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Sample records for plasmid preparation based

  1. PREPARATION OF Ag NANOPARTICLES BASED ON PHOTOIRRADIATION METHOD WITH PLASMID DNA-TEMPLATED%质粒DNA光化学反应制备Ag纳米颗粒

    Institute of Scientific and Technical Information of China (English)

    刘建华; 章锦丹; 张晓亮; 李松梅; 于美

    2012-01-01

    以碱基对数为7.5 kB的大肠杆菌环状质粒DNA为模板,通过紫外线辐射作用下的光化学反应方法制备了Ag纳米颗粒.采用紫外-可见光吸收光谱,TEM和EDS等方法研究了辐射时间、溶液浓度等工艺条件对Ag纳米颗粒组成、形貌和结构的影响.结果表明,获得的Ag纳米颗粒为fcc结构,颗粒直径为25-40 nm;一定条件下,Ag纳米颗粒为环状,中间孔洞直径为8-10 nm,Ag+与碱基对数摩尔比为4、辐射时间为40 min时,Ag纳米颗粒中环状结构最多.%Over the past two decades, interest has been focused on the synthesis of novel nano-materials with different sizes and different morphologies due to their intriguing chemical, electronic, biosensing, optical and catalytic properties. The biomacromolecules like nucleic acids, proteins, amino acids and peptides have been employed as template for nanomaterial design and synthesis. DNA, which could self-assemble into complex structures such as cubes and squares, is being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. In this work, the plasmid DNA with 7.5 kB base pairs separated from the bacillus was used as a template to prepare Ag nanoparticles based on the ultraviolet ray (UV) photoirradiation method. The effects of experimental conditions, such as the photoirradiation time and concentration of metallic Ag+ ions, on the composition, morphology, and structure of the obtained nanoparticles were detailed studied. The Ag nanoparticles were characterized by using UV-visible light absorption spectroscopy, TEM and EDS. It is found that Ag+ and UV are necessary in this preparation based on plasmid DNA. The resulted Ag nanoparticles are fcc structure, and the average diameter of obtained nanoparticles is 25-40 nm. The sizes of nanoparticles increased with the increasing of the photoirradiation time and the concentration of Ag+. When the mol ratio of Ag+ to base pairs is 4 :1 and the

  2. Method for the preparation of plasmid DNA suitable for physicochemical measurements.

    Science.gov (United States)

    Vojtísková, M; Lukásová, E; Palecek, E

    1985-01-01

    A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

  3. Polymerase chain reaction-based gene removal from plasmids

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    Vishnu Vardhan Krishnamurthy

    2015-09-01

    Full Text Available This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033, which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116. Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

  4. Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

    DEFF Research Database (Denmark)

    Merkey, Brian; Lardon, Laurent; Seoane, Jose Miguel;

    2011-01-01

    . By extending an individual‐based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid...... and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual......Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor...

  5. Optimum range of plasmid supercoiled DNA for preparation of ccompetent Top 10 E. coli

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    Muhammad Tahir Majeed

    2011-05-01

    Full Text Available Objectives: In-house preparation of chemically competent andelectrocompetent Top 10 E. coli is not only economical butmeets the needs for most of the molecular cloning work. Forsuch transformations an optimum range of plasmidsupercoiled DNA is needed. Therefore, the present studydescribes the modification of two protocols for the preparationof such cells, and optimization of the amount of plasmidsupercoiled DNA required for better efficiency.Materials and methods: As most of the available protocols torender bacterial cells competent need special media orchemicals and are time consuming, the methods from HelenDonis-Keller Laboratory Manual of Washington University inSt. Louis and Goldberg Laboratory Standard Protocols of theUnited States Department of Agriculture have been used aftermeticulous selection and with few modifications for preparingchemically competent and electrocompetent Top 10 E. coli,respectively. The transformation was carried out using pUC19supercoiled plasmid DNA.Results: The transformation efficiencies of chemicallycompetent and electrocompetent Top 10 E. coli were found tobe 1.1 x 106 and 7.88 x 107 tranformants/μg of DNA,respectively. Such efficiencies are slightly higher than therequired (105-106 transformants/μg DNA for most of thecloning experimentation.Conclusion: The results of the present study indicatethat for sufficient transformation competence rates theoptimum range of plasmid supercoiled DNA is 10 ng forchemically competent and 0.1 ng for electrocompetentTop 10 E. coli.

  6. Preparation and characterization of chitosan/β-cyclodextrin nanoparticles containing plasmid DNA encoding interleukin-12.

    Science.gov (United States)

    Nahaei, M; Valizadeh, H; Baradaran, B; Nahaei, M R; Asgari, D; Hallaj-Nezhadi, S; Dastmalchi, S; Lotfipour, F

    2013-01-01

    Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes.pUMVC3-hIL12 loaded Low Molecular Weight chitosan/β-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line.All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA.LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

  7. Influence of Single Base Change in Shine-Dalgarno Sequence on the Stability of B.Subtilis Plasmid PSM604

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    B.Subtilis expression plasmids generally require a stringent Shine-Dalgarno Sequence(SDS). Site-directed-mutagenesis was explored to change the Shine-Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.

  8. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

    Science.gov (United States)

    Diebold, S S; Schulz, O; Alexopoulou, L; Leitner, W W; Flavell, R A; Reis e Sousa, C

    2009-03-01

    Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

  9. Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells.

    Science.gov (United States)

    Faurez, Florence; Dory, Daniel; Henry, Aurélie; Bougeard, Stéphanie; Jestin, André

    2010-04-01

    In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes.

  10. Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

    Directory of Open Access Journals (Sweden)

    Sarker SR

    2013-04-01

    Full Text Available Satya Ranjan Sarker, Yumiko Aoshima, Ryosuke Hokama, Takafumi Inoue, Keitaro Sou, Shinji Takeoka Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns, Tokyo, Japan Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the

  11. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells.

    Science.gov (United States)

    Hallaj-Nezhadi, Somayeh; Valizadeh, Hadi; Dastmalchi, Siavoush; Baradaran, Behzad; Jalali, Mohammad Barzegar; Dobakhti, Faramarz; Lotfipour, Farzaneh

    2011-01-01

    Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. In this study, we aimed to prepare plasmid DNA loaded chitosan nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicity and transfection efficiency in Murine CT-26 colon carcinoma cells. Nanoparticles were prepared using a complex coacervation process at different N/P ratios and characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. Size and zeta potential varied from 76.73 to 867.03 nm and between 5.68 and 16.77 mV, respectively. Strong attachment of the DNA to chitosan was observed after polyplex preparation. Encapsulation efficiencies were high (72.97-94.87%). The transfection efficiencies of the prepared complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at (N/P = 16) with mean particle size of 381.83±82.77 nm (polydispersity index=0.44) indicating the improved transfection of pUMVC3-hIL12 about 2.80 times compared to that of the naked pUMVC3-hIL12. Prepared polyplexes were nontoxic to CT-26 cells. Chitosan-DNA nanoparticles at N/P = 16 with minimal cytotoxicity, can be used as suitable candidate for Il-12 delivery. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  12. Development of new plasmid DNA vaccine vectors with R1-based replicons

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    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  13. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

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    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

  14. Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene.

    Science.gov (United States)

    Ma, Tao; Jiang, Jin-Ling; Liu, Ying; Ye, Zheng-Bao; Zhang, Jun

    2014-09-01

    c-Myc plays a key role in glioma cancer stem cell maintenance. A drug delivery system, nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene (NPs-c-Myc-siRNA-pDNAs), for the treatment of glioma, has not previously been reported. NPs-c-Myc-siRNA-pDNAs were prepared and evaluated in vitro. Three kinds of c-Myc-siRNA fragments were separately synthesized and linked with empty siRNA expression vectors in the mole ratio of 3:1 by T4 DNA ligase. The linked products were then separately transfected into Escherichia coli. DH5α followed by extraction with Endofree plasmid Mega kit (Qiagen, Hilden, Germany) obtained c-Myc-siRNA-pDNAs. Finally, the recombinant c-Myc-siRNA3-pDNAs, generating the highest transfection efficiency and the greatest apoptotic ability, were chosen for encapsulation into NPs by the double-emulsion solvent-evaporation procedure, followed by stability, transfection efficiency, as well as qualitative and quantitative apoptosis evaluation. NPs-c-Myc-siRNA3-pDNAs were obtained with spherical shape in uniform size below 150 nm, with the zeta potential about -18 mV, the encapsulation efficiency and loading capacity as 76.3 ± 5.4% and 1.91 ± 0.06%, respectively. The stability results showed that c-Myc-siRNA3-pDNAs remained structurally and functionally stable after encapsulated into NPs, and NPs could prevent the loaded c-Myc-siRNA3-pDNAs from DNase degradation. The transfection efficiency of NPs-c-Myc-siRNA3-pDNAs was proven to be positive. Furthermore, NPs-c-Myc-siRNA3-pDNAs produced significant apoptosis with the apoptotic rate at 24.77 ± 5.39% and early apoptosis cells observed. Methoxy-poly-(ethylene-glycol)-poly-(lactide-co-glycolide) nanoparticles (MPEG-PLGA-NPs) are potential delivery carriers for c-Myc-siRNA3-pDNAs.

  15. Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

    Science.gov (United States)

    Zou, Weiwei; Liu, Chunxi; Chen, Zhijin; Zhang, Na

    2009-09-01

    The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

  16. Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

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    Zou Weiwei

    2009-01-01

    Full Text Available Abstract The purpose of the present work was to formulate and evaluate cationic poly(lactic acid-poly(ethylene glycol (PLA-PEG nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95% could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

  17. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

    OpenAIRE

    Al-Allaf, Faisal A.; Tolmachov, Oleg E.; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2012-01-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered ...

  18. A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Porse, Andreas; Sommer, Morten Otto Alexander

    2017-01-01

    tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have...... widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both...

  19. Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer.

    Science.gov (United States)

    Browne, C J; Pinyon, J L; Housley, D M; Crawford, E N; Lovell, N H; Klugmann, M; Housley, G D

    2016-04-01

    Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here, we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require ~100 × lower applied voltages for cell transduction than conventional electroporation. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level.

  20. Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells.

    Science.gov (United States)

    Schiffmacher, Andrew T; Keefer, Carol L

    2012-08-01

    Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.

  1. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  2. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  3. An approach towards bronchoscopic-based gene therapy using electrical field accelerated plasmid droplets.

    Science.gov (United States)

    Hradetzky, D; Boehringer, S; Geiser, Th; Gazdhar, A

    2012-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating disease affecting the distal lung, due to failure of the alveolar epithelium to heal after micro-injuries, leading to inefficient gas exchange and resulting in death. Therapeutic options are very limited. A new therapeutic approach based on gene therapy restores the self-healing process within the lung in the experimental setup. A basic requirement of this therapy is the successful transduction of genes into the alveolar epithelium in the distal part of the lung, for which a new therapeutic instrument is required. In this paper we present the concept and first experimental results of a device which uses an electrical field to accelerate the charged droplets of plasmid suspension toward the tissue and which overcomes cell membrane with its impact energy. The aim is to develop a therapeutic device capable of being integrated into minimally invasive procedures such as bronchoscopy.

