atomic force microscopy, superconducting quantum interference device, optical absorbance and photoluminescence spectroscopy. A superparamagnetic response revealed from the magnetisation measurements of the as synthesised magnetite nanoparticles was retained even after the growth of the CdS shell. From luminescence and high resolution atomic force microscopy measurements, it is shown that the core–shell structures advantageously combine magnetic as well as fluorescence response with a tendency towards self-organization.

  7. A new bifunctional hybrid nanostructure as an active platform for photothermal therapy and MR imaging.

    Science.gov (United States)

    Khafaji, Mona; Vossoughi, Manouchehr; Hormozi-Nezhad, M Reza; Dinarvand, Rassoul; Börrnert, Felix; Irajizad, Azam

    2016-01-01

    As a bi-functional cancer treatment agent, a new hybrid nanostructure is presented which can be used for photothermal therapy by exposure to one order of magnitude lower laser powers compared to similar nanostructures in addition to substantial enhancment in magnetic resonance imaging (MRI) contrast. This gold-iron oxide hybrid nanostructure (GIHN) is synthesized by a cost-effective and high yield water-based approach. The GIHN is sheilded by PEG. Therefore, it shows high hemo and biocompatibility and more than six month stability. Alongside earlier nanostructures, the heat generation rate of GIHN is compareable with surfactnat-capped gold nanorods (GNRs). Two reasons are behind this enhancement: Firstly the distance between GNRs and SPIONs is adjusted in a way that the surface plasmon resonance of the new nanostructure is similar to bare GNRs and secondly the fraction of GNRs is raised in the hybrid nanostructure. GIHN is then applied as a photothermal agent using laser irradiation with power as low as 0.5 W.cm(-2) and only 32% of human breast adenocarcinoma cells could survive. The GIHN also acts as a dose-dependent transvers relaxation time (T2) MRI contrast agent. The results show that the GINH can be considered as a good candidate for multimodal photothermal therapy and MRI. PMID:27297588

  8. Bifunctional separator as a polysulfide mediator for highly stable Li-S batteries

    KAUST Repository

    Abbas, Syed Ali

    2016-05-24

    The shuttling process involving lithium polysulfides is one of the major factors responsible for the degradation in capacity of lithium–sulfur batteries (LSBs). Herein, we demonstrate a novel and simple strategy—using a bifunctional separator, prepared by spraying poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) on pristine separator—to obtain long-cycle LSBs. The negatively charged SO3– groups present in PSS act as an electrostatic shield for soluble lithium polysulfides through mutual coulombic repulsion, whereas PEDOT provides chemical interactions with insoluble polysulfides (Li2S, Li2S2). The dual shielding effect can provide an efficient protection from the shuttling phenomenon by confining lithium polysulfides to the cathode side of the battery. Moreover, coating with PEDOT:PSS transforms the surface of the separator from hydrophobic to hydrophilic, thereby improving the electrochemical performance. We observed an ultralow decay of 0.0364% per cycle when we ran the battery for 1000 cycles at 0.25 C—far superior to that of the pristine separator and one of the lowest recorded values reported at a low current density. We examined the versatility of our separator by preparing a flexible battery that functioned well under various stress conditions; it displayed flawless performance. Accordingly, this economical and simple strategy appears to be an ideal platform for commercialization of LSBs.

  9. GST-TAT-SOD: Cell Permeable Bifunctional Antioxidant Enzyme-A Potential Selective Radioprotector.

    Science.gov (United States)

    Pan, Jianru; He, Huocong; Su, Ying; Zheng, Guangjin; Wu, Junxin; Liu, Shutao; Rao, Pingfan

    2016-01-01

    Superoxide dismutase (SOD) fusion of TAT was proved to be radioprotective in our previous work. On that basis, a bifunctional recombinant protein which was the fusion of glutathione S-transferase (GST), SOD, and TAT was constructed and named GST-TAT-SOD. Herein we report the investigation of the cytotoxicity, cell-penetrating activity, and in vitro radioprotective effect of GST-TAT-SOD compared with wild SOD, single-function recombinant protein SOD-TAT, and amifostine. We demonstrated that wild SOD had little radioprotective effect on irradiated L-02 and Hep G2 cells while amifostine was protective to both cell lines. SOD-TAT or GST-TAT-SOD pretreatment 3 h prior to radiation protects irradiated normal liver cells against radiation damage by eliminating intracellular excrescent superoxide, reducing cellular MDA level, enhancing cellular antioxidant ability and colony formation ability, and reducing apoptosis rate. Compared with SOD-TAT, GST-TAT-SOD was proved to have better protective effect on irradiated normal liver cells and minimal effect on irradiated hepatoma cells. Besides, GST-TAT-SOD was safe for normal cells and effectively transduced into different organs in mice, including the brain. The characteristics of this protein suggest that it may be a potential radioprotective agent in cancer therapy better than amifostine. Fusion of two antioxidant enzymes and cell-penetrating peptides is potentially valuable in the development of radioprotective agent. PMID:27313832

  10. GST-TAT-SOD: Cell Permeable Bifunctional Antioxidant Enzyme—A Potential Selective Radioprotector

    Science.gov (United States)

    Pan, Jianru; He, Huocong; Su, Ying; Zheng, Guangjin; Wu, Junxin; Liu, Shutao; Rao, Pingfan

    2016-01-01

    Superoxide dismutase (SOD) fusion of TAT was proved to be radioprotective in our previous work. On that basis, a bifunctional recombinant protein which was the fusion of glutathione S-transferase (GST), SOD, and TAT was constructed and named GST-TAT-SOD. Herein we report the investigation of the cytotoxicity, cell-penetrating activity, and in vitro radioprotective effect of GST-TAT-SOD compared with wild SOD, single-function recombinant protein SOD-TAT, and amifostine. We demonstrated that wild SOD had little radioprotective effect on irradiated L-02 and Hep G2 cells while amifostine was protective to both cell lines. SOD-TAT or GST-TAT-SOD pretreatment 3 h prior to radiation protects irradiated normal liver cells against radiation damage by eliminating intracellular excrescent superoxide, reducing cellular MDA level, enhancing cellular antioxidant ability and colony formation ability, and reducing apoptosis rate. Compared with SOD-TAT, GST-TAT-SOD was proved to have better protective effect on irradiated normal liver cells and minimal effect on irradiated hepatoma cells. Besides, GST-TAT-SOD was safe for normal cells and effectively transduced into different organs in mice, including the brain. The characteristics of this protein suggest that it may be a potential radioprotective agent in cancer therapy better than amifostine. Fusion of two antioxidant enzymes and cell-penetrating peptides is potentially valuable in the development of radioprotective agent. PMID:27313832

  11. GST-TAT-SOD: Cell Permeable Bifunctional Antioxidant Enzyme—A Potential Selective Radioprotector

    Directory of Open Access Journals (Sweden)

    Jianru Pan

    2016-01-01

    Full Text Available Superoxide dismutase (SOD fusion of TAT was proved to be radioprotective in our previous work. On that basis, a bifunctional recombinant protein which was the fusion of glutathione S-transferase (GST, SOD, and TAT was constructed and named GST-TAT-SOD. Herein we report the investigation of the cytotoxicity, cell-penetrating activity, and in vitro radioprotective effect of GST-TAT-SOD compared with wild SOD, single-function recombinant protein SOD-TAT, and amifostine. We demonstrated that wild SOD had little radioprotective effect on irradiated L-02 and Hep G2 cells while amifostine was protective to both cell lines. SOD-TAT or GST-TAT-SOD pretreatment 3 h prior to radiation protects irradiated normal liver cells against radiation damage by eliminating intracellular excrescent superoxide, reducing cellular MDA level, enhancing cellular antioxidant ability and colony formation ability, and reducing apoptosis rate. Compared with SOD-TAT, GST-TAT-SOD was proved to have better protective effect on irradiated normal liver cells and minimal effect on irradiated hepatoma cells. Besides, GST-TAT-SOD was safe for normal cells and effectively transduced into different organs in mice, including the brain. The characteristics of this protein suggest that it may be a potential radioprotective agent in cancer therapy better than amifostine. Fusion of two antioxidant enzymes and cell-penetrating peptides is potentially valuable in the development of radioprotective agent.

  12. Cobalt Phosphide Hollow Polyhedron as Efficient Bifunctional Electrocatalysts for the Evolution Reaction of Hydrogen and Oxygen.

    Science.gov (United States)

    Liu, Mengjia; Li, Jinghong

    2016-01-27

    The development of efficient and low-cost hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) electrocatalysts for renewable-energy conversion techniques is highly desired. A kind of hollow polyhedral cobalt phosphide (CoP hollow polyhedron) is developed as efficient bifunctional electrocatalysts for HER and OER templated by Co-centered metal-organic frameworks. The as-prepared CoP hollow polyhedron, which have large specific surface area and high porosity providing rich catalytic active sites, show excellent electrocatalytic performances for both HER and OER in acidic and alkaline media, respectively, with onset overpotentials of 35 and 300 mV, Tafel slopes of 59 and 57 mV dec(-1), and a current density of 10 mA cm(-2) at overpotentials of 159 and 400 mV for HER and OER, respectively, which are remarkably superior to those of particulate CoP (CoP particles) and comparable to those of commercial noble-metal catalysts. In addition, the CoP hollow polyhedron also show good durability after long-term operations. PMID:26711014

  13. Catalytic characterization of bi-functional catalysts derived from Pd–Mg–Al layered double hydroxides

    Indian Academy of Sciences (India)

    N N Das; S C Srivastava

    2002-08-01

    Hydrotalcite like precursors containing PdII–MgII–AlIII with varying molar ratios, (Pd + Mg)/Al ≈ 3 and Mg/Pd ≈ 750 to 35, were prepared by coprecipitation of metal nitrates at constant pH. Characterization of samples as synthesized and their calcined products by elemental analyses, powder XRD, TG–DTA, FT–IR spectroscopy, TPR and N2 physisorption indicated a well crystalline hydrotalcite like structure with incorporation of Pd2+ in the brucite layers. Thermal decomposition of hydrotalcite precursors at intermediate temperatures led to amorphous mixed oxides, Pd/MgAl(O), which on reduction yielded bi-functional catalyst, Pd°/MgAl(O). The resultant catalysts with acid, base and hydrogenating sites, were highly active and selective for one-step synthesis of methyl isobutyl ketone (MIBK) from acetone and hydrogen. The results showed an optimal balance between acid-base and metallic sites were required to increase the selectivity of MIBK and stability of the catalysts.

  14. The determination of the rate of conjugation immunoglobuline with bifunctional chelator

    Science.gov (United States)

    Málek, Z.; Miler, V.; Budský, F.

    2006-01-01

    The work was performed under the GACR project: "Technology of preparation of radionuclides and their labelled compounds for nuclear medicine and pharmacy with the use of the reactor LVR-15" reg. no. 104/03/0499. Imaging of cell’s antigens with the use of labelled immunoglobulines allows imaging of specific receptors on cell membrane and specific tumours. It is necessary to carry out the labelling of the immunoglobulines with radionuclides of suitable physical properties, which form cations (e.g., 111In, 90Y, 177Lu) that form very strong chelates of sufficiently high stability constant preventing the dissociation of complexes or the radionuclide under “in-vivo” conditions. The immunoglobuline must be conjugated with the bifunctional chelator (BCH), which contains both chelating unit and reactive group for binding to the immunoglobuline. In our laboratory we have conjugated human IgG and monoclonal antibody CD20 with diethylenetriamine pentaacetic acid dianhydride (cDTPAA). Radionuclides 90Y and 177Lu prepared on the LVR-15 reactor in NRI Rez were used for labelling. After conjugation and labelling the yields in relation to the amount of isotopic carrier have been determined.

  15. Bifunctional redox flow battery-1 V(III)/V(II)-glyoxal(O2) system

    International Nuclear Information System (INIS)

    Bifunctional redox flow batteries (BRFB) possess functions of both electricity storage and electrochemical preparation, having the potential for increasing the electrical energy utilization. A V(III)/V(II)-glyoxal(O2) system has been developed. Separators of the BRFB play a key role in BRFB performance. A Nafion solution was sprayed on a gas diffusion layer (GDL) at the Nafion loading of 2 mg cm-2, and the GDL was then hot-pressed onto a Nafion115 cation exchange membrane, obtaining a modified separator. This separator not only prevents the crossover of vanadium but also has favorable conductivity, obtaining optimal charge and organic electro-synthesis performance of the BRFB. The effects of the concentrations of glyoxal and HCl on the performance of BRFB were also investigated. It is shown that the optimal concentration of glyoxal and HCl should be 1.2 and 3 M, respectively. As a result, the current efficiency of organic electro-synthesis is further increased. An acceptable discharge performance is achieved for a period exceeding 20 h at the current density of 20 mA cm-2. The average discharge voltage of 0.73 V and the coulombic efficiency of 66% are obtained. It is demonstrated that the principle of the BRFB is feasible. However, further experiments are needed to improve the performance

  16. Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

    International Nuclear Information System (INIS)

    Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

  17. Bifunctional impedimetric sensors based on azodicarboxamide supported on modified graphene nanosheets.

    Science.gov (United States)

    Azadbakht, Azadeh; Roushani, Mahmoud; Abbasi, Amir Reza; Derikvand, Zohreh; Menati, Saeid

    2016-12-01

    Herein, gold-coated graphene oxide nanosheets hybrid material (GO/AuNPs) with exceptional physical and chemical properties has been utilized as a novel platform for electrode modification. The synthetic method of GO/AuNPs involves anon-covalent functionalization of exfoliated GO with AuNPs based on the reduction of the Au(III) complex by sodium citrate. The prepared GO/AuNPs hybrid exhibits the dispersion of high density AuNPs which were densely decorated on the large surface area of GO. The GO/AuNPs modified glassy carbon (GC) electrode was employed as a sensing platform to immobilize azodicarboxamide (ACA). The morphology, structure and electrochemical performance of the sensor were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results indicate that the modified electrode has a notable bifunctional catalytic activity. Electrocatalytic oxidations of cysteine and electrocatalytic reduction of iodate at the surface of modified electrode were investigated with different technique. PMID:27612707

  18. Intermolecular interactions in cyclic complexes with hydrogen bond of bifunctional N-containing compounds in solution

    International Nuclear Information System (INIS)

    The structure of molecular and ionic hydrogen-bonded complexes formed by bifunctional N-containing molecules, which can act as proton donor and proton acceptor simultaneously, and the nature of molecular interactions in these complexes were studied in solution. The spectroscopic, steric and thermodynamic characteristics of self-associates of 3,5-dimethylpyrazole, diphenylformamidine, diphenylguanidine and diphenyltriazene and complexes of these compounds with carboxylic acids were obtained. The quantum chemical calculations of the structure of complexes and the vibrational frequencies in IR spectra were carried out in the harmonic approximation and with the anharmonic effects taken into account. It was shown that under the interaction with weak carboxylic acids (HCOOH, CH3COOH, CH2ClCOOH) the complexes have molecular structure with two H-bonds NH...O=C and OH...N, and the interaction with strong acids (CHCl2COOH, CCl3COOH, CF3COOH) results in formation of cyclic hydrogen-bonded ionic pairs with proton transfer from hydroxyl group to the N atom of the azo-compound. (authors)

  19. Preparation of magnetic and fluorescent bifunctional chitosan nanoparticles for optical determination of copper ion

    International Nuclear Information System (INIS)

    We describe a simple method for the synthesis of highly magnetic and fluorescent bifunctional chitosan nanoparticles (MF-CSNPs). Water-soluble and magnetic Fe3O4-chitosan nanoparticles and CdSe quantum dots capped with thioglycolic acid were incorporated into a chitosan matrix via electrostatic interaction. The optical, magnetic, crystallographic and morphological properties of the new nanoparticles were studied by UV-visible, fluorescence, X-ray diffraction and transmission electron microscopy. In addition, MF-CSNPs are found to be a useful probe for the determination of copper ion which acts as a quencher of fluorescence. The relative fluorescence intensity of MF-CSNPs is linearly related to the concentration of copper ion in the 0.125 to 25 ng.mL-1 concentration range. The MF-CSNPs also are found to adsorb copper ion which therefore can be separated and enriched by manipulating them with an external magnetic field. Before enrichment, the limit of detection (LOD) for copper ion is 120 pg.mL-1, but after enrichment, the LOD is 46 pg.mL-1. (author)

  20. Cu/Ag-based bifunctional nanoparticles obtained by one-pot laser-assisted galvanic replacement

    International Nuclear Information System (INIS)

    We have prepared, for the first time, stable and uncapped Ag/Cu-based bifunctional nanoparticles (NPs) (BFNPs) in water, by combining ps laser ablation in liquid environment and galvanic replacement. The particles were obtained in a single step by 1,064 nm irradiation of a Cu target in water solutions of AgNO3 or AgNO2. Under proper salt concentration and irradiation conditions, the laser beam activates formation of deep orange colloids, which are positively charged and stable for weeks. High resolution transmission electron microscopy (HRTEM) analysis showed a predominance of composite crystalline nanostructures with size in the 1–15 nm range and consisting of fcc Ag and fcc Cu (or its oxides). While CuO tenorite crystalline phase was detected by HRTEM, X-ray photoelectron spectroscopy analysis permitted to observe also the Cu(I) oxidation state of Cu, being the Cu(I)/Cu(II) ratio different in the samples obtained in AgNO3 or AgNO2 baths. Functionalization with organic ligands and subsequent Raman tests demonstrated the SERS activity of the BFNPs and the existence of different complexing surface sites.

  1. NAD(H recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available A chimeric bifunctional enzyme composing of galactose dehydrogenase (galDH; from Pseudomonas fluorescens and lactate dehydrogenase (LDH; from Bacillus stearothermophilus was successfully constructed. The chimeric galDH/LDH possessed dual characteristics of both galactose dehydrogenase and lactate dehydrogenase activities while exhibiting hexameric rearrangement with a molecular weight of approximately 400 kDa. In vitro observations showed that the chimeric enzyme was able to recycle NAD with a continuous production of lactate without any externally added NADH. Two fold higher recycling rate (0.3 mM/h than that of the native enzyme was observed at pH values above 8.5. Proximity effects became especially pronounced during the recycling assay when diffusion hindrance was induced by polyethylene glycol. All these findings open up a high feasibility to apply the NAD(H recycling system for metabolic engineering purposes e.g. as a model to gain a better understanding on the molecular proximity process and as the routes for synthesizing of numerous high-value-added compounds.

  2. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly. PMID:26734760

  3. Exact functional derivative of the nonadditive kinetic-energy bifunctional in the long-distance limit

    Science.gov (United States)

    Jacob, Christoph R.; Beyhan, S. Maya; Visscher, Lucas

    2007-06-01

    We have investigated the functional derivative of the nonadditive kinetic-energy bifunctional, which appears in the embedding potential that is used in the frozen-density embedding formalism, in the limit that the separation of the subsystems is large. We have derived an exact expression for this kinetic-energy component of the embedding potential and have applied this expression to deduce its exact form in this limit. Comparing to the approximations currently in use, we find that while these approximations are correct at the nonfrozen subsystem, they fail completely at the frozen subsystem. Using test calculations on two model systems, a H2O⋯Li+ complex and a cluster of aminocoumarin C151 surrounded by 30 water molecules, we show that this failure leads to a wrong description of unoccupied orbitals, which can lead to convergence problems caused by too low-lying unoccupied orbitals and which can further have serious consequences for the calculation of response properties. Based on our results, a simple correction is proposed, and we show that this correction is able to fix the observed problems for the model systems studied.

  4. Bifunctional polyacrylonitrile fiber-mediated conversion of sucrose to 5-hydroxymethylfurfural in mixed-aqueous systems.

    Science.gov (United States)

    Shi, Xian-Lei; Zhang, Min; Lin, Huikun; Tao, Minli; Li, Yongdan; Zhang, Wenqin

    2015-03-01

    A highly efficient catalytic system composed of a bifunctional polyacrylonitrile fiber (PANF-PA[BnBr]) and a metal chloride was employed to produce 5-hydroxymethylfurfural (HMF) from sucrose in mixed-aqueous systems. The promoter of PANF-PA[BnBr] incorporates protonic acid groups that promote the hydrolysis of the glycosidic bond to convert sucrose into glucose and fructose, and then catalyzes fructose dehydration to HMF, while the ammonium moiety may promote synergetically with the metal chloride the isomerization of glucose to fructose and transfer HMF from the aqueous to the organic phase. The detailed characterization by elemental analysis, FTIR spectroscopy, and SEM confirmed the rangeability of the fiber promoter during the modification and utilization processes. Excellent results in terms of high yield (72.8%) of HMF, superior recyclability (6 cycles) of the process, and effective scale-up and simple separation procedures of the catalytic system were obtained. Moreover, the prominent features (high strength, good flexibility, etc.) of the fibers are very attractive for fix-bed reactor. PMID:25573698

  5. Impedimetric and amperometric bifunctional glucose biosensor based on hybrid organic-inorganic thin films.

    Science.gov (United States)

    Wang, Huihui; Ohnuki, Hitoshi; Endo, Hideaki; Izumi, Mitsuru

    2015-02-01

    A novel glucose biosensor with an immobilized mediator was studied using electrochemical impedance spectroscopy (EIS) and amperometry measurements. The biosensor has a characteristic ultrathin form and is composed of a self-assembled monolayer anchoring glucose oxidase (GOx) covered with Langmuir-Blodgett (LB) films of Prussian blue (PB). The immobilized PB in the LB films acts as a mediator and enables the biosensor to work under a low potential (0.0V vs. Ag/AgCl). In the EIS measurements, a dramatic decrease in charge transfer resistance (Rct) was observed with sequential addition of glucose, which can be attributed to enzymatic activity. The linearity of the biosensor response was observed by the variation of the sensor response (1/Rct) as a function of glucose concentration in the range 0 to 25mM. The sensor also showed linear amperometric response below 130mM glucose. The organic-inorganic system of GOx and PB nanoclusters demonstrated bifunctional sensing action, both amperometry and EIS modes, as well as long sensing stability for 4 days. PMID:25014167

  6. Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution.

    Science.gov (United States)

    Fan, Jiying; Wang, Zhanqing; Huang, Liying; Shen, Yaling

    2016-09-01

    VAS-TRAIL is a bifunctional fusion protein that combines anti-angiogenic activity with tumor-selective apoptotic activity for enhanced anti-tumor efficacy. VAS-TRAIL is expressed as inclusion body in Escherichia coli, but protein refolding is difficult to achieve and results in low yields of bioactive protein. In this study, we describe an efficient method for VAS-TRAIL refolding. The solubilization of aggregated VAS-TRAIL was achieved by a triple agent solution, which consists of an alkaline solution (pH 11.5) containing 0.4M l-arginine and 2M urea. The solubilized protein showed high purity and preserved secondary structure according to fluorescence properties. VAS-TRAIL refolding was performed through stepwise dialysis and resulted in more than 50% recovery of the soluble protein. The function of l-arginine was additive with alkaline pH, as shown by the significant improvement in refolding yield (≈30%) by l-arginine-containing solubilization solutions compared with alkaline solubilization solutions without l-arginine. The refolded VAS-TRAIL also showed β-sheet structures and the propensity for oligomerization. Bioassays showed that the refolded fusion protein exhibited the expected activities, including its apoptotic activities toward tumor and endothelial cells, which proposed its promising therapeutic potential. PMID:26358405

  7. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease Pl treatment

    International Nuclear Information System (INIS)

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo(a)pyrene diol epoxide I, nuclease P1 treatment resulted in a significant reduction in detection of major adducts 1-amino-6-nitropyrene, 1-amino-8-nitropyrene, 2-aminofluorene, 2-naphthylamine and 4-aminobiphenyl modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene modified DNA. These results suggest that at least initially, both modications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitro-aromatics and aromatic amines

  8. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease P1 treatment.

    Science.gov (United States)

    Gallagher, J E; Jackson, M A; George, M H; Lewtas, J; Robertson, I G

    1989-04-01

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo[a]pyrene diol epoxide I (BPDE-I), nuclease P1 treatment resulted in a significant reduction in detection of major adducts from 1-amino-6-nitropyrene (1-amino-6-NP), 1-amino-8-nitropyrene (1-amino-8-NP), 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 4-aminobiphenyl (4-ABP) modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene (2-AAF) modified DNA. These results suggest that, at least initially, both modifications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitroaromatics and aromatic amines. PMID:2540901

  9. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  10. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    International Nuclear Information System (INIS)

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  11. Production of a new non-specific nuclease from Yersinia enterocolitica subsp. palearctica: optimization of induction conditions using response surface methodology

    OpenAIRE

    Fang, Xiu-Juan; Tang, Zhen-Xing; Li, Zhen-Hua; Zhang, Zhi-Liang; Shi, Lu-E

    2014-01-01

    A new non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) was expressed in Escherichia coli (E. coli) BL 21 StarTM (DE3)plysS. Induction conditions, including isopropyl-β-D-thiogalactoside (IPTG) concentration, cell density (OD600), induction time and induction temperature, were optimized using response surface methodology. Statistical analysis of the results revealed that induction temperature and all the quadratic terms of variables had significant effects on enzy...

  12. The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

    OpenAIRE

    Chung, Betty Y; Hardcastle, Thomas J.; Joshua D Jones; Irigoyen, Nerea; Firth, Andrew E.; Baulcombe, David C.; Brierley, Ian

    2015-01-01

    Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenie...

  13. Interference of Co-Amplified Nuclear Mitochondrial DNA Sequences on the Determination of Human mtDNA Heteroplasmy by Using the SURVEYOR Nuclease and the WAVE HS System

    OpenAIRE

    Yen, Hsiu-Chuan; Li, Shiue-Li; Hsu, Wei-Chien; Tang, Petrus

    2014-01-01

    High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detectin...

  14. Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA.

    Science.gov (United States)

    Kumari, Rupa; Raghavan, Sathees C

    2015-01-01

    RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs. PMID:25327637

  15. FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

    Science.gov (United States)

    Chaudhury, Indrajit; Stroik, Daniel R.

    2014-01-01

    Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation. PMID:25135477

  16. Histidine 121 of staphylococcal nuclease. Correction of the Hδ21H NMR assignment and reinterpretation of the role this residue plays in conformation heterogeneity of the protein

    International Nuclear Information System (INIS)

    Heteronuclear two-dimensional NMR studies of wild-type staphylococcal nuclease containing histidine residues uniformly labeled with carbon-13 (26% isotope) have led to full analysis of the aromatic parts of the histidine 1H and 13C spin systems. The 1Hδ2 and 13Cδ2 resonances of His121 were found to be split as the result of the Nal right-equilibrium Na2 conformational equilibrium described previously and attributed to cis-trans isomerism about the Lys116-Pro117 peptide bond (Nal, cis; Na2, trans). The relative intensities of the pair of 1Hε1 peaks from the same residue. A soluble-mutant enzyme (nuclease G79S + H124L), which exhibited a drastically altered [Na1] to [Na2] ratio, provided additional evidence that the pair of 1Hδ2 peaks and the pair of 1Hε1 peaks of His121 report on the same conformational equilibrium (Na1 right-equilibrium Na2). The unusual chemical shift of the 1Hδ2 of His121 is attributed to diamagnetic shielding by the aromatic ring of Tyr91 as verified by ring-current calculations based on two X-ray structures for wild-type staphylococcal nuclease

  17. Efficient nanobiocatalytic systems of nuclease P1 immobilized on PEG-NH2 modified graphene oxide: effects of interface property heterogeneity.

    Science.gov (United States)

    Zhuang, Wei; He, Linjiao; Zhu, Jiahua; Zheng, Jianwei; Liu, Xiaojing; Dong, Yihui; Wu, Jinglan; Zhou, Jingwei; Chen, Yong; Ying, Hanjie

    2016-09-01

    The use of graphene oxide (GO) nanosheets for functional enzyme support has attracted intensive interest owing to their unique planar structure and intriguing physical and chemical properties. However, the detailed effects of the interface properties of GO and its functionalized derivatives on active biomolecules are not well understood. We immobilize nuclease P1, a common industrial nucleic acid production enzyme, on pristine and amino poly(ethylene glycol) (PEG-NH2) modified GO nanosheets with interface property heterogeneity using two approaches, physical adsorption and chemical crosslinking. It is demonstrated that nuclease P1 could be stable immobilized on the surface of pristine GO by physical adsorption and on the edge of modified GO nanosheets by chemical crosslinking. The resultant loading capacity of nuclease P1 on pristine GO is as high as 6.45mg/mg as a consequence of strong electrostatic and hydrophobic interactions between the enzyme and carrier. However, it is determined that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on PEG-NH2-modified GO are obviously improved compared to those of the enzyme immobilized on pristine GO. The enhanced catalytic behavior demonstrates that GO and its derivatives have great potential in efficient biocatalytic systems. PMID:27295495

  18. Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

    Science.gov (United States)

    Renaud, Jean-Baptiste; Boix, Charlotte; Charpentier, Marine; De Cian, Anne; Cochennec, Julien; Duvernois-Berthet, Evelyne; Perrouault, Loïc; Tesson, Laurent; Edouard, Joanne; Thinard, Reynald; Cherifi, Yacine; Menoret, Séverine; Fontanière, Sandra; de Crozé, Noémie; Fraichard, Alexandre; Sohm, Frédéric; Anegon, Ignacio; Concordet, Jean-Paul; Giovannangeli, Carine

    2016-03-01

    Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing. PMID:26923600

  19. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha

    2015-04-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  20. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

    Science.gov (United States)

    Kim, Daesik; Kim, Sojung; Kim, Sunghyun; Park, Jeongbin; Kim, Jin-Soo

    2016-01-01

    We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome. PMID:26786045

  1. A Mismatch EndoNuclease Array-Based Methodology (MENA for Identifying Known SNPs or Novel Point Mutations

    Directory of Open Access Journals (Sweden)

    Josep M. Comeron

    2016-04-01

    Full Text Available Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs, point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1 genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2 identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3 screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

  2. True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

    Directory of Open Access Journals (Sweden)

    Maia Gurushidze

    Full Text Available Transcription activator-like effector nucleases (TALENs are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

  3. Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Renaud

    2016-03-01

    Full Text Available Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

  4. Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

    Institute of Scientific and Technical Information of China (English)

    Dongshan Yang; Jiangtian Tian; Feng Li; Jifeng Zhang; Lin Chang; Duanqing Pei; Y Eugene Chen; Liangxue Lai; Huaqiang Yang; Wei Li; Bentian Zhao; Zhen Ouyang; Zhaoming Liu; Yu Zhao; Nana Fan; Jun Song

    2011-01-01

    @@ Dear Editor, Gene targeting in mouse embryonic stem (ES) cells has revolutionized the field of mouse genetics and allowed for the analysis of diverse aspects of gene function in vivo.For more than two decades,researchers have been actively searching for ES cells from large animals such as pigs and cattle.Unfortunately,to date,no ES cell lines from large animals have passed the crucial test of germ line contribution.The sole method of gene targeting to date in these species remains somatic cell nuclear transfer (SCNT) combined with DNA homologous recombination (HR).Due to the limited proliferation competency and extremely low frequency of HR in somatic cells (less than 10-6),this process is highly inefficient and only a few successful examples have been achieved,even though enrichment strategies such as positivenegative marker selection,promoter-trap and adenoassociated viral delivery were previously used [1-3].The low efficiency of gene targeting in cultured somatic cells is the main barrier for gene targeting in large animals.Recently,zinc-finger nuclease (ZFN) technology has emerged as a powerful tool for genome editing.The success of ZFN technology for gene targeting in fruit flies,zebra fish,rodents as well as human cell lines encouraged us to establish a high-efficiency gene-targeting platform in large animals such as pigs [4-8].