  4. An individual-based approach to explain plasmid invasion in bacterial populations

    DEFF Research Database (Denmark)

    Seoane, Jose Miguel; Yankelevich, Tatiana; Dechesne, Arnaud

    2011-01-01

    observe, however, that transient periods of elevated plasmid transfer in newly formed transconjugant cells are offset by unfavorable cell-to-cell contact mechanics, which ultimately precludes the pWWO TOL plasmid from fully invading tightly packed multicellular P. putida populations such as microcolonies...

  5. Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica.

    Science.gov (United States)

    Lindsey, Rebecca L; Frye, Jonathan G; Fedorka-Cray, Paula J; Meinersmann, Richard J

    2011-10-01

    In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.

  6. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

    Science.gov (United States)

    Al-Allaf, Faisal A; Tolmachov, Oleg E; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2013-02-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.

  7. Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Carvalho Flávia Maria de Souza

    2005-01-01

    Full Text Available Xanthomonas axonopodis pv. citri strains that cause disease in citrus were investigated by pulsed field and plasmid profile analysis. For the first method, genomic DNA was digested by the rare-cutting enzymes Xba I and Vsp I. The strains evaluated were collected in seven different States of Brazil and in Argentina, Bolivia, Paraguay and Uruguay. Genetic variability was found among strains of X. axonopodis pv. citri from different geographical areas Argentina, Bolivia and Uruguay, with similarities varying from 0.62 to 0.83. However, the strains collected in Brazil, despite being from different States, have shown a genetic similarity ranging from 0.83 to 1.00. Cluster analysis showed a relationship between genomic similarity and geographical origin of the strains. Plasmids were observed in all strains, with a total of five different plasmids, with sizes between 57.7 and 83.0 kilobases. The 72.6 kb plasmid was the most frequent, present in 15 out of 22 strains, while the 68.1 kb plasmid was observed in two strains only. Although the plasmid diversity detected in the present study was not very great, the X. axonopodis pv. citri strains evaluated showed a considerable degree of diversity with regard to this extrachromosomal genetic element.

  8. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  9. Donor plasmid design for codon and single base genome editing using zinc finger nucleases.

    Science.gov (United States)

    Pruett-Miller, Shondra M; Davis, Gregory D

    2015-01-01

    In recent years, CompoZr zinc finger nuclease (ZFN) technology has matured to the point that a user-defined double strand break (DSB) can be placed at virtually any location in the human genome within 50 bp of a desired site. Such high resolution ZFN engineering is well within the conversion tract limitations demarcated by the mammalian DNA repair machinery, resulting in a nearly universal ability to create point mutations throughout the human genome. Additionally, new architectures for targeted nuclease engineering have been rapidly developed, namely transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, further expanding options for placement of DSBs. This new capability has created a need to explore the practical limitations of delivering plasmid-based information to the sites of chromosomal double strand breaks so that nuclease-donor methods can be widely deployed in fundamental and therapeutic research. In this chapter, we explore a ZFN-compatible donor design in the context of codon changes at an endogenous locus encoding the human RSK2 kinase.

  10. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  11. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    Directory of Open Access Journals (Sweden)

    Trine Aakvik Strand

    Full Text Available Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  12. Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

    Science.gov (United States)

    Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

    2016-03-01

    The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves.

  13. Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

    Science.gov (United States)

    Kondo, T; Mukai, M; Kondo, Y

    1991-10-01

    We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

  14. Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-gene Library

    Science.gov (United States)

    2013-10-09

    various fragments from L plant arum. Cells were grown in LB medium with 5% (V/V) ethanol and starting optical density of 0.3. Survival comparison...in final titer is observed. Expression of the HSP plasmid appears to slow both growth and production throughout the entire fermentation . However...this may be an effect of overexpression of the HSPs during the early portions of the fermentation where there are low concentrations of ethanol and

  15. Procarcinogens ? Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

    OpenAIRE

    Bui, Ngoc; Nguyen, Thi Thu Huyen; Mai, Chi Thanh; Bettarel, Yvan; Hoang, Thi Yen; Trinh, Thi Thuy Linh; Truong, Nam Hai; CHU, HOANG HA; Nguyen, Vu Thanh Thanh; Nguyen, Huu Duc; W?lfl, Stefan

    2016-01-01

    In Vietnam, a great number of toxic substances, including carcinogens and procarcinogens, from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. In the present study, we report the development of novel yeast-based biosensor systems to determine both genotoxic carcinogens and procarcinogens by cotransformation with two plasmids. One plasmid is carrying human CPR and CYP (CYP3A4, CYP2B6, or CYP2D6) genes, while the other co...

  16. Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid-based veterinary vaccine

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority

    2013-05-01

    Full Text Available Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

  17. Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells.

    Science.gov (United States)

    Megges, Matthias; Oreffo, Richard O C; Adjaye, James

    2016-01-01

    Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

  18. Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices.

    Science.gov (United States)

    Sousa, Angela; Sousa, Fani; Queiroz, João A

    2010-09-01

    The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

  19. Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

    Science.gov (United States)

    Ma, Chunying; Zhang, Jin; Guo, Liwen; Du, Changguo; Song, Ping; Zhao, Baojing; Li, Ling; Li, Chao; Qiao, Renzhong

    2016-01-04

    Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

  20. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  1. Plasmid-based Stat3 siRNA delivered by hydroxyapatite nanoparticles suppresses mouse prostate tumour growth in vivo

    Institute of Scientific and Technical Information of China (English)

    Zuo-Wen Liang; Ling Zhang; Bao-Xue Yang; Bao-Feng Guo; Yang Li; Xiao-Jie Li; Xin Li; Li-Ting Zhao; Ii-Fang Gao; Hao Yu; Xue-Jian Zhao

    2011-01-01

    DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling and inhibits prostate tumour growth. However, the antitumour activity depends on the efficient delivery of si-Stat3. The effects on the growth of mouse prostate cancer cells of si-Stat3 delivered by hydroxyapatite were determined in this study. RM-1 tumour blocks were transplanted into C57BL/6 mice. CaCl2-modif ied hydroxyapatite carrying si-Stat3 plasmids were injected into tumours, and tumour growth and histology were determined. The expression levels of Stat3, pTyr-Stat3, Bcl-2, Bax, Caspase3, VEGF and cyclin Dl were measured by western blot analysis. Amounts of apoptosis in cancer cells were analysed with immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay. The results showed that hydroxyapatite-delivered si-Stat3 significantly suppressed tumour growth up to 74% (P<0.01). Stat3 expression was dramatically downregulated in the tumours. The immunohistochemistry and TUNEL results showed that si-Stet3-induced apoptosis (up to 42%, P<0.01). The Stat3 downstream genes Bcl-2, VEGFand cyclin Dl were also strongly downregulated in the tumour tissues that also displayed significant increases in Bax expression and Caspase3 activity. These results suggest that hydroxyapatite can be used for the in vivo delivery of plasmid-based siRNAs into tumours.

  2. Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology.

    Science.gov (United States)

    Kroll, Jens; Steinle, Anna; Reichelt, Rudolf; Ewering, Christian; Steinbüchel, Alexander

    2009-05-01

    Plasmid stability in recombinant microorganisms is a very important requirement for highly efficient plasmid-based production processes in biotechnology. To stably maintain plasmids, we developed in this study an efficient and stringent novel anabolism-based addiction system, which can be widely used. This novel addiction system is based on two components: (i) an Escherichia coli HMS174(DE3) knockout mutant of the ispH gene coding for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2) of the deoxyxylulose 5-phosphate (DXP) pathway, impairing the synthesis of isopentenyl pyrophosphate (IPP) and (ii) a completely synthetic and episomal mevalonate (MVA) pathway as an alternative supplier of essential IPP. The latter is encoded by a plasmid that contains the genes for HMG-CoA reductases from Lactococcus lactis and Staphylococcus aureus plus HMG-CoA-synthase, MVA kinase, MVP kinase and MVPP decarboxylase from S. aureus. This plasmid should then also harbor the genes for the protein or for the pathway that will be produced or that will be utilized for production of a chemical. To demonstrate the functionality of this addiction system, a mutated cyanophycin synthetase gene (cphA(6308)C595S) was used. To determine plasmid stabilities, flasks experiments in media supplied or not supplied with antibiotics were carried out with the knockout mutant and two control strains, one harboring plasmid pCOLADuet-1::MVA1-5::cphA(6308) and the other harboring a conventional expression plasmid pET-23a::cphA(6308). As revealed by measuring the colony-forming units of aliquots spread on solid media with or without antibiotics, the knockout mutant revealed a plasmid stability of 100% whereas the control strains exhibited plasmid stabilities of only 64% and 2%, respectively. Radiometric enzyme activity measurements for CphA revealed only 95% and 12.5% of the activity in the control strains harboring pCOLADuet-1::MVA1-5::cphA(6308) and pET-23a::cphA(6308), respectively, in

  3. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  4. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  5. Evaluation of Maltose-Based Cationic Liposomes with Different Hydrophobic Tails for Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Bo Li

    2017-03-01

    Full Text Available In this paper, three cationic glycolipids with different hydrophobic chains Malt-DiC12MA (IX a, Malt-DiC14MA (IX b and Malt-DiC16MA (IX c were constructed by using maltose as starting material via peracetylation, selective 1-O-deacetylation, trichloroacetimidation, glycosylation, azidation, deacetylation, Staudinger reaction, tertiary amination and quaternization. Target compounds and some intermediates were characterized by 1H-NMR, 13C-NMR, 1H-1H COSY and 1H-13C HSQC. The results of gel electrophoresis assay, atomic force microscopy images (AFM and dynamic light scattering (DLS demonstrate that all the liposomes could efficiently bind and compact DNA (N/P ratio less than 2 into nanoparticles with proper size (88 nm–146 nm, PDI < 0.4 and zeta potential (+15 mV–+26 mV. The transfection efficiency and cellular uptake of glycolipids in HEK293 cell were evaluated through the enhanced green fluorescent protein (EGFP expression and Cy3-labeled pEGFP-C1 (Enhanced Green Fluorescent Protein plasmid images, respectively. Importantly, it indicated that Malt-DiC14MA exhibited high gene transfer efficiency and better uptake capability at N/P ratios of 8:1. Additionally, the result of cell viability showed glycolipids exhibited low biotoxicity and good biocompatibility by thiazolyl blue tetrazolium bromide (MTT assay.

  6. Preparation of Ag/AgBr/TiO2 as Catalyst Carriers and Its Damage to Plasmid DNA andTetrahymena

    Institute of Scientific and Technical Information of China (English)

    LIU Liwei; ZHANG Yinlong

    2015-01-01

    The composites based on the TiO2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/AgBr/TiO2/Active carbon (AC) composites was studied on the plasmid DNA andTetrahymena membrane. The atomic force micrograph (AFM) images showed that, in the presence of the composites under illumination, most pUC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2− of the phospho-lipid phospho-diester onTetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the lfuorescence polarization of DPH was observed, relfecting a signiifcant decrease in membrane lfuidity ofTetrahymena.