  5. DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion.

    Science.gov (United States)

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2016-02-01

    Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage. PMID:26247322

  6. Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy.

    Science.gov (United States)

    Karakikes, Ioannis; Stillitano, Francesca; Nonnenmacher, Mathieu; Tzimas, Christos; Sanoudou, Despina; Termglinchan, Vittavat; Kong, Chi-Wing; Rushing, Stephanie; Hansen, Jens; Ceholski, Delaine; Kolokathis, Fotis; Kremastinos, Dimitrios; Katoulis, Alexandros; Ren, Lihuan; Cohen, Ninette; Gho, Johannes M I H; Tsiapras, Dimitrios; Vink, Aryan; Wu, Joseph C; Asselbergs, Folkert W; Li, Ronald A; Hulot, Jean-Sebastien; Kranias, Evangelia G; Hajjar, Roger J

    2015-01-01

    A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca(2+) handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies. PMID:25923014

  7. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka; Mahfouz, Magdy M

    2015-10-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. PMID:25907574

  8. Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Sylvia Merkert

    2014-01-01

    Full Text Available Genetic engineering of human induced pluripotent stem cells (hiPSCs via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs. Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

  9. Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (A)BC excision nuclease

    International Nuclear Information System (INIS)

    Human cell free extract prepared by the method of Manley et al. carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunits(s) of human excision nuclease

  10. Gadolinium and fluorescent bi-functionally labeling and in vitro MRI of rat bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Objective: To determine the feasibility of magnetically labeling and tracking mesenchymal stem cells (MSCs) in vitro by using a gadolinium and fluorescent bi-functionally transfection agent of polyethylenimine. Methods: A gadolinium bifunctional transfection reagent complex was obtained after the linear polyethylenimine derivative (JetPEI-FluoR) was incubated with Gd-DTPA. Mesenchymal stem cells isolated from the bone marrows of SD rats were cultured and expanded. The mesenchymal stem cells were incubated with the bi-functional labeling agents. After labeling, the MSCs were examined with fluoroscope and electron microscope and the biological characters were detected including trypan blue exclusion test, MTT, and apoptosis detection. On a 1.5 T MR system, the labeled MSCs were examined with spin echo T1WI and T2WI and T1 measurement with mixed sequence. After labeling, the cells were cultured and undergone routine passage. Prior MR examinations were repeated for each passage of labeled cells. All data was statistically prolessed with SPSS for Windows. Results: Of 5 x 105 MSCs incubated with the bi-functional agents, 4.25 x 105 MSCs were successfully labeled, the percentage of labeled MSCs was 85% fluoroscopically. The high density electron particles of gadolinium observed electron microscopically existed around cellular apparatuses, especially around Golgi apparatus. In trypan blue exclusion test, the exclusion rate of labeled MSCs with incubation duration of 3,6,12,24 h was (96.55±2.90)%, (94.17± 2.56)%, (97.16±3.12)% and (94.23±2.67)%, respectively. The corresponding exclusion rate of unlabeled MSCs was (95.86±2.67)%, (92.04±2.21)%, (93.38±3.64)% and (92.12±2.53)%, respectively. There was no statistical difference of trypan blue exclusion rate between labeled cells and control unlabeled cells within 24 hours of incubation (F=4.523, P>0.05). In the proliferation test, the optical absorption value of labeled MSC with 2.5, 5.0, 10.0, 20.0, 30.0 and 40.0

  11. Acid/base bifunctional carbonaceous nanomaterial with large surface area: Preparation, characterization, and adsorption properties for cationic and anionic compounds

    International Nuclear Information System (INIS)

    Nanostructured carbonaceous materials are extremely important in the nano field, yet developing simple, mild, and “green” methods that can make such materials possess large surface area and rich functional groups on their surfaces still remains a considerable challenge. Herein, a one-pot and environment-friendly method, i.e., thermal treatment (180 °C; 18 h) of water mixed with glucose and chitosan (CTS), has been proposed. The resultant carbonaceous nanomaterials were characterized by field emitting scanning electron microscope, N2 adsorption/desorption, Fourier transform infrared spectroscope, X-ray photoelectron spectroscopy, and zeta-potential analysis. It was found that, in contrast to the conventional hydrothermally carbonized product from pure glucose, with low surface area (9.3 m2 g−1) and pore volume (0.016 cm3 g−1), the CTS-added carbonaceous products showed satisfactory textural parameters (surface area and pore volume up to 254 m2 g−1 and 0.701 cm3 g−1, respectively). Moreover, it was also interestingly found that these CTS-added carbonaceous products possessed both acidic (–COOH) and basic (–NH2) groups on their surfaces. Taking the advantages of large surface area and –COOH/–NH2 bifunctional surface, the carbonaceous nanomaterials exhibited excellent performance for adsorptions of cationic compound (i.e., methylene blue) at pH 10 and anionic compound (i.e., acid red 18) at pH 2, respectively. This work not only provides a simple and green route to prepare acid/base bifunctional carbonaceous nanomaterials with large surface area but also well demonstrates their potential for application in adsorption. - Highlights: • A simple and green method was proposed to prepare carbon nanomaterials. • The carbon product showed acid/base bifunctional surface with large surface area. • The carbon material could efficiently adsorb both cationic and anionic compounds

  12. Facile preparation of Ag-Cu bifunctional electrocatalysts for zinc-air batteries

    International Nuclear Information System (INIS)

    Highlights: • Ag-Cu dendrites are observed for the first time to exhibit high catalytic activity for oxygen reduction reaction. • Ag-Cu dendrites are directly synthesized through galvanic displacement on the current collector layer made of Ni foams. • A bifunctional air cathode is fabricated using Ag-Cu dendrites as a carbon-free, binder-free catalyst layer. • Both the primary and rechargeable zinc–air batteries fabricated by Ag-Cu catalysts exhibit excellent performance. - ABSTRACT: An inexpensive, facile galvanic displacement reaction for the direct growth of silver–copper (Ag-Cu) catalysts on nickel foams is developed for the first time. The resulting Ag-Cu catalysts exhibit dendritic morphologies. Ag and Cu atoms are in their metallic state while the presence of CuO and Cu2O are limited on the surface of catalyst. The catalysts demonstrate high catalytic activity for oxygen reduction reaction (ORR) in alkaline solution, as evaluated by both linear scanning voltammetry and rotating disk electrode polarization measurements. The ORR catalysed by Ag-Cu catalyst in alkaline solution proceeds through a four-electron pathway. An air cathode is fabricated using Ag-Cu catalyst as a carbon-free, binder-free catalyst layer. Using this Ag-Cu catalyst based air cathode, both the primary and rechargeable zinc-air batteries show excellent battery performance. The specific capacity of the primary zinc-air battery is 572 mAh g−1. Especially, the rechargeable zinc-air battery shows high round-trip efficiency, appealing stability at a long charge-discharge cycle period

  13. Modification of bifunctional epoxy resin using CO{sub 2} fixation process and nanoclay

    Energy Technology Data Exchange (ETDEWEB)

    Khoshkish, Morteza; Bouhendi, Hosein, E-mail: H.boohendi@ippi.ac.ir; Vafayan, Mehdi

    2014-10-15

    A bifunctional epoxy resin was modified by using a CO{sub 2} fixation solution process in the presence of tetra n-butyl ammonium bromide (TBAB) as catalyst and the modified treated resin was treated by cloisite 30B as nano additive. The Unmodified epoxy resin (UME), CO{sub 2} fixated modified epoxy resin (CFME), and CFME/clay nano composite (CFMEN), were cured by diethylenetriamine (DETA). A cycloaliphatic compound as a reactive diluent was used to control the viscosity of high viscose CFME. The exfoliation of organoclay in UME and CFME was investigated by X-ray diffraction and activation energy was computed using the advanced integral isoconversional method. The activation energy dependency demonstrated that the mechanism of UME curing did not change in the presence of nanoclay. In contrast, the CO{sub 2} fixation results showed a significant change in the activation energy dependency. The Thermal stability parameters include the initial degradation temperature (IDT), the temperature at the maximum rate of weight loss (T{sub max}), and the decomposition activation energy (E{sub d}) were determined by thermal gravimetry analysis. Dynamic mechanical thermal analysis measurements showed that the presence of organoclay in CFME increases the T{sub g} of nano composite in contrast to UME. The fracture roughness of UME, CFME and CFNE were determined by scanning electron microscope. The exfoliated UME/1%clay nanocomposite was confirmed by TEM image. - Highlights: • A new epoxy resin was synthesized using CO{sub 2} fixation reaction. • The synthesized epoxy resin was modified by an organo nano-clay. • CO{sub 2} fixation noticeably changed the curing mechanism. • CO{sub 2} fixation reaction consumes CO{sub 2} which is a harmful greenhouse gas.

  14. Novel bifunctional anthracycline and nitrosourea chemotherapy for human bladder cancer: analysis in a preclinical survival model.

    Science.gov (United States)

    Glaves, D; Murray, M K; Raghavan, D

    1996-08-01

    A hybrid drug [N-2-chloroethylnitrosoureidodaunorubicin (AD312)] that combines structural and functional features of both anthracyclines and nitrosoureas was evaluated in a preclinical survival model of human bladder cancer. To measure the therapeutic activity of AD312, UCRU-BL13 transitional cell carcinoma cells were grown as xenografts in nude mice, and tumor growth rates were compared after i.v. administration of the drug at three dose levels. AD312 treatment at 45 and 60 mg/kg achieved 7-10-fold inhibition of tumor growth and increased host survival by 156 and 249%, respectively. Doses of 60 mg/kg showed optimal therapeutic efficacy, with sustained tumor growth inhibition, an over 2-fold increase in life span, and 40% of mice tumor free ("cured") at 120 days. Tumors were unresponsive to maximum tolerated doses of doxorubicin, a standard anthracycline used as a single agent and in combination therapies for bladder cancer. 1,3-Bis-[2-chloroethyl]-1-nitrosourea was used as a control for the apparently enhanced response of human tumors in murine hosts to nitrosoureas. 1, 3-Bis-[2-chloroethyl]-1-nitrosourea administered in three injections of 20 mg/kg did not cure mice but temporarily inhibited tumor growth by 70% and prolonged survival by 55%; its activity in this model suggests that it may be included in the repertoire of alkylating agents currently used for treatment of bladder cancers. AD312 showed increased antitumor activity with less toxicity than doxorubicin, and its bifunctional properties provide the opportunity for simultaneous treatment of individual cancer cells with two cytotoxic modalities as well as treatment of heterogeneous populations typical of bladder cancers. This novel cytotoxic drug cured doxorubicin-refractory disease and should be investigated for the clinical management of bladder cancer. PMID:9816302

  15. Cloning and Functional Analysis of the Bifunctional Agglutinin/Trypsin Inhibitor from Helianthus tuberosus L.

    Institute of Scientific and Technical Information of China (English)

    Tuanjie Chang; Hongli Zhai; Songbiao Chen; Guisheng Song; Honglin Xu; Xiaoli Wei; Zhen Zhu

    2006-01-01

    In order to find new insect resistance genes, four homologous cDNAs, hta-a, hta-b, hta-c and hta-d with lengths of 775, 718, 784 and 752 bp, respectively (GenBank accession numbers AF477031-AF477034), were isolated from a tuber cDNA expression library of Helianthus tuberosus L. Sequence analysis revealed that all four cDNAs contain an open reading frame of 444 bp, coding a polypeptide of 147 amino acid residues, and that the sequences of the cDNAs are very similar to those of the mannose-binding agglutinin genes of the jacalin-related family. In hemagglutination reactions and hapten inhibition assays, affinity-purified HTA (Helianthus tuberosus agglutinin) from induced Escherichia coli BL21(DE3) expressing GST-HTA shows hemagglutination ability and a higher carbohydrate-binding ability for mannose than other tested sugars.Trypsin inhibitory activity was detected in the crude extracts of induced E. coli BL21(DE3)expressing HTA,and was further verified by trypsin inhibitory activity staining on native polyacrylamide gel. The mechanism of interaction between HTA and trypsin was studied by molecular modeling. We found that plenty of hydrogen bonds and electrostatic interactions can be formed between the supposed binding sites of HTA-b and the active site of trypsin, and that a stable HTA/trypsin complex can be formed. The results above imply that HTA might be a bifunctional protein with carbohydrate-binding activity and trypsin inhibitory activity. Moreover,Northern blotting analysis demonstrated that hta is predominantly expressed in tubers of H. tuberosus, very weakly expressed in stems, but not expressed at all in other tissues. Southern blotting analysis indicated that hta is encoded by a multi-gene family. The insect resistance traits have been described in another paper.

  16. Novel configuration of bifunctional air electrodes for rechargeable zinc-air batteries

    Science.gov (United States)

    Li, Po-Chieh; Chien, Yu-Ju; Hu, Chi-Chang

    2016-05-01

    A novel configuration of two electrodes containing electrocatalysts for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) pressed into a bifunctional air electrode is designed for rechargeable Zn-air batteries. MOC/25BC carbon paper (MOC consisting of α-MnO2 and XC-72 carbon black) and Fe0.1Ni0.9Co2O4/Ti mesh on this air electrode mainly serve as the cathode for the ORR and the anode for the OER, respectively. The morphology and physicochemical properties of Fe0.1Ni0.9Co2O4 are investigated through scanning electron microscopy, inductively coupled plasma-mass spectrometry, and X-ray diffraction. Electrochemical studies comprise linear sweep voltammetry, rotating ring-disk electrode voltammetry, and the full-cell charge-discharge-cycling test. The discharge peak power density of the Zn-air battery with the unique air electrode reaches 88.8 mW cm-2 at 133.6 mA cm-2 and 0.66 V in an alkaline electrolyte under an ambient atmosphere. After 100 charge-discharge cycles at 10 mA cm-2, an increase of 0.3 V between charge and discharge cell voltages is observed. The deep charge-discharge curve (10 h in each step) indicates that the cell voltages of discharge (1.3 V) and charge (1.97 V) remain constant throughout the process. The performance of the proposed rechargeable Zn-air battery is superior to that of most other similar batteries reported in recent studies.

  17. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

    Science.gov (United States)

    Dou, Wenfang; Xu, Meijuan; Cai, Dongmei; Zhang, Xiaomei; Rao, Zhiming; Xu, Zhenghong

    2011-10-01

    Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the L-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and β-subunits. When the argJ regions corresponding to the α- and β-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on L: -arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of L: -arginine significantly (16.8%). PMID:21785983

  18. Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms.

    Science.gov (United States)

    Marc, F; Weigel, P; Legrain, C; Almeras, Y; Santrot, M; Glansdorff, N; Sakanyan, V

    2000-08-01

    The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences. PMID:10931207

  19. Bifunctional oligodeoxynucleotide/antagomiR constructs: evaluation of a new tool for microRNA silencing.

    Science.gov (United States)

    Ziegler, Saskia; Eberle, Mariel Esther; Wölfle, Sabine J; Heeg, Klaus; Bekeredjian-Ding, Isabelle

    2013-12-01

    MicroRNAs (miRNAs) are fine-tuners in cellular processes, including those of the immune response. To study their functions and effects in immune cells, it is necessary to achieve specific silencing of individual miRNAs. To date, introduction of antisense microRNAs (antagomiRs) into primary cells is based on electroporation, lipofection, and viral vectors. However, these techniques often compromise viability, proliferative capacity, and differentiation. Furthermore, efficiency varies depending on the cell type and some are not suitable for in vivo approaches. To overcome these limitations we exploited the property of phosphorothioate (PTO)-modified DNA oligodeoxynucleotides (ODN) to enter cells with high efficacy: we developed and evaluated ODN/antagomiR constructs that consist of a PTO-ODN carrier covalently linked to a fully methylated antagomiR RNA sequence. Using these constructs, we achieved transfection efficiency of approximately 99% in leukocytes-in particular, in B lymphocytes that are hard to transfect with other methods. Our data demonstrate that miRNA silencing by the antagomiR portion of the constructs was specific and efficient, which could be further confirmed by an increase in target protein under silencing conditions. The constructs were successfully tested in human B cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells, thus demonstrating their versatility. Moreover, introduction of stimulatory CpG sequences into the ODN portion conveys immune stimulatory quality when intended. Thus, bifunctional ODN/antagomiR constructs represent a highly efficient, versatile, and easy-to-handle tool to manipulate cellular miRNA expression levels and to allow the subsequent investigation of specific miRNA functions. PMID:24236889

  20. A metal-free bifunctional electrocatalyst for oxygen reduction and oxygen evolution reactions

    Science.gov (United States)

    Zhang, Jintao; Zhao, Zhenghang; Xia, Zhenhai; Dai, Liming

    2015-05-01

    The oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) are traditionally carried out with noble metals (such as Pt) and metal oxides (such as RuO2 and MnO2) as catalysts, respectively. However, these metal-based catalysts often suffer from multiple disadvantages, including high cost, low selectivity, poor stability and detrimental environmental effects. Here, we describe a mesoporous carbon foam co-doped with nitrogen and phosphorus that has a large surface area of ˜1,663 m2 g-1 and good electrocatalytic properties for both ORR and OER. This material was fabricated using a scalable, one-step process involving the pyrolysis of a polyaniline aerogel synthesized in the presence of phytic acid. We then tested the suitability of this N,P-doped carbon foam as an air electrode for primary and rechargeable Zn-air batteries. Primary batteries demonstrated an open-circuit potential of 1.48 V, a specific capacity of 735 mAh gZn-1 (corresponding to an energy density of 835 Wh kgZn-1), a peak power density of 55 mW cm-2, and stable operation for 240 h after mechanical recharging. Two-electrode rechargeable batteries could be cycled stably for 180 cycles at 2 mA cm-2. We also examine the activity of our carbon foam for both OER and ORR independently, in a three-electrode configuration, and discuss ways in which the Zn-air battery can be further improved. Finally, our density functional theory calculations reveal that the N,P co-doping and graphene edge effects are essential for the bifunctional electrocatalytic activity of our material.

  1. Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

    Science.gov (United States)

    Féthière, J.; Tsuda, Y.; Coulombe, R.; Konishi, Y.; Cygler, M.

    1996-01-01

    The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes. PMID:8762149

  2. Efficient Access to Chiral Benzhydrols via Asymmetric Transfer Hydrogenation of Unsymmetrical Benzophenones with Bifunctional Oxo-Tethered Ruthenium Catalysts.

    Science.gov (United States)

    Touge, Taichiro; Nara, Hideki; Fujiwhara, Mitsuhiko; Kayaki, Yoshihito; Ikariya, Takao

    2016-08-17

    A concise asymmetric transfer hydrogenation of diaryl ketones, promoted by bifunctional Ru complexes with an etherial linkage between 1,2-diphenylethylenediamine (DPEN) and η(6)-arene ligands, was successfully developed. Because of the effective discrimination of substituents at the ortho position on the aryl group, unsymmetrical benzophenones were smoothly reduced in a 5:2 mixture of formic acid and triethylamine with an unprecedented level of excellent enantioselectivity. For the non-ortho-substituted benzophenones, the oxo-tethered catalyst electronically discerned biased substrates, resulting in attractive performance yielding chiral diarylmethanols with >99% ee. PMID:27463264

  3. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    OpenAIRE

    Larissa Balabanova; Vasily Golotin; Svetlana Kovalchuk; Alexander Bulgakov; Galina Likhatskaya; Oksana Son; Valery Rasskazov

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa...

  4. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    OpenAIRE

    Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel

    2011-01-01

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast t...

  5. Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

    Full Text Available Abstract Background Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively. However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i to assemble and construct zinc finger arrays and nucleases (ZFN with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii to advance a model for pre-clinically testing lentiviral vectors (LV that deliver and transduce either ZFN genotype. Methods and Results First, we computationally generated the consensus sequences of (a 114 dsDNA-binding zinc finger (Zif arrays (ZFAs or ZifHIV-pol and (b two zinc-finger nucleases (ZFNs which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN. Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN. Second, a model for constructing lentiviral vectors (LVs that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively is proposed. Third, two preclinical models for controlled testing of

  6. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    Science.gov (United States)

    Balabanova, Larissa; Golotin, Vasily; Kovalchuk, Svetlana; Bulgakov, Alexander; Likhatskaya, Galina; Son, Oksana; Rasskazov, Valery

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens. PMID:25397876

  7. Preparation of IrO2 nanoparticles with SBA-15 template and its supported Pt nanocomposite as bifunctional oxygen catalyst

    Science.gov (United States)

    Kong, Fan-Dong; Liu, Jing; Ling, Ai-Xia; Xu, Zhi-Qiang; Wang, Hui-Yun; Kong, Qing-Sheng

    2015-12-01

    In the present work, we report the syntheses of IrO2 nanoparticles with SBA-15 template (s-IrO2), and s-IrO2 supported Pt nanocomposite (Pt/s-IrO2) as bifunctional oxygen catalyst. Physical characterizations including X-ray diffraction and transmission electron microscopy demonstrate that s-IrO2 catalyst has excellent uniformity and regularity in particle shape and much ordered distribution in geometric space, and Pt/s-IrO2 catalyst shows a uniform Pt dispersion on the surface of the s-IrO2 particles. Electrochemical analyses prove that s-IrO2 catalyst possesses superior OER activity at operating potentials; and that Pt/s-IrO2 catalyst, in comparison to Pt/commercial IrO2, has higher ESA value and ORR catalytic performance with a mechanism of four-electron pathway and a high ORR efficiency. And as a bifunctional oxygen catalyst, Pt/s-IrO2 also exhibits more remarkable OER performance than the commercial one. The s-IrO2 nanoparticles will be a promising active component (for OER), and suitable for Pt support (for ORR).

  8. Efficient bifunctional catalyst lipase/organophosphonic acid-functionalized silica for biodiesel synthesis by esterification of oleic acid with ethanol.

    Science.gov (United States)

    Yin, Ping; Chen, Wen; Liu, Wei; Chen, Hou; Qu, Rongjun; Liu, Xiguang; Tang, Qinghua; Xu, Qiang

    2013-07-01

    An efficient bifunctional catalyst lipase/organophosphonic acid-functionalized silica (SG-T-P-LS) has been successfully developed, and biodiesel production of fatty acid ethyl ester (FAEE) from free fatty acid (FFA) oleic acid with short-chain alcohol ethanol catalyzed by SG-T-P-LS was investigated. The process optimization using response surface methodology (RSM) was performed and the interactions between the operational variables were elucidated, and it was found that the molar ratio of alcohol to acid was the most significant factor. The optimum values for maximum conversion ratio can be obtained by using a Box-Behnken center-united design, and the conversion ratio could reach 89.94 ± 0.42% under the conditions that ethanol/acid molar ratio was 1.05:1 and SG-T-P-LS to FFA weight ratio was 14.9 wt.% at 28.6°C. The research results show that SG-T-P and LS-20 could work cooperatively to promote the esterification reaction, and the bifunctional catalyst SG-T-P-LS is a potential catalyst for biodiesel production. PMID:23688666

  9. Synthesis and Characterization of Bifunctional Organic-Glasses Based on Diphenylhydrazone and Barbituric Acid Derivative for Photorefractive Application

    Energy Technology Data Exchange (ETDEWEB)

    Park, Ki Hong [KIST, Seoul (Korea, Republic of); Lee, Sang Ho; Choi, Chil Sung; Kim, Nak Joong [Hanyang University, Seoul (Korea, Republic of); Choi, Dong Hoon [Kyunghee University, Youngin (Korea, Republic of)

    2003-12-15

    A series of amorphous molecules that possess both photoconductive and electro-optic properties was synthesized in order to investigate photorefractive properties of bifunctional organic-glasses. Diethylaminobenzaldehyde- diphenylhydrazone was covalently attached to 5-(4-diethylamino-benzylidene)-1,3-dimethylpyrimidine- 2,4,6-trione through a flexible alkyl chain (3, 4, 5, 6 and 10 carbons) containing two ether linkages. The longer linkage not only lowered the glass transition temperature (Tg) of the molecules, but also allowed faster orientation of the chromophore. To examine the photorefractive properties, a 50 μm-thick film was prepared from the mixture of a bifunctional molecule, butyl benzyl phthalate, and C{sup 60}. The photoconductivity of this composite was as high as 8.01 x 10{sup -12} S/cm at 60 V/μm, and the maximum diffraction efficiency (ηmax) of 50 μm-thick film was about 5% at 80 V/μm.

  10. A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

    Directory of Open Access Journals (Sweden)

    Larissa Balabanova

    Full Text Available A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

  11. Staphylococcal Nuclease and Tudor Domain Containing 1 (SND1 Protein) Promotes Hepatocarcinogenesis by Inhibiting Monoglyceride Lipase (MGLL).

    Science.gov (United States)

    Rajasekaran, Devaraja; Jariwala, Nidhi; Mendoza, Rachel G; Robertson, Chadia L; Akiel, Maaged A; Dozmorov, Mikhail; Fisher, Paul B; Sarkar, Devanand

    2016-05-13

    Staphylococcal nuclease and tudor domain containing 1 (SND1) is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC), and functions as an oncogene. This study was carried out to identify novel SND1-interacting proteins to better understand its molecular mechanism of action. SND1-interacting proteins were identified by a modified yeast two-hybrid assay. Protein-protein interaction was confirmed by co-immunoprecipitation analysis. Monoglyceride lipase (MGLL) expression was analyzed by quantitative RT-PCR, Western blot, and immunohistochemistry. MGLL-overexpressing clones were analyzed for cell proliferation and cell cycle analysis and in vivo tumorigenesis in nude mice. MGLL was identified as an SND1-interacting protein. Interaction of SND1 with MGLL resulted in ubiquitination and proteosomal degradation of MGLL. MGLL expression was detected in normal human hepatocytes and mouse liver, although it was undetected in human HCC cell lines. An inverse correlation between SND1 and MGLL levels was identified in a human HCC tissue microarray as well as in the TCGA database. Forced overexpression of MGLL in human HCC cells resulted in marked inhibition in cell proliferation with a significant delay in cell cycle progression and a marked decrease in tumor growth in nude mouse xenograft assays. MGLL overexpression inhibited Akt activation that is independent of enzymatic activity of MGLL and overexpression of a constitutively active Akt rescued cells from inhibition of proliferation and restored normal cell cycle progression. This study unravels a novel mechanism of SND1 function and identifies MGLL as a unique tumor suppressor for HCC. MGLL might function as a homeostatic regulator of Akt restraining its activation. PMID:26997225

  12. Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of Staphylococcal nuclease

    International Nuclear Information System (INIS)

    The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with conservative lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 104 and 105 relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site. Thus, the kinetic properties of R35K and R87K cannot be used to describe the roles of Arg 35 and Arg 87 in catalysis by the wild-type enzyme. Furthermore, on the basis of the effects of these conservative substitutions for Arg 35 and Arg 87. The authors predict that other substitutions for these residues, including neutral substitutions, are also likely to cause conformational alterations in the active site

  13. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

    Science.gov (United States)

    Fichorova, Raina N; Mendonca, Kevin; Yamamoto, Hidemi S; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. PMID:25818602

  14. Electrospinning preparation and properties of magnetic-photoluminescent bifunctional bistrand-aligned composite nanofibers bundles

    International Nuclear Information System (INIS)

    Fe3O4/PVP//Eu(BA)3phen/PVP magnetic-photoluminescent bifunctional bistrand-aligned composite nanofibers bundles based on ferroferric oxide(Fe3O4) nanoparticles and europium complex Eu(BA)3phen (BA = benzoic acid) were fabricated via electrospinning by employing a homemade parallel axial electrospinning setup with the side by side dual spinnerets for the first time. The structures, morphology, and properties of the as-prepared products were investigated in detail by X-ray diffraction, field-emission scanning electron microscopy, energy dispersive spectroscopy, transmission electron microscopy (TEM), a fluorescence spectrometer, and a vibrating sample magnetometer. SEM analysis showed the average diameter of the individual-strand fiber of the bistrand-aligned composite nanofibers bundles was 200 ± 25 nm. TEM image demonstrated that Fe3O4 nanoparticles were only dispersed into one strand of the bistrand-aligned composite nanofibers bundles, and the average diameter of Fe3O4 nanoparticles was about 15 nm. Under the excitation of 274 nm ultraviolet light, Fe3O4/PVP//Eu(BA)3phen/PVP bistrand-aligned composite nanofibers bundles exhibited red emissions of predominant peaks at 592 and 616 nm, which were respectively attributed to the 5D0 → 7F1 and 5D0 → 7F2 transitions of Eu3+ ions. The fluorescence intensity of the bistrand-aligned composite nanofibers bundles was higher than that of Fe3O4/Eu(BA)3phen/PVP composite nanofibers, and was decreased with the increase of the amounts of Fe3O4 nanoparticles. The saturation magnetizations of the bistrand-aligned composite nanofibers bundles and the Fe3O4/Eu(BA)3phen/PVP composite nanofibers were about equal when the two nanostructures contained the same mass ratios of Fe3O4 nanoparticles, but the saturation magnetizations of the bistrand-aligned composite nanofibers bundles were increased with the increase of the amounts of Fe3O4 nanoparticles. The new type Fe3O4/PVP//Eu(BA)3phen/PVP bistrand-aligned composite nanofibers