  7. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.

    Science.gov (United States)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

  8. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

    Science.gov (United States)

    Shareck, Julie; Choi, Young; Lee, Byong; Miguez, Carlos B

    2004-01-01

    Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

  9. Development and characterization of a new plasmid delivery system based on chitosan-sodium deoxycholate nanoparticles.

    Science.gov (United States)

    Cadete, A; Figueiredo, L; Lopes, R; Calado, C C R; Almeida, A J; Gonçalves, L M D

    2012-03-12

    Chitosan is one of the most promising polymers for drug delivery through the mucosal routes because of its polycationic, biocompatible, and biodegradable nature, and particularly due to its mucoadhesive and permeation-enhancing properties. Bile salts are known to interact with lipid membranes, increasing their permeability. The addition of bile salts to chitosan matrices may improve the delivery characteristics of the system, making it suitable for mucosal administration of bioactive substances. In the present study we have developed chitosan nanoparticles using sodium deoxycholate as a counter ion and evaluated their potential as gene delivery carriers. Chitosan-sodium deoxycholate nanoparticles (CS/DS) obtained via a mild ionic gelation procedure using different weight ratios were used to encapsulate plasmid DNA (pDNA) expressing a "humanized" secreted Gaussia Luciferase as reporter gene (pGLuc, 5.7 kDa). Mean particle size, polydispersity index and zeta potential were evaluated in order to select the best formulation for further in vitro studies. The nanoparticles presented an average size of 153-403 nm and a positive zeta potential ranging from +33.0 to +56.9 mV, for nanoparticles produced with CS/DS ratios from 1:4 to 1:0.6 (w:w), respectively. The pDNA was efficiently encapsulated and AFM studies showed that pDNA-loaded nanoparticles presented a more irregular surface due to the interaction between cationic chitosan and negatively charged pDNA which results in a more compact structure when compared to empty nanoparticles. Transfection efficiency of CS/DS-pDNA nanoparticles into moderately (AGS) and well differentiated (N87) gastric adenocarcinoma cell lines was determined by measuring the expression of luciferase, while cell viability was assessed using the MTT reduction. The CS/DS nanoparticles containing encapsulated pDNA were able to transfect both AGS and N87 cell lines, being more effective with AGS cells, the less differentiated cell line. The highest

  10. PREPARATION OF LEUCITE-BASED COMPOSITES

    Directory of Open Access Journals (Sweden)

    Alexandra Kloužková

    2012-12-01

    Full Text Available The aim of this study was to prepare leucite dental composites from two separately synthesized components - tetragonal leucite and glassy matrix. The newly developed procedure is based on the preparation of crystalline tetragonal leucite powder by relatively low temperature synthesis under hydrothermal conditions. Matrix powder was prepared by a classical melting process and subsequent milling of the quenched glass. The dental composites were prepared by mixing of 10 wt. %, 20 wt. % and 30 wt. % of synthesized tetragonal leucite with glass powder followed by pressing and firing. The sintering process was observed by optical microscope and the optimal firing temperature for each composite was determined. Optical and electron microscopy was used to characterise the microstructure of the composites, especially the distribution of the leucite particles in the matrix. Dilatometric measurements proved that the coefficient of thermal expansion of the composites increased up to 44 % in comparison with the basic matrix.

  11. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

    Directory of Open Access Journals (Sweden)

    Boerrigter Michaël

    2004-01-01

    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  12. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  13. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  14. Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.

    Science.gov (United States)

    Li, Zicong; Zeng, Fang; Meng, Fanming; Xu, Zhiqian; Zhang, Xianwei; Huang, Xiaoling; Tang, Fei; Gao, Wenchao; Shi, Junsong; He, Xiaoyan; Liu, Dewu; Wang, Chong; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2014-05-01

    The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

  15. DermaVir: a plasmid DNA-based nanomedicine therapeutic vaccine for the treatment of HIV/AIDS.

    Science.gov (United States)

    Lori, Franco

    2011-10-01

    The HIV global pandemic continues to rage with over 33 million people living with the disease. Although multidrug therapy has improved the prognosis for those infected by the virus, it has not eradicated the infection. Immunological therapies, including therapeutic vaccines, are needed to supplement drug therapy in the search for a 'functional cure' for HIV. DermaVir (Genetic Immunity Kft, Budapest, Hungary and McLean, Virginia, USA), an experimental HIV/AIDS therapeutic vaccine, combines three key elements of rational therapeutic vaccine design: a single plasmid DNA (pDNA) immunogen expressing 15 HIV antigens, a synthetic pDNA nanomedicine formulation and a dendritic cell-targeting topical-vaccine administration. DermaVir's novel mechanism of action, natural transport by epidermal Langerhans cells to the lymph nodes to express the pDNA-encoded HIV antigens and induce precursor/memory T cells with high proliferation capacity, has been consistently demonstrated in mouse, rabbit, primate and human subjects. Safety, immunogenicity and preliminary efficacy of DermaVir have been clinically demonstrated in HIV-infected human subjects. The DermaVir technology platform for dendritic cell-based therapeutic vaccination might offer a new treatment paradigm for cancer and infectious diseases.

  16. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    F Wegman

    2011-03-01

    Full Text Available Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2, which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.

  17. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    Science.gov (United States)

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

  18. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

    Science.gov (United States)

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela

    2015-06-01

    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.

  19. A new anti-tumor strategy based on in vivo tumstatin overexpression after plasmid electrotransfer in muscle

    Energy Technology Data Exchange (ETDEWEB)

    Thevenard, Jessica, E-mail: jessica.thevenard@univ-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France); Ramont, Laurent, E-mail: lramont@chu-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France); CHU de Reims, Avenue du Général Koenig, F-51092 Reims (France); Mir, Lluis M., E-mail: luis.mir@igr.fr [CNRS, UMR 8203, Institut Gustave Roussy, 114, Rue Edouard Vaillant, F-94805 Villejuif Cedex (France); Université Paris-Sud, UMR 8203, Institut Gustave Roussy, 114, Rue Edouard Vaillant, F-94405 Orsay Cedex (France); Dupont-Deshorgue, Aurélie, E-mail: aurelie.dupont@univ-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France); Maquart, François-Xavier, E-mail: fmaquart@chu-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France); CHU de Reims, Avenue du Général Koenig, F-51092 Reims (France); Monboisse, Jean-Claude, E-mail: jc.monboisse@univ-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France); CHU de Reims, Avenue du Général Koenig, F-51092 Reims (France); Brassart-Pasco, Sylvie, E-mail: sylvie.brassart-pasco@univ-reims.fr [FRE CNRS/URCA 3481, University of Reims Champagne-Ardenne, 51 rue Cognacq Jay, F-51095 Reims (France)

    2013-03-22

    Highlights: ► A new therapeutic strategy based on tumstatin in vivo overexpression is proposed. ► pVAX1©–tumstatin electrotransfer in muscle mediates protein expression in muscle. ► A substantial expression of tumstatin is detected in the serum of electrotransfected mice. ► Tumstatin overexpression decreases tumor growth and increases mouse survival. -- Abstract: The NC1 domains from the different α(IV) collagen chains were found to exert anti-tumorigenic and/or anti-angiogenic activities. A limitation to the therapeutic use of these matrikines is the large amount of purified recombinant proteins, in the milligram range in mice that should be administered daily throughout the experimental procedures. In the current study, we developed a new therapeutic approach based on tumstatin (NC1α3(IV)) overexpression in vivo in a mouse melanoma model. Gene electrotransfer of naked plasmid DNA (pDNA) is particularly attractive because of its simplicity, its lack of immune responsiveness and its safety. The pDNA electrotransfer in muscle mediates a substantial gene expression that lasts several months. A pVAX1© vector containing the tumstatin cDNA was injected into the legs of C57BL/6 mice and submitted to electrotranfer. Sera were collected at different times and tumstatin was quantified by ELISA. Tumstatin secretion reached a plateau at day 21 with an expression level of 12 μg/mL. For testing the effects of tumstatin expression on tumor growth in vivo, B16F1 melanoma cells were subcutaneously injected in mice 7 days after empty pVAX1© (Mock) or pVAX1©–tumstatin electrotransfer. Tumstatin expression triggered a large decrease in tumor growth and an increase in mouse survival. This new therapeutic approach seems promising to inhibit tumor progression in vivo.

  20. A simple and efficient method for assembling TALE protein based on plasmid library.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Zhang

    Full Text Available DNA binding domain of the transcription activator-like effectors (TALEs from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs and TALE transcription factors (TALE-TFs based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

  1. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    Science.gov (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  2. Preparation

    Directory of Open Access Journals (Sweden)

    M.M. Dardir

    2014-03-01

    Full Text Available Some hexanamide-mono and di-linoleniate esters were prepared by the reaction of linolenic acid and hexanamide (derived from the reaction of hexanoic acid and diethanolamine. The chemical structure for the newly prepared hexanamide-mono and di-linoleniate esters were elucidated using elemental analysis, (FTIR, H 1NMR and chemical ionization mass spectra (CI/Ms spectroscopic techniques. The results of the spectroscopic analysis indicated that they were prepared through the right method and they have high purity. The new prepared esters have high biodegradability and lower toxicity (environmentally friendly so they were evaluated as a synthetic-based mud (ester-based mud for oil-well drilling fluids. The evaluation included study of the rheological properties, filtration and thermal properties of the ester based-muds formulated with the newly prepared esters compared to the reference commercial synthetic-based mud.

  3. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  4. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  5. Broad host range plasmid-based gene transfer system in the cyanobacterium Gloeobacter violaceus which lacks thylakoids

    Institute of Scientific and Technical Information of China (English)

    GUO Haitao; XU Xudong

    2004-01-01

    Gloeobacter violaceus, a cyanobacterium lack of thylakoids, is refractory to genetic manipulations because its cells are enveloped by a thick gelatinous sheath and in colonial form.In this study, a large number of single cells were obtained by repeated pumping with a syringe with the gelatinous sheath removed.And an exogenous broad host range plasmid pKT210 was conjugatively transferred into G.violaceus.Analyses with dot-blot hybridization and restriction mapping showed that the exogenous plasmid pKT210 had been introduced into G.violaceus and stably maintained with no alteration in its structure.pKT210 extracted from G.violaceus exconjugants could be transformed into the mcr- mrr- E.coli strain DH10B but not the mcr+ mrr+ strain DH5α, which suggests that a methylase system may be present in G.violaceus.