  15. Robust Control of PEP Formation Rate in the Carbon Fixation Pathway of C4 Plants by a Bi-functional Enzyme

    Directory of Open Access Journals (Sweden)

    Hart Yuval

    2011-10-01

    Full Text Available Abstract Background C4 plants such as corn and sugarcane assimilate atmospheric CO2 into biomass by means of the C4 carbon fixation pathway. We asked how PEP formation rate, a key step in the carbon fixation pathway, might work at a precise rate, regulated by light, despite fluctuations in substrate and enzyme levels constituting and regulating this process. Results We present a putative mechanism for robustness in C4 carbon fixation, involving a key enzyme in the pathway, pyruvate orthophosphate dikinase (PPDK, which is regulated by a bifunctional enzyme, Regulatory Protein (RP. The robust mechanism is based on avidity of the bifunctional enzyme RP to its multimeric substrate PPDK, and on a product-inhibition feedback loop that couples the system output to the activity of the bifunctional regulator. The model provides an explanation for several unusual biochemical characteristics of the system and predicts that the system's output, phosphoenolpyruvate (PEP formation rate, is insensitive to fluctuations in enzyme levels (PPDK and RP, substrate levels (ATP and pyruvate and the catalytic rate of PPDK, while remaining sensitive to the system's input (light levels. Conclusions The presented PPDK mechanism is a new way to achieve robustness using product inhibition as a feedback loop on a bifunctional regulatory enzyme. This mechanism exhibits robustness to protein and metabolite levels as well as to catalytic rate changes. At the same time, the output of the system remains tuned to input levels.

  16. Microwave assisted facile one-pot synthesis of 188Re-complex using a tetrahydroborate exchange resin. A bifunctional chelating agent for radiopharmaceuticals

    International Nuclear Information System (INIS)

    A facile one-pot synthesis of 188Re-complex as a bifunctional chelating agent for the preparation of therapeutic radiopharmaceuticals was accomplished with good labeling yields and radiochemical purity by using a tetrahydroborate exchange resin as a reducing agent for a disulfide ligand as well as the [188Re] perrhenate ion under microwave irradiation. (author)

  17. Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

    DEFF Research Database (Denmark)

    Johansson, Eva; Fanø, Mathias; Bynck, Julie H;

    2005-01-01

    complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely...

  18. Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects

    OpenAIRE

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched doubl...

  19. Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination.

    Science.gov (United States)

    Futai, Kazuki; Sumaoka, Jun; Komiyama, Makoto

    2016-05-01

    By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids. PMID:27057646

  20. Investigation of centers sensitive to S1-nuclease in the genoma of the yeast S. cerevisiae after in-vivo exposure to gamma radiation

    International Nuclear Information System (INIS)

    The structure, distribution and repair of basal damage in DNS after exposure to 60Co gamma radiation were investigated in S. cerevisiae cells. Small DNS regions with mispaired or unpaired bases of rather high stability were found whose rate of incidence and linear dose dependence appear to be similar to those of double strand breaks. In contrast to double strand breaks, they showed no statistical' distribution pattern across the genoma. Liquid holding experiments showed that centers sensitive to S1-nuclease will be repaired in S. cerevisiae by a combined process of recombination and postreplication repair; the gene products of the genes RAD50 and RAD18 are involved. (orig./AJ)

  1. Comparison of Culture and a Novel 5′ Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

    OpenAIRE

    McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J.; Lew, Ala E.

    2006-01-01

    A Campylobacter fetus subsp. venerealis-specific 5′ Taq nuclease PCR assay using a 3′ minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5′ Taq nucleas...

  2. Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Feder Marcin

    2007-07-01

    Full Text Available Abstract Background The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases exhibit a common PD-(D/EXK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI and half-pipe (R.PabI, and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. Results Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. Conclusion Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our

  3. Preparation and characteristics of Fe{sub 3}O{sub 4}-YVO{sub 4}:Eu{sup 3+} bifunctional magnetic-luminescent nanocomposites

    Energy Technology Data Exchange (ETDEWEB)

    Peng Hongxia [School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022 (China); Liu Guixia, E-mail: liuguixia22@yahoo.com.cn [School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022 (China); Dong Xiangting; Wang Jinxian; Xu Jia; Yu Wensheng [School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022 (China)

    2011-06-16

    Graphical abstract: Highlights: > Bifunctional magnetic-luminescent nanocomposites with Fe{sub 3}O{sub 4} nanoparticles as the core and YVO{sub 4}:Eu{sup 3+} as the shell. > A cubic spinel structrue of Fe{sub 3}O{sub 4} core and a tetragonal phase of YVO{sub 4} shell were obtained. > The nanocomposites displayed a strong red emission and superparamagnetic behavior at room temperature. - Abstract: A facile direct precipitation method has been developed for the synthesis of bifunctional magnetic-luminescent nanocomposites with Fe{sub 3}O{sub 4} nanoparticles as the core and YVO{sub 4}:Eu{sup 3+} as the shell. Transmission electron microscopy (TEM) images revealed that the obtained bifunctional nanocomposites had a core-shell structure and a spherical morphology. The average size was {approx}150 nm, and the thickness of the shell was {approx}15 nm. The X-ray diffraction (XRD) patterns showed that a cubic spinel structure of Fe{sub 3}O{sub 4} core and a tetragonal phase of YVO{sub 4} shell were obtained. Fourier transform infrared (FT-IR) spectra confirmed that the YVO{sub 4}:Eu{sup 3+} had been successfully deposited on the surface of Fe{sub 3}O{sub 4} nanoparticles. Photoluminescence (PL) spectra indicated that the nanocomposites displayed a strong red characteristic emission of Eu{sup 3+}. Magnetic measurements showed that the obtained bifunctional nanocomposites exhibited superparamagnetic behavior at room temperature. Therefore, the bifunctional nanocomposites are expected to develop many potential applications in biomedical fields.

  4. Acid/base bifunctional carbonaceous nanomaterial with large surface area: Preparation, characterization, and adsorption properties for cationic and anionic compounds

    Energy Technology Data Exchange (ETDEWEB)

    Li, Kai; Ma, Chun–Fang; Ling, Yuan; Li, Meng [Department of Chemistry, Faculty of Material Science and Chemistry, China University of Geosciences, Wuhan 430074 (China); Gao, Qiang, E-mail: gaoqiang@cug.edu.cn [Department of Chemistry, Faculty of Material Science and Chemistry, China University of Geosciences, Wuhan 430074 (China); Engineering Research Center of Nano-Geo Materials of Ministry of Education, China University of Geosciences, Wuhan 430074 (China); Luo, Wen–Jun, E-mail: heartnohome@yahoo.com.cn [Department of Chemistry, Faculty of Material Science and Chemistry, China University of Geosciences, Wuhan 430074 (China)

    2015-07-15

    Nanostructured carbonaceous materials are extremely important in the nano field, yet developing simple, mild, and “green” methods that can make such materials possess large surface area and rich functional groups on their surfaces still remains a considerable challenge. Herein, a one-pot and environment-friendly method, i.e., thermal treatment (180 °C; 18 h) of water mixed with glucose and chitosan (CTS), has been proposed. The resultant carbonaceous nanomaterials were characterized by field emitting scanning electron microscope, N{sub 2} adsorption/desorption, Fourier transform infrared spectroscope, X-ray photoelectron spectroscopy, and zeta-potential analysis. It was found that, in contrast to the conventional hydrothermally carbonized product from pure glucose, with low surface area (9.3 m{sup 2} g{sup −1}) and pore volume (0.016 cm{sup 3} g{sup −1}), the CTS-added carbonaceous products showed satisfactory textural parameters (surface area and pore volume up to 254 m{sup 2} g{sup −1} and 0.701 cm{sup 3} g{sup −1}, respectively). Moreover, it was also interestingly found that these CTS-added carbonaceous products possessed both acidic (–COOH) and basic (–NH{sub 2}) groups on their surfaces. Taking the advantages of large surface area and –COOH/–NH{sub 2} bifunctional surface, the carbonaceous nanomaterials exhibited excellent performance for adsorptions of cationic compound (i.e., methylene blue) at pH 10 and anionic compound (i.e., acid red 18) at pH 2, respectively. This work not only provides a simple and green route to prepare acid/base bifunctional carbonaceous nanomaterials with large surface area but also well demonstrates their potential for application in adsorption. - Highlights: • A simple and green method was proposed to prepare carbon nanomaterials. • The carbon product showed acid/base bifunctional surface with large surface area. • The carbon material could efficiently adsorb both cationic and anionic compounds.

  5. Synthesis and evaluation of a new bifunctional NETA chelate for molecular targeted radiotherapy using90Y or177Lu

    International Nuclear Information System (INIS)

    Introduction: Therapeutic potential of β-emitting cytotoxic radionuclides 90Y and 177Lu has been demonstrated in numerous preclinical and clinical trials. A bifunctional chelate that can effectively complex with the radioisotopes is a critical component for molecular targeted radiotherapy 90Y and 177Lu. A new bifunctional chelate 5p-C-NETA with a relatively long alkyl spacer between the chelating backbone and the functional unit for conjugation to a tumor targeting moiety was synthesized. 5p-C-NETA was conjugated to a model targeting moiety, a cyclic Arg-Gly-Asp-D-Tyr-Lys (RGDyK) peptide binding integrin αvβ3 protein overexpressed on various cancers. 5p-C-NETA was conjugated to c(RGDyK) peptide and evaluated for potential use in molecular targeted radiotherapy of 90Y and 177Lu. Methods: 5p-C-NETA conjugated with c(RGDyK) was evaluated in vitro for radiolabeling, serum stability, binding affinity, and the result of the in vitro studies of 5p-C-NETA-c(RGDyK) was compared to that of 3p-C-NETA-c(RGDyK). 177Lu-5p-C-NETA-c(RGDyK) was further evaluated for in vivo biodistribution using gliobastoma bearing mice. Result: The new chelate rapidly and tightly bound to a cytotoxic radioisotope for cancer therapy, 90Y or 177Lu with excellent radiolabeling efficiency and maximum specific activity under mild condition (> 99%, RT, < 1 min). 90Y- and 177Lu-radiolabeled complexes of the new chelator remained stable in human serum without any loss of the radiolanthanide for 14 days. Introduction of the tumor targeting RGD moiety to the new chelator made little impact on complexation kinetics and stability with 90Y or 177Lu. 177Lu-radiolabeled 5p-C-NETA-c(RGDyK) conjugate was shown to target tumors in mice and produced a favorable in vivo stability profile. Conclusion: The results of in vitro and in vivo evaluation suggest that 5p-C-NETA is an effective bifunctional chelate of 90Y and 177Lu that can be applied for generation of versatile molecular targeted radiopharmaceuticals

  6. Mislocalization of XPF-ERCC1 nuclease contributes to reduced DNA repair in XP-F patients.

    Directory of Open Access Journals (Sweden)

    Anwaar Ahmad

    2010-03-01

    Full Text Available Xeroderma pigmentosum (XP is caused by defects in the nucleotide excision repair (NER pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF(R153P were compared to an XP-causing mutation (XPF(R799W in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF(R153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF(R153P-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV-induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially

  7. Influence of some exo nucleases in response to the induced genetic damage in Escherichia coli by alpha radiation

    International Nuclear Information System (INIS)

    Within the strategies with those that E. coli counts to overcome to the genetic damage there is the SOS response, a group of genes that participate in repair and/or tolerance that it confers to the bacteria major opportunities of surviving. These genes are repressed and its only are expressed when it happens genetic damage. So that this system is activated it is necessary that DNA of a band exists and in this sense the double ruptures (RDB) its are not able to induce this response unless there is a previous processing. In stumps with defects in certain genes that have to do with repair of RDB (as recO, recJ and xonA) the activity of SOS is smaller than in a wild stump what suggests that these participate in the previous processes to the activation of the response. The ionizing radiation produce among other many lesions, RDB in greater or smaller proportion, depending on the ionization capacity. A parameter to evaluate this capacity is the lineal energy transfer (LET), defined as the average energy given by unit of distance travelled. In general the LET of the corpuscular radiations is a lot but high that of the electromagnetic one, for what produces bigger quantity of ionizations inside a restricted zone and it increases by this way the probability that RDB has been generated. This work has for object to infer the participation of xonA and recJ in this response and to evaluate the damage produced by ionizing radiation of different LET (alpha particles of different energies) in a stump with all the functional repair mechanisms. Its were considered two parameters: the survival and the activity of SOS evaluated by means of the chromo test. The results indicate that the activity of these exo nucleases is necessary for the repair of RDB as well as for the processing of lesions foresaw to the activation of SOS. As for the treatment with alphas of different energies is observed that so much the survival like the activity of SOS vary as the LET of the radiation changes

  8. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

    Science.gov (United States)

    Wang, Jianbin; DeClercq, Joshua J; Hayward, Samuel B; Li, Patrick Wai-Lun; Shivak, David A; Gregory, Philip D; Lee, Gary; Holmes, Michael C

    2016-02-18

    The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy. PMID:26527725

  9. Differential reaction kinetics, cleavage complex formation, and nonamer binding domain dependence dictate the structure-specific and sequence-specific nuclease activity of RAGs.

    Science.gov (United States)

    Naik, Abani Kanta; Raghavan, Sathees C

    2012-01-20

    During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease. PMID:22119487

  10. Fe3O4@Ag magnetic nanoparticles for microRNA capture and duplex-specific nuclease signal amplification based SERS detection in cancer cells.

    Science.gov (United States)

    Pang, Yuanfeng; Wang, Chongwen; Wang, Jing; Sun, Zhiwei; Xiao, Rui; Wang, Shengqi

    2016-05-15

    A functionalized Fe3O4@Ag magnetic nanoparticle (NP) biosensor for microRNA (miRNA) capture and ultrasensitive detection in total RNA extract from cancer cells was reported in this paper. Herein, Raman tags-DNA probes modified Fe3O4@Ag NPs were designed both as surface-enhanced Raman scattering (SERS) SERS and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, target miRNAs were captured to the surface of Fe3O4@Ag NPs through DNA/RNA hybridization. In the presence of endonuclease duplex specific nuclease (DSN), one target miRNA molecule could rehybrid thousands of DNA probes to trigger the signal-amplifying recycling. Base on the superparamagnetic of Fe3O4@Ag NPs, target miRNA let-7b can be captured, concentrated and direct quantified within a PE tube without any PCR preamplification treatment. The detection limit was 0.3fM (15 zeptomole, 50μL), nearly 3 orders of magnitude lower than conventional fluorescence based DSN biosensors for miRNA(∼100fM), even single-base difference between the let-7 family members can be discriminated. The result provides a novel proposal to combine the perfect single-base recognition and signal-amplifying ability of the endonuclease DSN with cost-effective SERS strategy for miRNA point-of-care (POC) clinical diagnostics. PMID:26749099

  11. Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and neurokinin A-encoding messenger ribonucleic acids

    International Nuclear Information System (INIS)

    In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay

  12. The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

    Directory of Open Access Journals (Sweden)

    Sandra Ebert

    2010-04-01

    Full Text Available The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochon­drial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

  13. Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

    Directory of Open Access Journals (Sweden)

    Nico Rublack

    2014-08-01

    Full Text Available Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated.

  14. Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

    DEFF Research Database (Denmark)

    Yao, Shuo; Just Mikkelsen, Marie

    2010-01-01

    ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major......Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (Adh......B), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce...

  15. The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

    Directory of Open Access Journals (Sweden)

    Tiedtke Arno

    2006-03-01

    Full Text Available Abstract Background Dihydrofolate reductase (DHFR and thymidylate synthase (TS are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. Results Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. Conclusion This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications.

  16. The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation.

    Directory of Open Access Journals (Sweden)

    Sarah B Guttenplan

    Full Text Available Many bacteria inhibit motility concomitant with the synthesis of an extracellular polysaccharide matrix and the formation of biofilm aggregates. In Bacillus subtilis biofilms, motility is inhibited by EpsE, which acts as a clutch on the flagella rotor to inhibit motility, and which is encoded within the 15 gene eps operon required for EPS production. EpsE shows sequence similarity to the glycosyltransferase family of enzymes, and we demonstrate that the conserved active site motif is required for EPS biosynthesis. We also screen for residues specifically required for either clutch or enzymatic activity and demonstrate that the two functions are genetically separable. Finally, we show that, whereas EPS synthesis activity is dominant for biofilm formation, both functions of EpsE synergize to stabilize cell aggregates and relieve selective pressure to abolish motility by genetic mutation. Thus, the transition from motility to biofilm formation may be governed by a single bifunctional enzyme.

  17. Nonlinearity of the Bifunctional of the Nonadditive Kinetic Energy: Numerical Consequences in Orbital-Free Embedding Calculations

    OpenAIRE

    Dulak, Marcin; Wesolowski, Tomasz Adam

    2006-01-01

    The bifunctional of the nonadditive kinetic energy in the reference system of noninteracting electrons ( [ρA, ρB] = Ts[ρA + ρB] − Ts[ρA] − Ts[ρB]) is the key quantity in orbital-free embedding calculations because they hinge on approximations to [ρA,ρB]. Since [ρA,ρB] is not linear in ρA, the associated potential (functional derivative) [ρ,ρB]/δρ|ρ=ρA(r) changes if ρA varies. In this work, for two approximations to [ρA,ρB], which are nonlinear in ρA (gradient-free and gradient-dependent), the...

  18. RNA Secondary Structure Modulates FMRP’s Bi-Functional Role in the MicroRNA Pathway

    Directory of Open Access Journals (Sweden)

    Phillip Kenny

    2016-06-01

    Full Text Available MicroRNAs act by post-transcriptionally regulating the gene expression of 30%–60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP’s interaction with the RNA helicase Moloney leukemia virus 10 (MOV10, which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex.

  19. N-Terminal Modification with Pseudo-Bifunctional PEG-Hexadecane Markedly Improves the Pharmacological Profile of Human Growth Hormone.

    Science.gov (United States)

    Wu, Ling; Ji, Shaoyang; Hu, Tao

    2015-05-01

    Human growth hormone (hGH) has been used to treat children with short stature, renal failure, and Turner's syndrome. However, clinical application of hGH suffers from its short plasma half-life and low bioavailability. PEGylation and albumin binding are two of the most effective approaches to prolong the plasma half-life of hGH. However, the steric shielding effects of polyethylene glycol (PEG) and albumin can drastically decrease the bioactivity of hGH, which is opposite to the increased pharmacokinetics (PK). In the present study, a long-acting hGH with markedly improved pharmacological profile was rationally designed and prepared by N-terminal modification of hGH with pseudo-bifunctional PEG-hexadecane by using PEG (3.5 kDa or 10 kDa) as the linker. PEGylation and albumin binding with hexadecane can increase the hydrodynamic volume and decrease the immunogenicity of hGH, which thereby markedly increases the PK of hGH. Since N-terminus is far from the bioactive domain of hGH, N-terminal modification of hGH can minimize the steric shielding effects on the bioactive domain of hGH. Hexadecane-bound albumin can be slowly released from hGH during the in vivo circulation, which can slowly restore the bioactivity of hGH. Thus, the high bioactivity of PEG-hexadecane modified hGH (hGH-PEG-HD) was synergistically achieved by N-terminal modification with pseudo-bifunctional PEG-hexadecane and slow-release of albumin. The high pharmacodynamics (PD) of hGH-PEG-HD was due to the synergistic effect of the high bioactivity and the overall increased PK. PMID:25849255

  20. Promising bifunctional chelators for copper 64-PET imaging: practical (64)Cu radiolabeling and high in vitro and in vivo complex stability.

    Science.gov (United States)

    Wu, Ningjie; Kang, Chi Soo; Sin, Inseok; Ren, Siyuan; Liu, Dijie; Ruthengael, Varyanna C; Lewis, Michael R; Chong, Hyun-Soon

    2016-04-01

    Positron emission tomography (PET) using copper-64 is a sensitive and non-invasive imaging technique for diagnosis and staging of cancer. A bifunctional chelator that can present rapid radiolabeling kinetics and high complex stability with (64)Cu is a critical component for targeted PET imaging. Bifunctional chelates 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA were evaluated for complexation kinetics and stability with (64)Cu in vitro and in vivo. Hexadentate 3p-C-NOTA and heptadentate 3p-C-NE3TA possess a smaller TACN-based macrocyclic backbone, while nonadentate 3p-C-DE4TA is constructed on a larger CYCLEN-based ring. The frequently explored chelates of (64)Cu, octadentate C-DOTA and hexadentate C-NOTA were also comparatively evaluated. Radiolabeling kinetics of bifunctional chelators with (64)Cu was assessed under mild conditions. All bifunctional chelates instantly bound to (64)Cu in excellent radiolabeling efficiency at room temperature. C-DOTA was less efficient in binding (64)Cu than all other chelates. All (64)Cu-radiolabeled bifunctional chelates remained stable in human serum without any loss of (64)Cu for 2 days. When challenged by an excess amount of EDTA, (64)Cu complexes of C-NOTA, 3p-C-NE3TA and 3p-C-NOTA were shown to be more stable than (64)Cu-C-DOTA and (64)Cu-3p-C-DE4TA. (64)Cu complexes of the new chelates 3p-C-NE3TA and 3p-C-NOTA displayed comparable in vitro and in vivo complex stability to (64)Cu-C-NOTA. In vivo biodistribution result indicates that the (64)Cu-radiolabeled complexes of 3p-C-NOTA and 3p-C-NE3TA possess excellent in vivo complex stability, while (64)Cu-3p-C-DE4TA was dissociated as evidenced by high renal and liver retention in mice. The results of in vitro and in vivo studies suggest that the bifunctional chelates 3p-C-NE3TA and 3p-C-NOTA offer excellent chelation chemistry with (64)Cu for potential PET imaging applications. PMID:26666778

  1. One-Dimensional RuO2/Mn2O3 Hollow Architectures as Efficient Bifunctional Catalysts for Lithium-Oxygen Batteries.

    Science.gov (United States)

    Yoon, Ki Ro; Lee, Gil Yong; Jung, Ji-Won; Kim, Nam-Hoon; Kim, Sang Ouk; Kim, Il-Doo

    2016-03-01

    Rational design and massive production of bifunctional catalysts with fast oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) kinetics are critical to the realization of highly efficient lithium-oxygen (Li-O2) batteries. Here, we first exploit two types of double-walled RuO2 and Mn2O3 composite fibers, i.e., (i) phase separated RuO2/Mn2O3 fiber-in-tube (RM-FIT) and (ii) multicomposite RuO2/Mn2O3 tube-in-tube (RM-TIT), by controlling ramping rate during electrospinning process. Both RM-FIT and RM-TIT exhibited excellent bifunctional electrocatalytic activities in alkaline media. The air electrodes using RM-FIT and RM-TIT showed enhanced overpotential characteristics and stable cyclability over 100 cycles in the Li-O2 cells, demonstrating high potential as efficient OER and ORR catalysts. PMID:26821307

  2. Study of application properties of novel trisazo hetero bi-functional reactive dyes based on j-acid derivatives for cotton

    International Nuclear Information System (INIS)

    Three novel trisazo hetero bi-functional reactive dyes based on J-acid derivatives were prepared using the diazonium salt of [4-(4-sulphophenylazo-)-2,5-dimethylazobenzene-2-sulphonic acid] and a hetero bi-functional coupling component, derived from 1-hydroxy-6-aminonapthalene-3-sulphonic acid (J-acid), 1-hydroxy-6- methylaminonapthalene-3-sulphonic acid (methyl J-acid), and 1-hydroxy-6-aminonaphthalene-3,5-disulphonic acid (sulpho J-acid). On balance, the dye derived from sulpho J-acid displayed the most attractive set of technical properties, building up and fixing more efficiently than those derived from J-acid and methyl J-acid. In addition, the sulpho J-acid based dye offered better migration and, therefore, level dyeing and ease of wash off. (author)

  3. A bifunctional chromogenic and fluorogenic probe for F{sup −} and Al{sup 3+} based on azo-benzimidazole conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Iniya, Murugan; Jeyanthi, Dharmaraj; Krishnaveni, Karuppiah; Chellappa, Duraisamy, E-mail: dcmku123@gmail.com

    2015-01-15

    A versatile bifunctional probe has been reported for selective individual detection of Al{sup 3+} and F{sup −} through “turn-on” chromogenic and fluorogenic dual modes. The photonic behaviors of probe upon addition of analyte have been determined using UV–vis absorption, fluorescence emission, quantum yield and fluorescence life-time measurement. Density functional theory calculations have been performed to establish the nature of interaction between probe and Al{sup 3+}/F{sup −}. - Highlights: • First report on azo based bifunctional chromogenic and fluorogenic chemosensor for Al{sup 3+} and F{sup −} with varied responses. • The probe displays simple method of synthesis, reversibility towards Al{sup 3+} and high stability. • TD–DFT calculations were performed to characterize the nature of fluorescent behavior of probe upon addition of Al{sup 3+} and F{sup −}.

  4. The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

    Science.gov (United States)

    Ohtsubo, K; Richardson, M

    1992-08-31

    A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds. PMID:1511747

  5. Coincident resection at both ends of random, γ-induced double-strand breaks requires MRX (MRN, Sae2 (Ctp1, and Mre11-nuclease.

    Directory of Open Access Journals (Sweden)

    James W Westmoreland

    2013-03-01

    Full Text Available Resection is an early step in homology-directed recombinational repair (HDRR of DNA double-strand breaks (DSBs. Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced "dirty" DSBs or even enzyme-induced "clean" DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility ("PFGE-shift". Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at "dirty" and clean DSB ends. These approaches apply to resection at

  6. Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard

    International Nuclear Information System (INIS)

    Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal–epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure. - Highlights: • Bifunctional anti-inflammatory prodrug (NDH4338) tested on SM exposed mouse skin • The prodrug NDH4338 was designed to target COX2 and acetylcholinesterase. • The application of NDH4338 improved cutaneous wound repair after SM induced injury. • NDH4338 treatment demonstrated a reduction in COX2 expression on SM injured skin. • Changes of skin repair

  7. Catalytic Asymmetric Synthesis of Both Enantiomers of 4‑Substituted 1,4-Dihydropyridines with the Use of Bifunctional Thiourea-Ammonium Salts Bearing Different Counterions

    Directory of Open Access Journals (Sweden)

    Kohzo Yoshida

    2010-11-01

    Full Text Available Organoammonium salts composed of a Brønsted acid and an anilinothiourea promoted the Michael addition of β-keto esters and α,β-unsaturated aldehydes in the presence of primary amines to give functionalized 1,4-dihydropyridines enantioselectively. With the use of the different Brønsted acids such as DFA and HBF4 with the same bifunctional thiourea, both enantiomers of 4-substituted 1,4-dihydropyridine were synthesized from the same starting materials.

  8. In-situ X-Ray Absorption Spectroscopy (XAS) Investigation of a Bifunctional Manganese Oxide Catalyst with High Activity for Electrochemical Water Oxidation and Oxygen Reduction

    OpenAIRE

    Gorlin, Yelena; Lassalle-Kaiser, Benedikt; Benck, Jesse D.; Gul, Sheraz; Webb, Samuel M; Yachandra, Vittal K.; Yano, Junko; Jaramillo, Thomas F.

    2013-01-01

    In-situ x-ray absorption spectroscopy (XAS) is a powerful technique that can be applied to electrochemical systems, with the ability to elucidate the chemical nature of electrocatalysts under reaction conditions. In this study, we perform in-situ XAS measurements on a bifunctional manganese oxide (MnOx) catalyst with high electrochemical activity for the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER). Using x-ray absorption near edge structure (XANES) and extended x-r...

  9. Identification of catalytic sites for oxygen reduction and oxygen evolution in N-doped graphene materials: Development of highly efficient metal-free bifunctional electrocatalyst

    OpenAIRE

    Yang, Hong Bin; Miao, Jianwei; Hung, Sung-Fu; Chen, Jiazang; Tao, Hua Bing; Wang, Xizu; Zhang, Liping; Chen, Rong; Gao, Jiajian; Chen, Hao Ming; Dai, Liming; Liu, Bin

    2016-01-01

    Oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) are critical to renewable energy conversion and storage technologies. Heteroatom-doped carbon nanomaterials have been reported to be efficient metal-free electrocatalysts for ORR in fuel cells for energy conversion, as well as ORR and OER in metal-air batteries for energy storage. We reported that metal-free three-dimensional (3D) graphene nanoribbon networks (N-GRW) doped with nitrogen exhibited superb bifunctional electroca...

  10. Synthesis, metal complexation and biological evaluation of a novel semi-rigid bifunctional chelating agent for 99mTc labelling

    OpenAIRE

    Le Gal, Julien; Michaud, Sandra; Gressier, Marie; Coulais, Yvon; Benoist, Eric

    2006-01-01

    A novel bifunctional chelating agent bearing an aromatic ring has been synthesised and characterised. This ligand formed well-defined oxorhenium complexes. The analogous 99mTcO-complex was obtained in an excellent yield with high radiochemical purity (>95%). The biodistribution of the 99mTo-complex after intravenous injection studied in normal rats showed that the activity was excreted mainly via renal-urinary pathway indicating its use for labelling peptides with 99mTc.