  6. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela;

    2012-01-01

    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

  7. Bacteriophages limit the existence conditions for conjugative plasmids.

    Science.gov (United States)

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A

    2015-06-02

    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  8. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  9. Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production

    Directory of Open Access Journals (Sweden)

    Marta Irla

    2016-09-01

    Full Text Available Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 g/L to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

  10. First report on rapid screening of nanomaterial-based antimicrobial agents against β-lactamase resistance using pGLO plasmid transformed Escherichia coli HB 101 K-12

    Science.gov (United States)

    Raj, M. Alpha; Muralidhar, Y.; Sravanthi, M.; Prasad, T. N. V. K. V.; Nissipriya, M.; Reddy, P. Sirisha; Neelima, T. Shoba; Reddy, G. Dilip; Adilaxmamma, K.; Kumar, P. Anand; Krishna, T. Giridhara

    2016-08-01

    Combating antibiotic resistance requires discovery of novel antimicrobials effective against resistant bacteria. Herein, we present for the first time, pGLO plasmid transformed Escherichia coli HB 101 K 12 as novel model for screening of nanomaterial-based antimicrobial agents against β-lactamase resistance. E. coli HB 101 was transformed by pGLO plasmid in the presence of calcium chloride (50 mM; pH 6.1) aided by heat shock (0-42-0 °C). The transformed bacteria were grown on Luria-Bertani agar containing ampicillin (amp) and arabinose (ara). The transformed culture was able to grow in the presence of ampicillin and also exhibited fluorescence under UV light. Both untransformed and transformed bacteria were used for screening citrate-mediated nanosilver (CNS), aloin-mediated nanosilver (ANS), 11-α-keto-boswellic acid (AKBA)-mediated nanosilver (BNS); nanozinc oxide, nanomanganese oxide (NMO) and phytochemicals such as aloin and AKBA. Minimum inhibitory concentrations (MIC) were obtained by microplate method using ρ-iodo nitro tetrazolium indicator. All the compounds were effective against transformed bacteria except NMO and AKBA. Transformed bacteria exhibited reverse cross resistance against aloin. ANS showed the highest antibacterial activity with a MIC of 0.32 ppm followed by BNS (10.32 ppm), CNS (20.64 ppm) and NZO (34.83 ppm). Thus, pGLO plasmid can be used to induce resistance against β-lactam antibiotics and the model can be used for rapid screening of new antibacterial agents effective against resistant bacteria.

  11. Characterization of CTX-M-producing Escherichia coli by repetitive sequence-based PCR and real-time PCR-based replicon typing of CTX-M-15 plasmids.

    Science.gov (United States)

    Önnberg, Anna; Söderquist, Bo; Persson, Katarina; Mölling, Paula

    2014-11-01

    The emergence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a major global concern. CTX-M is the dominating ESBL type worldwide, and CTX-M-15 is the most widespread CTX-M type. The dissemination of CTX-M appears to be in part due to global spread of the Escherichia coli clone O25b-ST131. However, the gene-encoding CTX-M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX-M genes. In this study, 152 CTX-M-producing E. coli isolated in 1999-2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence-based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real-time PCR-based plasmid replicon typing was performed on 82 CTX-M-15-producing E. coli isolates. In general, the CTX-M-producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The blaCTX -M-15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep-PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying blaCTX -M-15 may have occurred between different E. coli strains.

  12. Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice

    Science.gov (United States)

    Gómez, Leonardo A.; Alvarez, Francisco I.; Fernández, Pablo A.; Flores, Manuel R.; Molina, Raúl E.; Coloma, Roberto F.; Oñate, Angel A.

    2016-01-01

    Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis. PMID:27747197

  13. Bacteriophage-based Probiotic Preparation for Managing Shigella Infections

    Science.gov (United States)

    2015-04-16

    for a probiotic preparationbased on naturally occurring bacteriophages – as a way to condition the GI tract’s microflora gently and favorably...The preparation (designated “ShigActive”) is a bacteriophage cocktail that specifically targets Shigella spp. (significant diarrhea-causing pathogens...10-Apr-2013 Approved for Public Release; Distribution Unlimited Final Report: Bacteriophage -based Probiotic Preparation for Managing Shigella

  14. Increasing plasmid transformation efficiency of natural spizizen method in Bacillus Subtilis by a cell permeable peptide

    Directory of Open Access Journals (Sweden)

    Mehrdad Moosazadeh Moghaddam

    2013-01-01

    Full Text Available Introduction: Some of bacterial species are able to uptake DNA molecule from environment, the yield of this process depends on some conditions such as plasmid size and host type. In the case of Bacillus subtilis, DNA uptake has low efficacy. Using Spizizen minimal medium is common method in plasmid transformation into B. subtilis, but rate of this process is not suitable and noteworthy. The aim of this study was investigation of novel method for improvement of DNA transformation into B. subtilis based on CM11 cationic peptide as a membrane permeable agent.Materials and methods: In this study, for optimization of pWB980 plasmid transformation into B. subtilis, the CM11 cationic peptide was used. For this purpose, B. subtilis competent cell preparation in the present of different concentration of peptide was implemented by two methods. In the first method, after treatment of bacteria with different amount of peptide for 14h, plasmid was added. In the second method, several concentration of peptide with plasmid was exposed to bacteria simultaneously. Bacteria that uptake DNA were screened on LB agar medium containing kanamycin. The total transformed bacteria per microgram of DNA was calculated and compared with the control.Results: Plasmid transformation in best conditions was 6.5 folds higher than the control. This result was statistically significant (P value <0.001.Discussion and conclusion: This study showed that CM11 cationic peptide as a membrane permeable agent was able to increase plasmid transformation rate into B. subtilis. This property was useful for resolution of low transformation efficacy.

  15. Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet

    OpenAIRE

    Tett, Adrian; Spiers, Andrew J.; Crossman, Lisa C.; Ager, Duane; Ciric, Lena; Dow, J. Maxwell; Fry, John C.; Harris,David; Lilley, Andrew; Oliver, Anna; Parkhill, Julian; Quail, Michael A; Paul. B. Rainey; Saunders, Nigel J.; Seeger, Kathy

    2007-01-01

    The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome contain...

  16. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

    Directory of Open Access Journals (Sweden)

    Val F Lanza

    2014-12-01

    Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  17. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    Science.gov (United States)

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  18. Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Yan-Bo Liu1; De-Qi Xu; Ling Zhang1; Ya-Xiong Guo; Li-Fang Gao; Xi-Chun Liu; Li-Juan Zhao; Bao-Feng Guo; Li-Jing Zhao; Xue-Jian Zhao

    2012-01-01

    Persistent activation of Survivin and its overexpression contribute to the formation,progression and metastasis of several different tumor types.Therefore,Survivin is an ideal target for RNA interference mediated-growth inhibition.Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth.RNA interference does not fully ablate target gene expression,owing to the idiosyncrasies associated with shRNAs and their targets.To enhance the therapeutic efficacy of Survivin-specific shRNA,we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19).Then,the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S.typhimurium) to treat prostate cancer in vitroand in vivo.We found that the co-expressed Survivin-specific shRNA and GRIM-19synergistically and more effectively inhibited prostate tumor proliferation and survival,when compared with treatment with either single agent alone in vitro and in vivo.This study has provided a novel cancer gene therapeutic approach for prostate cancer.

  19. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  20. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  1. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  2. Preparations, Characterizations and Applications of Chitosan-based Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Chitosan is a natural polysaccharide prepared by the N-deacetylation of chitin. In this paper we have reviewed the methods of preparation of chitosan-based nanoparticles and their pharmaceutical applications. There are five methods of their preparations: emulsion cross-linking, emulsion-droplet coalescence, ionic gelation, reverse micellar method and chemically modified chitosan method. Chitosan nanoparticles are used as carriers for low molecular weight drug, vaccines and DNA. Releasing characteristics, biodistribution and applications are also summarized.

  3. Preparations, characterizations and applications of chitosan-based nanoparticles

    Science.gov (United States)

    Liu, Chenguang; Tan, Yulong; Liu, Chengsheng; Chen, Xiguang; Yu, Lejun

    2007-07-01

    Chitosan is a natural polysaccharide prepared by the N-deacetylation of chitin. In this paper we have reviewed the methods of preparation of chitosan-based nanoparticles and their pharmaceutical applications. There are five methods of their preparations: emulsion cross-linking, emulsion-droplet coalescence, ionic gelation, reverse micellar method and chemically modified chitosan method. Chitosan nanoparticles are used as carriers for low molecular weight drug, vaccines and DNA. Releasing characteristics, biodistribution and applications are also summarized.

  4. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  5. New methods for preparing mercury-based ferrofluids

    DEFF Research Database (Denmark)

    Linderoth, Søren; Rasmussen, L.H.; Mørup, Steen

    1991-01-01

    Metallic ferrofluids based on magnetic particles in mercury have been produced by two new methods. Alloy particles of Fe-B, Fe-Co-B, Fe-Ni-B, and Co-B were prepared by reduction of the transition metal ions in aqueous solutions by NaBH4 and subsequently suspended in mercury. In one preparation...

  6. Competency-Based Reading Preparation for Secondary Teachers.

    Science.gov (United States)

    Getz, Howard G.; Kennedy, Larry D.

    The reading preparation program at Illinois State University represents a fundamental shift in the instructional focus for preservice preparation of secondary level teachers. The competency-based program in reading is largely self-directed and self-paced. Large groups of students are assigned to an instructional team, and the faculty provides…

  7. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  8. [Isolation of the R'his plasmids of Vibrio cholerae].

    Science.gov (United States)

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G

    1987-06-01

    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  9. Development and application of automated systems for plasmid-based functional proteomics to improve syntheitc biology of engineered industrial microbes for high level expression of proteases for biofertilizer production

    Science.gov (United States)

    In addition to microarray technology, which provides a robust method to study protein function in a rapid, economical, and proteome-wide fashion, plasmid-based functional proteomics is an important technology for rapidly obtaining large quantities of protein and determining protein function across a...

  10. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  11. Teacher Preparation: Assessing Teacher Quality, Administrative Support, Standards-Based Teacher Preparation.

    Science.gov (United States)

    Poliakoff, Anne Rogers, Ed.

    2002-01-01

    This collection of papers focuses on the preparation of teachers by university-based education programs. The papers are: "The STEPS That Support P-12 Learning and Achievement" (Diana Rigden), which describes the Standards-based Teacher Education Project (STEP) for improving student learning; "The Responsibility for Assessing Beginning Teachers"…

  12. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Capillarity-based preparation system for optical colorimetric sensor arrays

    Science.gov (United States)

    Luo, Xiao-gang; Yi, Xin; Bu, Xiang-nan; Hou, Chang-jun; Huo, Dan-qun; Yang, Mei; Fa, Huan-bao; Lei, Jin-can

    2017-03-01

    In recent years, optical colorimetric sensor arrays have demonstrated beneficial features, including rapid response, high selectivity, and high specificity; as a result, it has been extensively applied in food inspection and chemical studies, among other fields. There are instruments in the current market available for the preparation of an optical colorimetric sensor array, but it lacks the corresponding research of the preparation mechanism. Therefore, in connection with the main features of this kind of sensor array such as consistency, based on the preparation method of contact spotting, combined with a capillary fluid model, Washburn equation, Laplace equation, etc., this paper develops a diffusion model of an optical colorimetric sensor array during its preparation and sets up an optical colorimetric sensor array preparation system based on this diffusion model. Finally, this paper compares and evaluates the sensor arrays prepared by the system and prepared manually in three aspects such as the quality of array point, response of array, and response result, and the results show that the performance index of the sensor array prepared by a system under this diffusion model is better than that of the sensor array of manual spotting, which meets the needs of the experiment.