  11. Discovery of Bifunctional Oncogenic Target Inhibitors against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K).

    Science.gov (United States)

    Van Dort, Marcian E; Hong, Hao; Wang, Hanxiao; Nino, Charles A; Lombardi, Rachel L; Blanks, Avery E; Galbán, Stefanie; Ross, Brian D

    2016-03-24

    The synthesis of a series of single entity, bifunctional MEK1/PI3K inhibitors achieved by covalent linking of structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive MEK inhibitor PD0325901 is described. Inhibitors displayed potent in vitro inhibition of MEK1 (0.015 strategy toward combined MEK1/PI3K inhibition. PMID:26943489

  12. All-optical OR/NOR Bi-functional logic gate by using cross-gain modulation in semiconductor optical amplifiers

    International Nuclear Information System (INIS)

    An OR/NOR bi-functional all-optical logic gate has been experimentally demonstrated at 10 Gbit/s by using cross-gain modulation (XGM) in only 2 semiconductor optical amplifiers (SOAs). One SOA was used for NOR operation and the other SOA was used for inversion to obtain OR operation. Numerical simulation has also been performed, which coincided well with the experimental results.

  13. Organocatalytic asymmetric Henry reaction of 1H-pyrrole-2,3-diones with bifunctional amine-thiourea catalysts bearing multiple hydrogen-bond donors.

    Science.gov (United States)

    Zhang, Ming-Liang; Yue, Deng-Feng; Wang, Zhen-Hua; Luo, Yuan; Xu, Xiao-Ying; Zhang, Xiao-Mei; Yuan, Wei-Cheng

    2016-01-01

    For the first time, a catalytic asymmetric Henry reaction of 1H-pyrrole-2,3-diones was achieved with a chiral bifunctional amine-thiourea as a catalyst possessing multiple hydrogen-bond donors. With this developed method, a range of 3-hydroxy-3-nitromethyl-1H-pyrrol-2(3H)-ones bearing quaternary stereocenters were obtained in acceptable yield (up to 75%) and enantioselectivity (up to 73% ee). PMID:26977188

  14. Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Yoke-Chen; Wang, James D.; Hahn, Rita A.; Gordon, Marion K.; Joseph, Laurie B. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Science, New York Medical College, Valhalla, NY (United States); Heindel, Ned D. [Department of Chemistry, Lehigh University, Bethlehem, PA (United States); Young, Sherri C. [Department of Chemistry, Muhlenberg College, Allentown, PA (United States); Sinko, Patrick J. [Department of Pharmaceutics, Rutgers University, Piscataway, NJ (United States); Casillas, Robert P. [MRIGlobal, Kansas City, MO (United States); Laskin, Jeffrey D. [Environmental and Occupational Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Gerecke, Donald R., E-mail: gerecke@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States)

    2014-10-15

    Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal–epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure. - Highlights: • Bifunctional anti-inflammatory prodrug (NDH4338) tested on SM exposed mouse skin • The prodrug NDH4338 was designed to target COX2 and acetylcholinesterase. • The application of NDH4338 improved cutaneous wound repair after SM induced injury. • NDH4338 treatment demonstrated a reduction in COX2 expression on SM injured skin. • Changes of skin repair

  15. Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

    OpenAIRE

    Osman, Erkan Y; Yen, Pei-Fen; Lorson, Christian L.

    2011-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifi...

  16. Self-assembly of cobalt-centered metal organic framework and multiwalled carbon nanotubes hybrids as a highly active and corrosion-resistant bifunctional oxygen catalyst

    Science.gov (United States)

    Fang, Yiyun; Li, Xinzhe; Li, Feng; Lin, Xiaoqing; Tian, Min; Long, Xuefeng; An, Xingcai; Fu, Yan; Jin, Jun; Ma, Jiantai

    2016-09-01

    Metal organic frameworks (MOF) derived carbonaceous materials have emerged as promising bifunctional oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) catalysts for electrochemical energy conversion and storage. But previous attempts to overcome the poor electrical conductivity of MOFs hybrids involve a harsh high-template pyrolytic process to in situ form carbon, which suffer from extremely complex operation and inevitable carbon corrosion at high positive potentials when OER is operated. Herein, a self-assembly approach is presented to synthesize a non-precious metal-based, high active and strong durable Co-MOF@CNTs bifunctional catalyst for OER and ORR. CNTs not only improve the transportation of the electrons but also can sustain the harsh oxidative environment of OER without carbon corrosion. Meanwhile, the unique 3D hierarchical structure offers a large surface area and stable anchoring sites for active centers and CNTs, which enables the superior durability of hybrid. Moreover, a synergistic catalysis of Co(II), organic ligands and CNTs will enhance the bifunctional electrocatalytic performance. Impressively, the hybrid exhibits comparable OER and ORR catalytic activity to RuO2 and 20 wt% Pt/C catalysts and superior stability. This facile and versatile strategy to fabricating MOF-based hybrids may be extended to other electrode materials for fuel cell and water splitting applications.

  17. A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids.

    Science.gov (United States)

    Sayanova, Olga; Haslam, Richard; Guschina, Irina; Lloyd, David; Christie, William W; Harwood, John L; Napier, Johnathan A

    2006-12-01

    The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist. PMID:16950768

  18. An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA.

    Science.gov (United States)

    Zhang, Jing; Wu, Dong-Zhi; Cai, Shu-Xian; Chen, Mei; Xia, Yao-Kun; Wu, Fang; Chen, Jing-Hua

    2016-01-15

    An immobilization-free electrochemical impedance biosensor for microRNA detection was developed in this work, which was based on both the duplex-specific nuclease assisted target recycling (DSNATR) and capture probes (Cps) enriched from the solution to electrode surface via magnetic beads (MBs). In the absence of miR-21, Cps cannot be hydrolyzed due to the low activity of duplex-specific nuclease (DSN) against ssDNA. Therefore, the intact Cps could be attached to the surface of magnetic glass carbon electrode (MGCE), resulting in a compact negatively charged layer as well as a large charge-transfer resistance. While in the presence of miR-21, it hybridized with Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzed the target-binding part of the Cp while liberating the intact miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. In this way, a single miR-21 was able to trigger the permanent hydrolysis of multiple Cps. Finally, all Cps were digested. Thus, the negatively charged layer could not be formed, resulting in a small charge-transfer resistance. By employing the above strategy, the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with a detection limit of 60aM. Meanwhile, the method showed little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human serum samples of breast cancer patients. PMID:26363493

  19. Exfoliated Pd/HNb3O8 nanosheet as highly efficient bifunctional catalyst for one-pot cascade reaction

    Science.gov (United States)

    Lee, Nahaeng; Chung, Young-Min

    2016-05-01

    Ultrathin two-dimensional metal oxide nanosheets have drawn attention as potential solid acid catalysts owing to their strong acidity, attributed to the bridged OH groups formed on the nanosheets. In this study, a new class of bifunctional acid-metal catalyst was realized by the deposition of Pd on layered niobium oxide (KNb3O8 and HNb3O8) or its exfoliated nanosheet (Pd/HNb3O8-NS) and applied to one-pot cascade deacetalization and hydrogenation. It was found that the acid strength of the support exerted a large influence not only on the promotion of the first deacetalization step, but also on the acceleration of the subsequent hydrogenation step. Comparative experiments using a series of Pd/HZSM-5 catalysts with different acidities reconfirmed the crucial role of acid strength on hydrogenation. However, the superior catalytic activity of Pd/HNb3O8-NS for hydrogenation compared to that of Pd/HZSM-5 of similar acidity suggests a more efficient ensemble effect of the strong acid sites with the nearby metal sites on the nanosheet surface. Among the catalysts used, Pd/HNb3O8-NS showed the best catalytic performance for one-pot cascade reaction affording the desired product (benzyl alcohol) in approximately 92% yield, which was 7.1 and 1.2 times higher than that of layered Pd/KNb3O8 or Pd/HNb3O8, respectively. The excellent catalytic performance of Pd/HNb3O8-NS may result from the characteristic features of nanosheets: (i) the synergistic cooperation between the bifunctional active sites and (ii) the two-dimensional open surface offering easier access of the reactants to the active sites. Although the use of NaBH4 as hydrogen source was effective in improving the initial reaction performance, the basic nature of NaBH4 adversely resulted in weakening the acid strength of the catalyst, and consequently led to a reduction in catalytic activity.

  20. Imaging cancer using PET - the effect of the bifunctional chelator on the biodistribution of a 64Cu-labeled antibody

    International Nuclear Information System (INIS)

    Introduction: Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the 64Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with 64Cu using these chelators in tumor-bearing mice. Methods: The chelators [S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA): 6-[p-(bromoacetamido)benzyl]-1, 4, 8, 11-tetraazacyclotetradecane-N, N', N'', N'''-tetraacetic acid (BAT-6): S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododocane tetraacetic acid (p-NH2-Bn-DOTA): 1,4,7,10-tetraazacyclododocane-N, N', N', N''-tetraacetic acid (DOTA): and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1, 8-diamine (SarAr)] were conjugated to the anti-GD2 antibody ch14.18, and the modified antibody was labeled with 64Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from positron emission tomography images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. Results: The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p 64Cu]ch14.18-p-NH2-Bn-NOTA was 4.74 ± 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [64Cu]ch14.18-SarAr was 8.06 ± 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. Conclusions: The results of this study indicate that differences in the thermodynamic stability of these chelator-Cu(II) complexes were not associated with significant differences in uptake of the tracer by the tumor

  1. Spherical nitrogen-doped hollow mesoporous carbon as an efficient bifunctional electrocatalyst for Zn-air batteries

    Science.gov (United States)

    Hadidi, Lida; Davari, Elaheh; Iqbal, Muhammad; Purkait, Tapas K.; Ivey, Douglas G.; Veinot, Jonathan G. C.

    2015-12-01

    Materials based upon porous carbon have gained considerable attention due to their high surface area, electric conductivity, thermal and chemical stability, low density, and availability. These superior properties make them ideal for diverse applications. Doping these carbon nanostructures holds promise of designing the properties of these structures and opening the door to practical applications. Herein, we report the preparation of hollow N-doped mesoporous carbon (HMC) spheres fabricated via polymerization and carbonization of dopamine on a sacrificial spherical SiO2 template that is removed upon hydrofluoric acid etching. The morphology and structural features of these HMCs were evaluated using scanning electron microscopy and transmission electron microscopy and the N-doping (7.1 at%) was confirmed by X-ray photoelectron spectroscopy (XPS). The oxygen reduction/evolution reaction (ORR/OER) performance of N-doped HMC was evaluated using rotating disk electrode (RDE) voltammetry in an alkaline electrolyte. N-doped HMC demonstrated a high ORR onset potential of -0.055 V (vs. Hg/HgO) and excellent stability. The outstanding bifunctional activity was implemented in a practical Zn-air battery (ZAB), which exhibited a small charge-discharge voltage polarization of 0.89 V and high stability over repeated cycling.Materials based upon porous carbon have gained considerable attention due to their high surface area, electric conductivity, thermal and chemical stability, low density, and availability. These superior properties make them ideal for diverse applications. Doping these carbon nanostructures holds promise of designing the properties of these structures and opening the door to practical applications. Herein, we report the preparation of hollow N-doped mesoporous carbon (HMC) spheres fabricated via polymerization and carbonization of dopamine on a sacrificial spherical SiO2 template that is removed upon hydrofluoric acid etching. The morphology and structural

  2. Y2O3: Eu3+, Tb3+ spherical particles based anti-reflection and wavelength conversion bi-functional films: Synthesis and application to solar cells

    International Nuclear Information System (INIS)

    Highlights: • Eu3+ and Tb3+ co-doped Y2O3 particles were successfully prepared. The as prepared particles can convert UV region photos to visible photons between 460 nm and 640 nm, which just matched the spectral response of most solar cells. • Y2O3 is not only a good photoluminescence host material, but also it has high corrosion resistivity, thermal stability, and transparency from violet to infrared light. Cooperated with SiO2 sols, it could realize a better anti-reflection property. • As a proof-of-concept application, the as prepared bi-functional films could effectively improve the photoelectric conversion efficiency by 0.23% compared to pure SiO2 AR coating film and 0.55% compared to glass. - Abstract: In this study, Eu3+ and Tb3+ co-doped Y2O3 particles were prepared via the simple, cost-effective urea homogeneous precipitation method without additives. The chosen particles were added in the SiO2 sols to get anti-reflection (AR) and wavelength conversion bi-functional films. Careful investigations were carried out to find the optimum preparation conditions and proper morphology. SEM images showed that the particle sizes reduced as metal ion/urea ratio decreased. Additionally, the extracted particles turned from sphere to lamellar type when the deionized water, which was used as solvent, reduced to a certain extent. The mechanisms of the morphology formation and diversification were proposed as well. The as prepared materials can convert UV region photos to visible photons between 460 nm and 640 nm, which just matched the spectral response of most solar cells. The spherical sample showed better luminescence performance than the one with lamellar morphology. In addition, the optical transmittance spectra indicated that the films adding spherical particles had better anti-reflective performance, and the best adding amount was 0.08 g. Finally, As a proof-of-concept application, the as prepared bi-functional films were used to test the standard

  3. Plasmodium AdoMetDC/ODC bifunctional enzyme is essential for male sexual stage development and mosquito transmission.

    Science.gov (United States)

    Hart, Robert J; Ghaffar, Atif; Abdalal, Shaymaa; Perrin, Benjamin; Aly, Ahmed S I

    2016-01-01

    Polyamines are positively-charged organic molecules that are important for cellular growth and division. Polyamines and their synthesizing enzymes are particularly abundant in rapidly proliferating eukaryotic cells such as parasitic protozoa and cancer cells. Polyamine biosynthesis inhibitors, such as Elfornithine, are now being considered for cancer prevention and have been used effectively against Trypanosoma brucei Inhibitors of polyamine biosynthesis have caused growth arrest of Plasmodium falciparum blood stages in vitro, but in P. berghei only partial inhibition has been observed. While polyamine biosynthesis enzymes are characterized and conserved in Plasmodium spp., little is known on the biological roles of these enzymes inside malaria parasite hosts. The bifunctional polyamine biosynthesis enzyme S-adenosyl methionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) was targeted for deletion in P. yoelii Deletion of AdoMetDC/ODC significantly reduced blood stage parasitemia but Anopheles transmission was completely blocked. We showed that male gametocytogenesis and male gamete exflagellation were abolished and consequently no ookinetes or oocyst sporozoites could be generated from adometdc/odc(-) parasites. Supplementation of putrescine and spermidine did not rescue the defective phenotypes of male gametocytes and gametes of the knockout parasites. These results highlight the crucial role of polyamine homeostasis in the development and functions of Plasmodium erythrocytic stages in the blood and in the mosquito vector and validate polyamine biosynthesis pathway enzymes as drug targeting candidates for malaria parasite transmission blocking. PMID:27387533

  4. The synthesis and properties of bifunctional and intelligent Fe3O4@titanium oxide core/shell nanoparticles.

    Science.gov (United States)

    Yin, Yichao; Liu, Chenjie; Wang, Baoxiang; Yu, Shoushan; Chen, Kezheng

    2013-05-21

    A simple, one-pot solvothermal method has been demonstrated for the preparation of bifunctional Fe3O4@titanium oxide core/shell nanoparticles. In a typical procedure, tetraalkoxyl titanium Ti(OC4H9)4 and FeCl3 as precursors were added into ethylene glycol and further solvothermal treatment was used to synthesize the core/shell particles. The core/shell particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), SQUID MPMS and rheometry. The morphological results showed titanium oxide nanorods with 100-200 nm length and 10-20 nm diameter coated on the surface of 200-300 nm Fe3O4 submicrospheres. Reaction time, the titanium source, the barium salt etc. have an influence on the morphology of core/shell particles. The core/shell particles can not only respond to an external magnetic field, but also to an electric field--a novel application of electrorheological fluid. PMID:23532087

  5. Hierarchical NiCo2 O4 Hollow Microcuboids as Bifunctional Electrocatalysts for Overall Water-Splitting.

    Science.gov (United States)

    Gao, Xuehui; Zhang, Hongxiu; Li, Quanguo; Yu, Xuegong; Hong, Zhanglian; Zhang, Xingwang; Liang, Chengdu; Lin, Zhan

    2016-05-17

    Bifunctional electrocatalysts for the oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) in alkaline electrolyte may improve the efficiency of overall water splitting. Nickel cobaltite (NiCo2 O4 ) has been considered a promising electrode material for the OER. However, NiCo2 O4 that can be used as an electrocatalyst in HER has not been studied yet. Herein, we report self-assembled hierarchical NiCo2 O4 hollow microcuboids for overall water splitting including both the HER and OER reactions. The NiCo2 O4 electrode shows excellent activity toward overall water splitting, with 10 mA cm(-2) water-splitting current reached by applying just 1.65 V and 20 mA cm(-2) by applying just 1.74 V across the two electrodes. The synthesis of NiCo2 O4 microflowers confirms the importance of structural features for high-performance overall water splitting. PMID:27061909

  6. Comprehensive Study of an Earth-Abundant Bifunctional 3D Electrode for Efficient Water Electrolysis in Alkaline Medium.

    Science.gov (United States)

    Sharifi, Tiva; Gracia-Espino, Eduardo; Jia, Xueen; Sandström, Robin; Wågberg, Thomas

    2015-12-30

    We report efficient electrolysis of both water-splitting half reactions in the same medium by a bifunctional 3D electrode comprising Co3O4 nanospheres nucleated on the surface of nitrogen-doped carbon nanotubes (NCNTs) that in turn are grown on conductive carbon paper (CP). The resulting electrode exhibits high stability and large electrochemical activity for both oxygen and hydrogen evolution reactions (OER and HER). We obtain a current density of 10 mA/cm(2) in 0.1 M KOH solution at overpotentials of only 0.47 and 0.38 V for OER and HER, respectively. Additionally, the experimental observations are understood and supported by analyzing the Co3O4:NCNT and NCNT:CP interfaces by ab initio calculations. Both the experimental and the theoretical studies indicate that firm and well-established interfaces along the electrode play a crucial role on the stability and electrochemical activity for both OER and HER. PMID:26629887

  7. Preparation and characterization of bifunctional oxidative and acidic catalysts Nb2O5/TS-1 for synthesis of diols

    International Nuclear Information System (INIS)

    Bifunctional oxidative and acidic catalyst was prepared by incorporation of titanium ion (Ti4+) and niobic acid in zeolite molecular-sieve. The catalysts being active both in oxidation reactions due to the presence of tetrahedral Ti4+, and acid-catalyzed reactions due to the presence of niobic acid. Nb/TS-1 was prepared by hydrothermal synthesis of TS-1, calcination in air and subsequent impregnation of niobium into TS-1. The sample was characterized by XRD, FTIR, UV-vis DR, TPR and pyridine adsorption techniques. The XRD analysis of Nb/TS-1 revealed that the MFI structure of the TS-1 support was found to be intact upon incorporation of niobium. The infrared spectra showed that the tetrahedral titanium in the TS-1 is still remained after impregnation with niobium while based on the UV-vis DR result, the niobium species are in the octahedral structure. On the basis TPR and infrared of hydroxyl groups results, it is concluded that niobium species interacted with the silanols on the surface of TS-1. Pyridine adsorption study shows both Bronsted and Lewis acid sites are present in Nb/TS-1. The catalytic results in the transformation of 1-octene to 1,2-octanediol through the formation of 1,2-epoxyoctane by using Nb/TS-1 indicate that the production of epoxide and diol was correlated with the presence of oxidative and Bronsted acidic sites in the catalyst

  8. Simultaneous H2 Generation and Biomass Upgrading in Water by an Efficient Noble-Metal-Free Bifunctional Electrocatalyst.

    Science.gov (United States)

    You, Bo; Jiang, Nan; Liu, Xuan; Sun, Yujie

    2016-08-16

    As an environmentally friendly approach to generate H2 , electrocatalytic water splitting has attracted worldwide interest. However, its broad employment has been inhibited by costly catalysts and low energy conversion efficiency, mainly due to the sluggish anodic half reaction, the O2 evolution reaction (OER), whose product O2 is not of significant value. Herein, we report an efficient strategy to replace OER with a thermodynamically more favorable reaction, the oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA), catalyzed by 3D Ni2 P nanoparticle arrays on nickel foam (Ni2 P NPA/NF). HMF is one of the primary dehydration intermediates of raw biomass and FDCA is of many industrial applications. As a bifunctional electrocatalyst, Ni2 P NPA/NF is not only active for HMF oxidation but also competent for H2 evolution. In fact, a two-electrode electrolyzer employing Ni2 P NPA/NF for simultaneous H2 and FDCA production required a voltage at least 200 mV smaller compared with pure water splitting to achieve the same current density, as well as exhibiting robust stability and nearly unity Faradaic efficiencies. PMID:27417546

  9. Asymmetric Michael Additions of 4-Hydroxycoumarin to β-Nitrostyrenes with Chiral, Bifunctional Hydrogen-Bonding Catalysts.

    Science.gov (United States)

    Wolf, Florian F; Klare, Helge; Goldfuss, Bernd

    2016-03-01

    Enantioselective Michael additions of 4-hydroxycoumarin to β-nitrostyrenes are catalyzed by different chiral, bifunctional hydrogen-bonding catalysts, based on thiourea- and squaramide motifs. The scope of the catalysis is tested by employing a series of substituted β-nitrostyrenes as well as different solvents. The 3,5-bis(trifluoromethyl)phenyl- and quinine-substituted squaramide catalyst is shown to be the most selective catalyst, resulting in 78% yield and 81% ee. Computational analyses of transition structures with different binding modes show that the most favored transition structure exhibits squaramide (NH)2 binding to an oxygen atom of the enolate nucleophile, while the nitroalkene coordinates via hydrogen bonding to the ammonium function of the quinuclidine unit of the catalyst. Hence, the canted directionality of the squaramide (NH)2 motif, favoring one-atom binding, might be decisive for the selectivity of the reaction. The absolute configuration of the major (-)-(R) enantiomer of the product is assigned computationally according to its optical rotation. PMID:26815792

  10. The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices.

    Science.gov (United States)

    Chen, J-S; Noah, E M; Pallua, N; Steffens, G C M

    2002-11-01

    The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins - horse radish peroxidase (HRP) was used as a model protein - were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP-PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices. PMID:15348172

  11. Synthesis and evaluation of two new bifunctional carboxymethylated tetraazamacrocyclic chelating agents for protein labeling with indium-111

    Energy Technology Data Exchange (ETDEWEB)

    Ruser, G.; Ritter, W.; Maecke, H.R. (Univ. Hospital Basel (Switzerland))

    1990-09-01

    The synthesis of two new N- and C-functionalized tetraazamacrocyclic ligands intended to be covalently linked to biomolecules like monoclonal antibodies and to bind the gamma-emitting isotope indium-111 in a thermodynamically and/or kinetically inert way is described. 12-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraa cetic acid (L1) was synthesized by means of bimolecular cyclization with the appropriate malonic acid diethyl ester and triethylenetetraamine, followed by reduction with diborane and alkylation of the cyclic tetraamine with bromoacetic acid. The corresponding triscarboxymethylated ligand L2 was made by statistical alkylation of the tetraamine. Both ligands fulfill the criteria for antibody labeling using the bifunctional chelate approach, namely fast chelate formation, high radiochemical yield, and high stability under physiological conditions. Surprisingly the heptadentate ligand L2 confers higher stability to In3+ and exhibits faster complex formation than octadentate L1. 13C NMR spectra in solution indicate that the difference in stability is not due to incomplete coordination of all four carboxylate groups in In-L1.

  12. Catalytic Upgrading of 5-Hydroxymethylfurfural to Drop-in Biofuels by Solid Base and Bifunctional Metal-Acid Catalysts.

    Science.gov (United States)

    Bohre, Ashish; Saha, Basudeb; Abu-Omar, Mahdi M

    2015-12-01

    Design and synthesis of effective heterogeneous catalysts for the conversion of biomass intermediates into long chain hydrocarbon precursors and their subsequent deoxygenation to hydrocarbons is a viable strategy for upgrading lignocellulose into distillate range drop-in biofuels. Herein, we report a two-step process for upgrading 5-hydroxymethylfurfural (HMF) to C9 and C11 fuels with high yield and selectivity. The first step involves aldol condensation of HMF and acetone with a water tolerant solid base catalyst, zirconium carbonate (Zr(CO3 )x ), which gave 92 % C9 -aldol product with high selectivity at nearly 100 % HMF conversion. The as-synthesised Zr(CO3 )x was analysed by several analytical methods for elucidating its structural properties. Recyclability studies of Zr(CO3 )x revealed a negligible loss of its activity after five consecutive cycles over 120 h of operation. Isolated aldol product from the first step was hydrodeoxygenated with a bifunctional Pd/Zeolite-β catalyst in ethanol, which showed quantitative conversion of the aldol product to n-nonane and 1-ethoxynonane with 40 and 56 % selectivity, respectively. 1-Ethoxynonane, a low oxygenate diesel range fuel, which we report for the first time in this paper, is believed to form through etherification of the hydroxymethyl group of the aldol product with ethanol followed by opening of the furan ring and hydrodeoxygenation of the ether intermediate. PMID:26549016

  13. Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

    Directory of Open Access Journals (Sweden)

    Gudiña Eduardo

    2008-02-01

    Full Text Available Abstract Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3' positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review.

  14. Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

    Science.gov (United States)

    Tsuji, Kohsei; Yoon, Ki-Seok; Ogo, Seiji

    2016-03-01

    Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHES77). Interestingly, the ADHES77 was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH4)2SO4 without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration. PMID:26216639

  15. Key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes.

    Science.gov (United States)

    Serrano, Ana; Frago, Susana; Herguedas, Beatriz; Martínez-Júlvez, Marta; Velázquez-Campoy, Adrián; Medina, Milagros

    2013-01-01

    Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis. PMID:22892871

  16. Self-activating nuclease and anticancer activities of copper(II) complexes with aryl-modified 2,6-di(thiazol-2-yl)pyridine.

    Science.gov (United States)

    Li, Lüying; Du, Kejie; Wang, Yi; Jia, Haina; Hou, Xiaojuan; Chao, Hui; Ji, Liangnian

    2013-08-28

    Three mononuclear copper complexes [Cu(PDTP)Cl2] (PDTP = 4-phenyl-2,6-di(thiazole-2-yl)pyridine, CuPDTP), [Cu(ADTP)Cl2] (ADTP = 4-(anthracen-9-yl)-2,6-di(thiazole-2-yl)pyridine, CuADTP) and [Cu(BFDTP)Cl2] (BFDTP = 4-(benzofuran-2-yl)-2,6-di(thiazole-2-yl)pyridine, CuBFDTP) were synthesized and characterized. The X-ray single crystallography results indicated that the Cu(II) ions showed slightly distorted square pyramid coordination environments, and the ligands deviated from ideal planarity in all three compounds. Based on the DNA binding studies, it was demonstrated that these three complexes exhibited weak DNA binding strengths, which were most likely groove binding modes. CuPDTP, CuADTP and CuBFDTP induced efficient DNA cleavage in the dark without the addition of external catalysts (oxidant or reductant). In contrast, in the presence of reducing or oxidizing agents, the nuclease activities increased more than 10-fold. Mechanistic investigations revealed the participation of reactive oxygen species, which can be trapped by ROS radical scavengers and ROS sensors. In the same experimental conditions, the free ligands and CuCl2 did not display any DNA cleaving activity. This result indicates that the complexes, rather than their components, play a significant role in the nuclease reaction process and that DNA cleavage may be initiated in an oxidative pattern. The proposed mechanism was attributed to the in situ activation of molecular oxygen by the oxidation of the copper complexes. In the MTT cytotoxicity studies, the three Cu(II) complexes exhibited an antitumor activity against the HeLa, BEL-7402 and HepG2 tumor cell lines. The HeLa cells treated with Cu(II) complexes demonstrated marked changes in their nuclear morphology, which were detected by Hoechst 33258 nuclear staining and acridine orange/ethidium bromide (AO/EB) staining assays. Nuclear chromatin cleavage also was observed from alkaline single-cell gel electrophoresis (comet assay). PMID:23843095

  17. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 106-3 x 108 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 106 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  18. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

  19. Removing organic contaminants with bifunctional iron modified rectorite as efficient adsorbent and visible light photo-Fenton catalyst

    International Nuclear Information System (INIS)

    Highlights: ► Rectorite was modified by ultrasonic-assisted ion-exchange and hydrolysis. ► The pillaring increased the layer-to-layer spacing of rectorite. ► The iron-modified rectorite was found to be an excellent adsorbent. ► The iron-modified rectorite showed good visible light photocatalytic ability. ► FeR was highly chemically stable with a wide operating range of pH. - Abstract: Iron-modified rectorite (FeR) was prepared as both adsorbent and catalyst. The iron modification increased layer-to-layer spacing and surface area of rectorite, leading to much increased adsorption of Rhodamine B (RhB) on rectorite. The maximum adsorption capacity of RhB on FeR reached 101 mg g−1 at pH 4.5, being 11 folds of that on the unmodified one. The iron modification also enabled rectorite to have efficient visible light photocatalytic ability. The apparent rate constant for the degradation of RhB (80 μM) at 298 K and pH 4.5 in the presence of H2O2 (6.0 mM) and FeR (0.4 g L−1) was evaluated to be 0.0413 min−1 under visible light and 0.122 min−1 under sunlight, respectively. The analysis with electron spin resonance spin-trapping technique supported that the iron modified rectorite effectively catalyzed the decomposition of H2O2 into hydroxyl radicals. On the basis of the characterization and analysis, the new bifunctional material was well clarified as both adsorbent and photocatalyst in the removing of organic pollutants.

  20. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel (Maryland); (GWU); (Georgia)

    2012-05-24

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.