  14. Capillarity-based preparation system for optical colorimetric sensor arrays.

    Science.gov (United States)

    Luo, Xiao-Gang; Yi, Xin; Bu, Xiang-Nan; Hou, Chang-Jun; Huo, Dan-Qun; Yang, Mei; Fa, Huan-Bao; Lei, Jin-Can

    2017-03-01

    In recent years, optical colorimetric sensor arrays have demonstrated beneficial features, including rapid response, high selectivity, and high specificity; as a result, it has been extensively applied in food inspection and chemical studies, among other fields. There are instruments in the current market available for the preparation of an optical colorimetric sensor array, but it lacks the corresponding research of the preparation mechanism. Therefore, in connection with the main features of this kind of sensor array such as consistency, based on the preparation method of contact spotting, combined with a capillary fluid model, Washburn equation, Laplace equation, etc., this paper develops a diffusion model of an optical colorimetric sensor array during its preparation and sets up an optical colorimetric sensor array preparation system based on this diffusion model. Finally, this paper compares and evaluates the sensor arrays prepared by the system and prepared manually in three aspects such as the quality of array point, response of array, and response result, and the results show that the performance index of the sensor array prepared by a system under this diffusion model is better than that of the sensor array of manual spotting, which meets the needs of the experiment.

  15. Chondrogenic effect of cell-based scaffold of self-assembling peptides/PLGA-PLL loading the hTGFβ3 plasmid DNA.

    Science.gov (United States)

    Pan, Qiyong; Li, Wenkai; Yuan, Xuefeng; Rakhmanov, Yeltay; Wang, Pengcheng; Lu, Rui; Mao, Zekai; Shang, Xiaobin; You, Hongbo

    2016-01-01

    With the application of tissue engineering to tissue regeneration, additional new complexes have been made in response to the challenge of cartilage-injury repair. This study was performed to construct a rat precartilaginous stem cells-based scaffold of self-assembling peptides RADA16-I/PLGA-PLL (poly-L-lysine coated PLGA) as extracellular matrix loading the NLS-TAT as a peptide-based carrier for a plasmid DNA containing hTGFβ3. After composites were cultured for 1, 2, 3 and 4 weeks, respectively, the results showed that the levels of chondrogenic-related gene expression were higher in the experimental group with and hTGFβ3 gene by reverse transcription-polymerase chain reaction, and with higher histochemical and immunohistochemical expression. hTGFβ3 protein expression had increased at 4 weeks based on western blot analysis. The results of this study show that a complex may be a suitable scaffold for cartilage repair and offer a strategy for tissue regeneration through the use of tissue engineering.

  16. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

    2000-01-01

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  17. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Yonghua Zhang

    2002-05-27

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  18. Characterization of Multidrug-Resistant Escherichia coli by Plasmid Replicon Typing and Pulsed-Field Gel Electrophoresis

    Science.gov (United States)

    Background: Characterization of plasmids has particular clinical significance because genes encoding important traits such as antimicrobial resistance are frequently present in plasmids. Plasmid replicon typing is a multiplex PCR based method that can be used to classify 18 of the 26 known plasmid t...

  19. [A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance].

    Science.gov (United States)

    Sahin, Fikret; Karasartova, Djursun; Gerçeker, Devran; Aysev, A Derya; Erdem, Birsel

    2008-07-01

    In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

  20. Preparation and Properties of Tung Oil-Based Polyurethane

    Institute of Scientific and Technical Information of China (English)

    袁才登; 赵晓明; 邵丽英; 唐克华

    2014-01-01

    Tung oil-based polyols were synthesized by the esterification and transesterification between Tung oil-based anhydride and butanediol. The hydroxyl values of the polyols prepared were tested and discussed. Polyurethane was prepared by using Tung oil-based polyols and/or poly(propylene glycol) as polyols and by using isophorone diisocyanate as isocyanate. The effect of the ratio of Tung oil-based polyols to poly(propylene glycol) on the proper-ties of polyurethane prepared was investigated by the water resistance, alcohol resistance and hardness tests. The re-sults show that Tung oil-based polyols are effective to improve the hardness, water resistance and alcohol resistance of polyurethane.

  1. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  2. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  3. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    YiNi; KeMa; JingNi; XiujuanZheng; YingWang; SidongXiong

    2004-01-01

    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs. Cellular & Molecular Immunology.

  4. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    Yi Ni; Ke Ma; Jing Ni; Xiujuan Zheng; Ying Wang; Sidong Xiong

    2004-01-01

    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.

  5. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  6. A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.

    Science.gov (United States)

    Misra, Santosh K; Naz, Sarwat; Kondaiah, Paturu; Bhattacharya, Santanu

    2014-01-01

    The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet.

    Science.gov (United States)

    Tett, Adrian; Spiers, Andrew J; Crossman, Lisa C; Ager, Duane; Ciric, Lena; Dow, J Maxwell; Fry, John C; Harris, David; Lilley, Andrew; Oliver, Anna; Parkhill, Julian; Quail, Michael A; Rainey, Paul B; Saunders, Nigel J; Seeger, Kathy; Snyder, Lori A S; Squares, Rob; Thomas, Christopher M; Turner, Sarah L; Zhang, Xue-Xian; Field, Dawn; Bailey, Mark J

    2007-08-01

    The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome containing 478 CDSs and an exceptional degree of genetic novelty; 80% of predicted coding sequences cannot be ascribed a function and 60% are orphans. Of those to which function could be assigned, 40% bore greatest similarity to sequences from Pseudomonas spp, and the majority of the remainder showed similarity to other gamma-proteobacterial genera and plasmids. pQBR103 has identifiable regions presumed responsible for replication and partitioning, but despite being tra+ lacks the full complement of any previously described conjugal transfer functions. The DNA sequence provided few insights into the functional significance of plant-induced transcriptional regions, but suggests that 14% of CDSs may be expressed (11 CDSs with functional annotation and 54 without), further highlighting the ecological importance of these novel CDSs. Comparative analysis indicates that pQBR103 shares significant regions of sequence with other plasmids isolated from sugar beet plants grown at the same geographic location. These plasmid sequences indicate there is more novelty in the mobile DNA pool accessible to phytosphere pseudomonas than is currently appreciated or understood.

  8. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Directory of Open Access Journals (Sweden)

    Bryon A Nicholson

    Full Text Available Neonatal Meningitis Escherichia coli (NMEC is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

  9. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system.

    Science.gov (United States)

    Szczepanowski, Rafael; Krahn, Irene; Linke, Burkhard; Goesmann, Alexander; Pühler, Alfred; Schlüter, Andreas

    2004-11-01

    Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP

  10. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  11. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  12. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  13. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    larger than previously assumed. I was able to show abundant plasmid transfer from the Gram negative donor strains to a wide diversity of Gram positive soil bacteria, formerly thought to constitute distinct clusters of gene transfer. Moreover, among the observed transconjugants, I identified a core super...... environmental factors that modulate plasmid transfer in soil microbial communities. In order to attain these goals, I developed a high-throughput method that enabled me to evaluate the permissiveness of bacterial communities towards introduced plasmids. This new approach is based on the introduction...... fraction of soil the bacteria (up to 1 in 10,000) were able to take up any of these broad host range conjugal plasmids. The transconjugal pools comprised 11 bacterial phyla. This finding indicates that the realized transfer range of broad host range plasmids in environmental microbial communities is much...

  14. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  15. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  16. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  17. Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA.

    Science.gov (United States)

    Jadhav, Vaibhav; Maiti, Souvik; Dasgupta, Antara; Das, Prasanta Kumar; Dias, Rita S; Miguel, Maria G; Lindman, Björn

    2008-07-01

    The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; thus, the accessibility of DNA to small molecules is preserved to a certain extent. The presence of more hydrophobic groups surrounding the positive amino charge also gave rise to a significantly lower cytotoxicity. The surfactant self-assembly pattern is quite different without and with DNA, illustrating the roles of electrostatic and steric effects in determining the effective shape of a surfactant molecule.

  18. Preparation and characterization of keratin-based biocomposite hydrogels prepared by electron beam irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Mira; Kim, Byoung-Suhk [Department of Organic Materials and Fiber Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shin, Hye Kyoung [Department of Chemistry, Inha University, 100 Inharo, Incheon 402-751 (Korea, Republic of); Park, Soo-Jin, E-mail: sjpark@inha.ac.kr [Department of Chemistry, Inha University, 100 Inharo, Incheon 402-751 (Korea, Republic of); Kim, Hak-Yong, E-mail: khy@jbnu.ac.kr [Department of Organic Materials and Fiber Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2013-12-01

    The biocompatible and highly porous keratin-based hydrogels were prepared using electron beam irradiation (EBI). The conditions for keratin-based hydrogel formation were investigated depending on several conditions, including the presence of poly(vinyl alcohol) (PVA), concentration of keratin solution, EBI dose, and poly(ethylene imine) (PEI) additives. The pure keratin (human hair and wool) aqueous solution was not gelled by EBI, while the aqueous keratin solutions blended with PVA were gelled at an EBI dose of more than 90 kGy. Furthermore, in the presence of PEI, the aqueous keratin solution blended with PVA could be gelled at a considerably lower EBI dose, even at 10 kGy. This finding suggests that the PEI additives significantly influence the rate of gelation and that PEIs function as an accelerator during gelation. The resulting keratin-based hydrogels were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), gel fraction, degree of swelling, gel strength, and kinetics of swelling analyses. - Highlights: • The biocompatible and highly porous keratin-based hydrogels were prepared using EBI. • The conditions for keratin-based hydrogel formation were examined. • PEI would play an accelerator role in the formation of keratin-based hydrogels. • The resulting keratin-based hydrogels are expected to be more environmentally friendly.

  19. Polymer ring resonator based devices prepared by DLW

    Science.gov (United States)

    Jandura, D.; Pudis, D.; Gaso, P.; Goraus, M.

    2017-05-01

    In this paper, the fabrication method of waveguide structures and devices as ring resonators for different waveguide applications based on polymer material is presented. The structures were designed in computer-aided design (CAD) software and two-photon polymerization lithography system was used for preparation of desired devices. Morphological properties of prepared devices were investigated using scanning electron microscope (SEM) and confocal microscope. Finally, we performed measurement of optical spectrum characteristics in telecommunication wavelengths range. The results corresponds to calculated parameters. Final polymer devices are promising for lab on a chip and sensing applications due to unique elastic and chemical properties.

  20. Complex nature of enterococcal pheromone-responsive plasmids.

    Science.gov (United States)

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria

    2010-01-01

    Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

  1. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

    NARCIS (Netherlands)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolo

  2. 基于色谱法的超螺旋质粒DNA纯化与分析进展%Advances on Purification Process and Analysis of Supercoiled Plasmid DNA Based on Chromatography

    Institute of Scientific and Technical Information of China (English)

    李亮; 柳方方; 宛煜嵩; 金芜军

    2014-01-01

    质粒DNA含有独立复制的遗传结构,是基因工程的常用工具,广泛应用于分子生物学基础研究、农业转基因检测、医疗诊断与基因治疗等领域。质粒DNA的构型一般分为超螺旋、开口环状及线性3种。初步纯化的质粒DNA溶液中常混有3种构型,并且掺杂一定量的蛋白质、RNA、内毒素以及宿主基因组DNA,这些杂质会影响后续的应用,因此质粒DNA需要进一步精细纯化。该文对质粒DNA的新应用领域、基于色谱的精细纯化技术及产物质量分析体系进行了综述,并展望了高纯度质粒DNA精细纯化及产物分析的发展方向。%Plasmid DNA,a common tool of genetic engineering containing the independent genetic structure of the replication,is widely used in basic research of molecular biology,genetically modi-fied detection,medical diagnosis and gene therapy. Plasmid DNA conformations are generally divided into three types:supercoiled,open circle and linear. Crude plasmid DNA often mixes with three configurations,and the amount of protein,RNA,endotoxin with the host genome DNA. These im-purities need to be further refined as they affect subsequent applications. The new applications of plasmid DNA,refined technologies and products based on chromatography system,and quality anal-ysis of purified plasmid DNA is reviewed in this paper. Finally,the trends of technology of high pu-rity plasmid DNA and analysis of refined product are presented.

  3. Fuel cell membrane preparation: effects of base polymer

    Energy Technology Data Exchange (ETDEWEB)

    Brack, H.P.; Scherer, G.G. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1997-06-01

    Radiation grafted films and membranes prepared from the partially fluorinated base copolymer poly(ethylene-alt-tetrafluoroethylene) or ETFE have better mechanical properties than those prepared from poly(tetrafluoroethylene-co-hexafluoropropylene) or FEP. The influence of the base copolymer film type on the grafting rate and yields is reported in the present investigation. An understanding of the effects of these parameters is important so that the grafting process can be carried out reproducibly in as short a time as possible. The grafting rate and yield as a function of the irradiation dose has been found to be much higher for the partially fluorinated base copolymer ETFE. (author) 2 figs., 1 tab., 5 refs.

  4. Preparation and characterization of crosslinked chitosan-based nanofibers

    Institute of Scientific and Technical Information of China (English)

    Ying Shan Zhou; Dong Zhi Yang; Jun Nie

    2007-01-01

    Crosslinked chitosan-based nanofibers were successfully prepared via electrospinning technique with heat mediated chemical crosslinking followed. The structure, morphology and mechanical property of nanofibers were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), Instron machine, respectively. The results showed that, nanofibers exhibited a smooth surface and regular morphology, and tensile strength of nanofibers improved with increasing of triethylene glycol dimethacrylate (TEGDMA) content.

  5. Preparation of bacterial cellulose based hydrogels and their viscoelastic behavior

    OpenAIRE

    2015-01-01

    Bacterial cellulose (BC) based hydrogels have been prepared in blended with carboxymethylcellulose and polyvinyl pyrrolidone by using heat treatment. The properties of BC-CMC and BC-PVP hydrogels were compared with pure BC, CMC and PVP hydrogels. These hydrogels were investigated by measuring their structural, morphological and viscoelastic properties. Through the morphological images, alignment of the porous flake like structures could be seen clearly within the inter-polymeric network of th...

  6. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  7. Chitosan-Based Matrices Prepared by Gamma Irradiation for Tissue Regeneration: Structural Properties vs. Preparation Method.

    Science.gov (United States)

    Casimiro, Maria Helena; Lancastre, Joana J H; Rodrigues, Alexandra P; Gomes, Susana R; Rodrigues, Gabriela; Ferreira, Luís M

    2017-02-01

    In the last decade, new generations of biopolymer-based materials have attracted attention, aiming its application as scaffolds for tissue engineering. These engineered three-dimensional scaffolds are designed to improve or replace damaged, missing, or otherwise compromised tissues or organs. Despite the number of promising methods that can be used to generate 3D cell-instructive matrices, the innovative nature of the present work relies on the application of ionizing radiation technology to form and modify surfaces and matrices with advantage over more conventional technologies (room temperature reaction, absence of harmful initiators or solvents, high penetration through the bulk materials, etc.), and the possibility of preparation and sterilization in one single step. The current chapter summarizes the work done by the authors in the gamma radiation processing of biocompatible and biodegradable chitosan-based matrices for skin regeneration. Particular attention is given to the correlation between the different preparation conditions and the final polymeric matrices' properties. We therefore expect to demonstrate that instructive matrices produced and improved by radiation technology bring to the field of skin regenerative medicine a supplemental advantage over more conservative techniques.

  8. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

    Science.gov (United States)

    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  9. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  10. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    Science.gov (United States)

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  11. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  12. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    Science.gov (United States)

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  13. Preparation and properties of copper-oil-based nanofluids

    Directory of Open Access Journals (Sweden)

    Xie Wenjie

    2011-01-01

    Full Text Available Abstract In this study, the lipophilic Cu nanoparticles were synthesized by surface modification method to improve their dispersion stability in hydrophobic organic media. The oil-based nanofluids were prepared with the lipophilic Cu nanoparticles. The transport properties, viscosity, and thermal conductivity of the nanofluids have been measured. The viscosities and thermal conductivities of the nanofluids with the surface-modified nanoparticles have higher values than the base fluids do. The composition has more significant effects on the thermal conductivity than on the viscosity. It is valuable to prepare an appropriate oil-based nanofluid for enhancing the heat-transfer capacity of a hydrophobic system. The effects of adding Cu nanoparticles on the thermal oxidation stability of the fluids were investigated by measuring the hydroperoxide concentration in the Cu/kerosene nanofluids. The hydroperoxide concentrations are observed to be clearly lower in the Cu nanofluids than in their base fluids. Appropriate amounts of metal nanoparticles added in a hydrocarbon fuel can enhance the thermal oxidation stability.

  14. Preparation and properties of copper-oil-based nanofluids.

    Science.gov (United States)

    Li, Dan; Xie, Wenjie; Fang, Wenjun

    2011-05-05

    In this study, the lipophilic Cu nanoparticles were synthesized by surface modification method to improve their dispersion stability in hydrophobic organic media. The oil-based nanofluids were prepared with the lipophilic Cu nanoparticles. The transport properties, viscosity, and thermal conductivity of the nanofluids have been measured. The viscosities and thermal conductivities of the nanofluids with the surface-modified nanoparticles have higher values than the base fluids do. The composition has more significant effects on the thermal conductivity than on the viscosity. It is valuable to prepare an appropriate oil-based nanofluid for enhancing the heat-transfer capacity of a hydrophobic system. The effects of adding Cu nanoparticles on the thermal oxidation stability of the fluids were investigated by measuring the hydroperoxide concentration in the Cu/kerosene nanofluids. The hydroperoxide concentrations are observed to be clearly lower in the Cu nanofluids than in their base fluids. Appropriate amounts of metal nanoparticles added in a hydrocarbon fuel can enhance the thermal oxidation stability.

  15. Preparation and Characterization of Sugar Based Catalyst on Various Supports

    Directory of Open Access Journals (Sweden)

    Jidon Adrian Janaun

    2017-04-01

    Full Text Available A novel structured carbon-based acid catalyst was prepared by depositing the carbon precursor onto glass, ceramic and aluminum supports via dip-coating method, followed by carbonization process for converting the d-glucose layer into black carbon char in an inert nitrogen environment at 400 °C. Then, the –SO3H group was introduced into the framework of the carbon char by multiple vapor phase sulfonation. Four different carbonization methods were carried out (dry pyrolysis and hydrothermal carbonization with or without pressurized in the catalyst preparation while among the carbonization methods, the samples which prepared from dry pyrolysis without pressurized process showed the strong acidity due to highest adsorption of acid group in the catalyst surface although the catalyst attached onto the support was the least compared to other preparation methods. Among the catalysts, the sulfonated carbon-base catalyst that is attached on the ceramic support exhibited the highest aci-dity (1.327 mmol/g followed by the catalyst deposited on the glass (0.917 mmol/g and aluminum (0.321 mmol/g supports. The porous structure of ceramic surface, allowed a better interaction between reactants and –SO3H site in the carbon. Through the FT-IR analysis, it was observed that the functional groups –COOH, –OH, and –SO3H were present in the active sites of the catalysts. The surface areas of  glass (Si–SC, ceramic (Ce–SC and aluminum (Al–SC catalysts were larger than 1 m2/g, whereas the pore size belongs to macroporous as the average pore size is more than 50 nm. It is also stable within the temperature of 400 °C as there was less than 10% weight loss revealed from the TGA analysis. Copyright © 2017 BCREC GROUP. All rights reserved Received: 20th April 2016; Revised: 14th October 2016; Accepted: 17th October 2016 How to Cite: Janaun, J.A., Mey, T.J., Bono, A., Krishnaiah, D. (2017. Preparation and Characterization of Sugar Based Catalyst on Various

  16. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

    Science.gov (United States)

    Kananavičiūtė, Rūta; Butaitė, Elena; Citavičius, Donaldas

    2014-01-01

    We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

  17. Industrial Preparation of Bauxite-based Mullite Raw Material

    Institute of Scientific and Technical Information of China (English)

    YANG Zhongzheng; DING Baohua

    2008-01-01

    Bauxite-based mullite grogs were prepared with grade II bauxite(≤3mm)and coal gangue(≤3mm)as starting materials with formula of w(Al2O3)=68%~72%.Mill the mixture to particle size≤0.044mm by wet milling to homogenize chemical composition. After shaping by wet extrusion and drying, the materials were fired at 1550℃, 1600℃ and 1700℃ for 6 hours respectively. After cooling apparent porosity and bulk density of fired materials were tested. The results show as follows: the appropriate sintering temperature for bauxite-based mullite with homogeneous and consistent composition and microstructure and properties is 1600℃,at which the mullite has apparent porosity≤1.5%,bulk density≥2.81g·cm-3and refractoriness under load is 1610-1650℃.Compared with the laboratory results, the sintering temperature is lowered about 100℃,bulk density increased 0.06g?cm-3.The industrial feasibility of preparing bauxite-based mullite grogs with the processing is confirmed.

  18. Preparation of perlite-based carbon dioxide absorbent.

    Science.gov (United States)

    He, H; Wu, L; Zhu, J; Yu, B

    1994-02-01

    A new highly efficient carbon dioxide absorbent consisting of sodium hydroxide, expanded perlite and acid-base indicator was prepared. The absorption efficiency, absorption capacity, flow resistance and color indication for the absorbent were tested and compared with some commercial products. The absorbent can reduce the carbon dioxide content in gases to 3.3 ppb (v/v) and absorbs not less than 35% of its weight of carbon dioxide. Besides its large capacity and sharp color indication, the absorbent has an outstanding advantage of small flow resistance in comparison with other commercial carbon dioxide absorbents. Applications in gas analysis and purification were also investigated.

  19. Plant Growth Promoting Bacteria from Cow Dung Based Biodynamic Preparations

    OpenAIRE

    Radha, T. K.; RAO, D. L. N.

    2014-01-01

    Indigenous formulations based on cow dung fermentation are commonly used in organic farming. Three biodynamic preparations viz., Panchagavya (PG), BD500 and ‘Cow pat pit’ (CPP) showed high counts of lactobacilli (109 ml−1) and yeasts (104 ml−1). Actinomycetes were present only in CPP (104 ml−1) and absent in the other two. Seven bacterial isolates from these ferments were identified by a polyphasic approach: Bacillus safensis (PG1), Bacillus cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysi...

  20. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    Science.gov (United States)

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  1. Preparation and Evaluation of Inhalable Itraconazole Chitosan Based Polymeric Micelles

    Directory of Open Access Journals (Sweden)

    Esmaeil Moazeni

    2012-12-01

    Full Text Available Background: This study evaluated the potential of chitosan based polymeric micelles as a nanocarrier system for pulmonary delivery of itraconazole (ITRA.Methods: Hydrophobically modified chitosan were synthesized by conjugation of stearic acid to the hydrophilic depolymerized chitosan. FTIR and 1HNMR were used to prove the chemical structure and physical properties of the depolymerized and the stearic acid grafted chitosan. ITRA was entrapped into the micelles and physicochemical properties of the micelles were investigated. Fluorescence spectroscopy, dynamic laser light scattering andtransmission electron microscopy were used to characterize the physicochemical properties of the prepared micelles. The in vitro pulmonary profile of polymeric micelles was studied by an air-jet nebulizer connected to a twin stage impinger.Results: The polymeric micelles prepared in this study could entrap up to 43.2±2.27 μg of ITRA per milliliter. All micelles showed mean diameter between 120–200 nm. The critical micelle concentration of the stearic acid grafted chitosan was found to be 1.58×10-2 mg/ml. The nebulization efficiency was up to 89% and the fine particle fraction (FPF varied from 38% to 47%. The micelles had enough stability to remain encapsulation of the drug during nebulization process.Conclusions: In vitro data showed that stearic acid grafted chitosan based polymeric micelles has a potential to be used as nanocarriers for delivery of itraconazole through inhalation.

  2. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

    Science.gov (United States)

    Wood, Whitney N; Smith, Kyle D; Ream, Jennifer A; Kevin Lewis, L

    2017-02-01

    Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Polymer/Carbon-Based Hybrid Aerogels: Preparation, Properties and Applications

    Directory of Open Access Journals (Sweden)

    Lizeng Zuo

    2015-10-01

    Full Text Available Aerogels are synthetic porous materials derived from sol-gel materials in which the liquid component has been replaced with gas to leave intact solid nanostructures without pore collapse. Recently, aerogels based on natural or synthetic polymers, called polymer or organic aerogels, have been widely explored due to their porous structures and unique properties, such as high specific surface area, low density, low thermal conductivity and dielectric constant. This paper gives a comprehensive review about the most recent progresses in preparation, structures and properties of polymer and their derived carbon-based aerogels, as well as their potential applications in various fields including energy storage, adsorption, thermal insulation and flame retardancy. To facilitate further research and development, the technical challenges are discussed, and several future research directions are also suggested in this review.

  4. Preparation and electrical properties of oil-based magnetic fluids

    Science.gov (United States)

    Sartoratto, P. P. C.; Neto, A. V. S.; Lima, E. C. D.; Rodrigues de Sá, A. L. C.; Morais, P. C.

    2005-05-01

    This paper describes an improvement in the preparation of magnetic fluids for electrical transformers. The samples are based on surface-coated maghemite nanoparticles dispersed in transformer insulating oil. Colloidal stability at 90°C was higher for oleate-grafted maghemite-based magnetic fluid, whereas decanoate and dodecanoate-grafted samples were very unstable. Electrical properties were evaluated for samples containing 0.80%-0.0040% maghemite volume fractions. Relative permittivity varied from 8.8 to 2.1 and the minimum value of the loss factor was 12% for the most diluted sample. The resistivity falls in the range of 0.7-2.5×1010Ωm, whereas the ac dielectric strength varied from 70to79kV. These physical characteristics reveal remarkable step forward in the properties of the magnetic fluid samples and may result in better operation of electrical transformers.

  5. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  6. In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina.

    Science.gov (United States)

    Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra

    2016-11-22

    The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

  7. In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina

    Directory of Open Access Journals (Sweden)

    Maria Carmela Latella

    2016-01-01

    Full Text Available The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

  8. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  10. Plant growth promoting bacteria from cow dung based biodynamic preparations.

    Science.gov (United States)

    Radha, T K; Rao, D L N

    2014-12-01

    Indigenous formulations based on cow dung fermentation are commonly used in organic farming. Three biodynamic preparations viz., Panchagavya (PG), BD500 and 'Cow pat pit' (CPP) showed high counts of lactobacilli (10(9) ml(-1)) and yeasts (10(4) ml(-1)). Actinomycetes were present only in CPP (10(4) ml(-1)) and absent in the other two. Seven bacterial isolates from these ferments were identified by a polyphasic approach: Bacillus safensis (PG1), Bacillus cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysinibacillus xylanilyticus (BD3) and Bacillus licheniformis (CPP1). This is the first report of L. xylanilyticus and B. licheniformis in biodynamic preparations. Only three carbon sources-dextrose, sucrose and trehalose out of 21 tested were utilized by all the bacteria. None could utilize arabinose, dulcitol, galactose, inositol, inulin, melibiose, raffinose, rhamnose and sorbitol. All the strains produced indole acetic acid (1.8-3.7 μg ml(-1) culture filtrate) and ammonia. None could fix nitrogen; but all except B. safensis and B. licheniformis could solubilize phosphorous from insoluble tri-calcium phosphate. All the strains except L. xylaniliticus exhibited antagonism to the plant pathogen Rhizoctonia bataticola whereas none could inhibit Sclerotium rolfsi. In green house experiment in soil microcosms, bacterial inoculation significantly promoted growth of maize; plant dry weight increased by ~21 % due to inoculation with B. cereus (PG2). Results provide a basis for understanding the beneficial effects of biodynamic preparations and industrial deployment of the strains.

  11. Preparation of carboxymethyl cellulose based microgels for cell encapsulation

    Directory of Open Access Journals (Sweden)

    Y. Ke

    2014-11-01

    Full Text Available Biocompatible and biodegradable carboxymethyl cellulose (CMC has been modified with 4-hydroxybenzylamine (CMC-Ph in order to prepare CMC-based microgels through the horseradish peroxidise/hydrogen peroxide enzymatic reaction. CMC-Ph was identified as a blend, and the amount of the grafted 4-hydroxybenzylamine per 100 units of CMC was between 17 and 23 according to the molecular weight of CMC. Through a special designed co-flowing microfluidic device, CMC-Ph microgels were prepared with the radius from 100 to 500 μm via adjusting the flow rates of the disperse phase and the continuous phase, respectively. The chondrocytic cell line ATDC5 was encapsulated in the CMC-Ph microgels. The cell-laden microgels were cultured for up to 40 days, illustrating the biocompatibility of CMC-Ph and the microfluidic approach through the enzymatic crosslinking reaction primarily. CMC-Ph showed a great promise to encapsulate the cells for further fabrication of the injectable scaffolds.

  12. Preparation and properties of pitch carbon based supercapacitor

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Using the mesophase pitch as precursor, KOH and CO2 as activated agents, the activated carbon electrode material was fabricated by physical-chemical combined activated technique for supercapacitor. The influence of activated process on the pore structure of activated carbon was analyzed and 14 F supercapacitor with working voltage of 2.5 V was prepared. The charge and discharge behaviors, the properties of cyclic voltammetry, specific capacitance, equivalent serials resistance (ESR), cycle properties, and temperature properties of prepared supercapacitor were examined. The cyclic voltammetry curve results indicate that the carbon based supercapacitor using the self-made activated carbon as electrode materials shows the desired capacitance properties. In 1 mol/L Et4NBFVAN electrolyte, the capacitance and ESR of the supercapacitor are 14.7 F and 60 mΩ, respectively. The specific capacitance of activated carbon electrode materials is 99.6 F/g; its energy density can reach 2.96 W·h/kg under the large current discharge condition. There is no obvious capacitance decay that can be observed after 5000 cycles. The leakage current is below 0.2 mA after keeping the voltage at 2.5 V for 1 h. Meanwhile, the supercapacitor shows desired temperature property; it can be operated normally in the temperature ranging from -40 ℃ to 70 ℃.

  13. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  14. Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack blaCMY in the A/C plasmid backbone

    Science.gov (United States)

    The objective of this study was to gain a better understanding of the conjugative transfer of antimicrobial resistance plasmids from 205 Salmonella enterica strains, isolated from cattle to E. coli or Salmonella recipients. PCR-based replicon typing (PBRT) was used to type incompatibility plasmid r...

  15. Construction and identification of plasmid-based replicon vector system derived from Alphavirus%质粒型甲病毒复制子载体系统的构建及其功能鉴定

    Institute of Scientific and Technical Information of China (English)

    张凤娟; 唐丽; 尉研; 王焕琴; 杨蕴芝; 吴萌; 张鹏; 梁国栋; 朱武洋

    2014-01-01

    目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.%Objective To construct a plasmid-based replicon vector system derived from Alphavirus,and its functions were identified.Methods based on XJ-160 viral infectious cDNA clone,the virus non-structural gene sequence were devided into three fragments to amplify and then cloned into the eukaryotic expression vector pVAX1 CMV promoter downstream by step clone.A multiple cloning site sequence replacing the virus structural gene,plasmid-based replicon vector derived from XJ-160 virus was constructed.Virus nucleoprotein gene and envelope glycoprotein gene were respectively cloned into pVAX1 CMV promoter downstream,constructing two helper plasmid vectors.Then by the expression of green fluorescent protein reporter gene and renilla luciferase reporter gene we test the function of the plasmid-based replicon vector system.Results The plasmid-based replicon vector system derived from XJ-160 virus was successfully constructed,and green fluorescent protein reporter gene and renilla luciferase reporter gene were successfully expressed

  16. [Peculicidal activity of plant essential oils and their based preparations].

    Science.gov (United States)

    Lopatina, Iu V; Eremina, O Iu

    2014-01-01

    The peculicidal activity of eight plant essential oils in 75% isopropyl alcohol was in vitro investigated. Of them, the substances that were most active against lice were tea tree (Melaleuca), eucalyptus, neem, citronella (Cymbopogon nardus), and clove (Syzygium aromaticum) oils; KT50 was not more than 3 minutes on average; KT95 was 4 minutes. After evaporating the solvent, only five (tea tree, cassia, clove, anise (Anisum vulgare), and Japanese star anise (Illicium anisatum) oils) of the eight test botanical substances were active against lice. At the same time, KT50 and KT95 showed 1.5-5-fold increases. Citronella and anise oils had incomplete ovicidal activity. Since the lice were permethrin-resistant, the efficacy of preparations based on essential oils was much higher than permethrin.

  17. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  18. The Native Plasmid pML21 Plays a Role in Stress Tolerance in Enterococcus faecalis ML21, as Analyzed by Plasmid Curing Using Plasmid Incompatibility.

    Science.gov (United States)

    Zuo, Fang-Lei; Chen, Li-Li; Zeng, Zhu; Feng, Xiu-Juan; Yu, Rui; Lu, Xiao-Ming; Ma, Hui-Qin; Chen, Shang-Wu

    2016-02-01

    To investigate the role of the native plasmid pML21 in Enterococcus faecalis ML21's response to abiotic stresses, the plasmid pML21 was cured based on the principle of plasmid incompatibility and segregational instability, generating E. faecalis mutant strain ML0. The mutant and the wild strains were exposed to abiotic stresses: bile salts, low pH, H2O2, ethanol, heat, and NaCl, and their survival rate was measured. We found that curing of pML21 lead to reduced tolerance to stress in E. faecalis ML0, especially oxidative and osmotic stress. Complementation analysis suggested that the genes from pML21 played different role in stress tolerance. The result indicated that pML21 plays a role in E. faecalis ML21's response to abiotic stresses.

  19. Preparation of bacterial cellulose based hydrogels and their viscoelastic behavior

    Science.gov (United States)

    Shah, Rushita; Vyroubal, Radek; Fei, Haojei; Saha, Nabanita; Kitano, Takeshi; Saha, Petr

    2015-04-01

    Bacterial cellulose (BC) based hydrogels have been prepared in blended with carboxymethylcellulose and polyvinyl pyrrolidone by using heat treatment. The properties of BC-CMC and BC-PVP hydrogels were compared with pure BC, CMC and PVP hydrogels. These hydrogels were investigated by measuring their structural, morphological and viscoelastic properties. Through the morphological images, alignment of the porous flake like structures could be seen clearly within the inter-polymeric network of the hydrogels. Also, the detail structure analysis of the polymers blended during the hydrogel formation confirms their interactions with each other were studied. Further, the viscoelastic behavior of all the hydrogels in terms of elastic and viscous property was studied. It is observed that at 1% strain, including CMC and PVP hydrogels, all the BC based hydrogels exhibited the linear trend throughout. Also the elastic nature of the material remains high compared to viscous nature. Moreover, the changes could be noticed in case of blended polymer based hydrogels. The values of complex viscosity (η*) decreases with increase in angular frequency within the range of ω = 0.1-100 rad.s-1.

  20. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  1. Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

    Science.gov (United States)

    Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

    2015-01-01

    Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements.

  2. INVESTIGATION OF POLYDL-LACTIDE-b-POLY(ETHYLENE GLYCOL)-b-POLYDL-LACTIDE MICROSPHERES CONTAINING PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    Yu Liu; Long Huang; Xiao-rong Qiao; Xian-mo Deng; Wen-xiang Jia; Xiao-hong Li

    2004-01-01

    PolyDL-lactide (PDLLA) and the block copolymer, polyDL-lactide-b-poly(ethylene glycol)-b-polyDL-lactide (PELA) were used as the microsphere matrix to encapsulate plasmid DNA. The PDLLA, PELA, pBR322-1oaded PDLLA and pBR322-1oaded PELA microspheres were prepared by solvent extraction method based on the formation of multiple w1/o/w2 emulsion. The microspheres were characterized by surface morphology, mean particle size, particle size distribution and loading efficiency. The integrity of DNA molecules after being extracted from microspheres was determined by agarose gel electrophoresis. The result suggested that plasmid DNA molecules could retain their integrity after being encapsulated by PELA. The PELA microspheres could prevent plasmid DNA from being digested by DNase. The in vitro degradation and release profiles of plasmid DNA-loaded microspheres were measured in pH = 7.4 buffer solution at 37℃. The in vitro degradation profiles of the microspheres were evaluated by the deterioration in microspheres surface morphology, the molecular weight reduction of polymer, the mass loss of microspheres, the changes of pH values of degradation medium, and the changes of particle size. The in vitro release profiles of the microspheres were assessed by measurement of the amount of DNA presented in the release medium at determined intervals. The release profiles were correlation with the degradation profiles. The release of plasmid DNA from PELA microspheres showed a similar biphasic trend, that is, an initial burst release was followed by a slow, but sustained release.

  3. A classification system for plasmids from Enterococci and other Gram-positive bacteria

    DEFF Research Database (Denmark)

    Jensen, Lars Bogø; Garcia-Migura, Lourdes; Valenzuela, Antonio Jesus Sanchez

    2010-01-01

    A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiati...

  4. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    Science.gov (United States)

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  5. PREPARATION AND CHARACTERIZATION OF AMIDATED PECTIN BASED POLYMER ELECTROLYTE MEMBRANES

    Institute of Scientific and Technical Information of China (English)

    R.K.Mishra; A.Anis; S.Mondal; M.Dutt; A.K.Banthia

    2009-01-01

    The work presents the synthesis and characterization of ami dated pectin(AP)based polymer electrolyte membranes(PEM)crosslinked with glutaraldehyde(GA).The prepared membranes are characterized by Fourier transform infrared spectroscopy(FTIR),organic elemental analysis,X-ray diffraction studies(XRD),thermogravimetric analysis (TGA)and impedance spectroscopy.Mechanical properties of the membranes are evaluated by tensile tests.The degree of amidation(DA),molar and mass reaction yields(YM and YN)are calculated based on the results of organic elemental analysis.FTIR spectroscopy indicated the presence of primary and secondary amide absorption bands.XRD pattern of membranes clearly indicates that there is a considerable increase in crystallinity as compared to parent pectin.TGA studies indicate that AP is less thermally stable than reference pectin.A maximum room temperature conductivity of 1.098×10-3 Scm-1 is obtained in the membrane,which is designated as AP-3.These properties make them good candidates for low cost biopolymer electrolyte membranes for fuel cell applications.

  6. Plasmid Isolation in Legionella pneumophila and Legionella-like Organisms.

    Science.gov (United States)

    1980-08-22

    834. 14. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen. 1978. A multiple plasmic-containing Escherichi coli strain...smaller 20 Mdal cryptic plasmid and was used as a control marker with the screening procedure. Escherichia coli V517 was supplied by E. M. Lederberg...Tris-borate buffer. This purified preparation was suitable for electrophoresis. Molecular weight estimates. Escherichia coli V517 was employed as an

  7. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

    Directory of Open Access Journals (Sweden)

    Olson Daniel G

    2012-05-01

    Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

  8. A New Fluorescence Immunosensing Method Based on Aptamer-plasmid Complex Amplification%基于核酸适配体-质粒DNA复合物信号放大的荧光免疫传感技术

    Institute of Scientific and Technical Information of China (English)

    朱静; 黄勇; 蒋小平; 谭钟扬; 蒋健晖; 沈国励; 俞汝勤

    2009-01-01

    提出了一种简便、高灵敏的荧光免疫传感新技术,通过抗体/抗原/核酸适配体-质粒DNA复合物的特异性识别与双链质粒DNA与荧光染料SYBR Green Ⅰ的嵌合作用,实现对血小板衍生增长因子BB(PDGF-BB)的检测.生物识别反应在微孔板中进行,PDGF-BB抗原与微孔板底部预包被的PDGF-BB抗体免疫反应后,加入核酸适配体-质粒DNA复合物与抗原形成夹心复合物.加入DNA双链嵌合染料SYBR Green Ⅰ与夹心复合物的双链DNA部分结合可产生强荧光,其荧光强度可用于定量测定PDGF-BB浓度.实验考察了离子浓度、核酸适配体的延伸引物片段与质粒PUC19的反应比例、染料SYBR Green Ⅰ浓度等分析条件对荧光信号的影响.在优化反应条件下,PDGF-BB检测的线性范围为0.2~200 μg/L,检出限为0.1 μg/L,并且实现了对人血清中PDGF-BB的定量检测.%A novel simple,sensitive fluorescence immunosensing method based on aptamer-plasmid complex amplification was developed. This method utilized the specific recognition between antibody and antigen as well as aptamer-plasmid complex and the intercalation of fluorescence dye SYBR Green Ⅰ in the groove of duplex plasmid DNA in detection of Platelet-Derived Growth Factor BB (PDGF-BB). The immunoassay was performed in the microtiter wells in which rabbit anti PDGF-BB antibody was immobilized. The PDGF BB analyte was captured by the primary antibody and then sandwiched by the aptamer-plasmid DNA complex. The introduction of fluorescence dye SYBR Green Ⅰ allows for the detection of the sandwiched immunocomplex of antibody/anigen/aptamer-plasmid complex. Under the optimized conditions of salt concentration,ratio of aptamer to PUC19,and SYBR Green Ⅰ concentration,the proposed method offers a linear detection range from 0.2 μg/L to 200 μg/L with a detection limit of 0.1μg/L.

  9. Screening large numbers of recombinant plasmids: modifications and additions to alkaline lysis for greater efficiency

    Institute of Scientific and Technical Information of China (English)

    XU Yibing; N.V. CHANDRASEKHARAN; Daniel L. SIMMONS

    2006-01-01

    Selecting bacteria transformed with recombinant plasmid is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. We describe here modifications to the ultra fast plasmid preparation method described previously by Law and Crickmore. The modified method is coupled to an efficient PCR step to rapidly determine orientation of the inserts. Compared to traditional methods of analysis requiring growth of overnight cultures, plasmid isolation and restriction enzyme digestion to determine orientation this procedure allows for the analysis and storage of a large number of recombinants within a few hours.

  10. A bimetallic nanocomposite electrode for direct and rapid biosensing of p53 DNA plasmid

    Indian Academy of Sciences (India)

    Ezat Hamidi-Asl; Jahan-Bakhsh Raoof; Nahid Naghizadeh; Simin Sharifi; Mohammad Saeid Hejazi

    2015-09-01

    A new label-free electrochemical DNA biosensor is presented based on carbon paste electrode (CPE) modified with gold (Au) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode. The proposed sensor was made by immobilization of 15-mer single stranded oligonucleotide probe related to p53 gene for detection of DNA plasmid samples. The hybridization detection relied on the alternation in the guanine oxidation signal following hybridization of the probe with complementary genomic DNA.The technique of differential pulse voltammetry (DPV) was used for monitoring guanine oxidation. To optimize the performance of the modified CPE, different electrodes were prepared in various percentages of Au and Pt nanoparticles. The modified electrode containing 15% Au/Pt bimetallic nanoparticles (15% Au/Pt-MCPE) was selected as the best working electrode. The selectivity of the sensor was investigated using plasmid samples containing non-complementary oligonucleotides. The detection limit of the biosensor was studied and calculated to be 53.10 pg L−1.

  11. Preparation of collagen-based materials for wound dressing

    Institute of Scientific and Technical Information of China (English)

    吴志谷; 盛志勇; 孙同柱; 耿淼; 黎君友; 姚咏明; 黄祖琇

    2003-01-01

    Objective To describe the methods which were used to develop collagen-based materials for wound dressing.Methods Fresh frozen bovine tendon was treated with 0.05 mol/L acetic acid at pH 3.2 for 48-72 hours, homogenized, filtered, mixed with 8% chondroitin sulphate, for creating a deaerated 1.5%-2.5% collagen solution. The solution was lyophilized in either a pre-frozen or non-pre-frozen mould. The collagen sponge was then cross-linked with 0.25% glutaraldehyde for 24 hours. Three other types of wound dressings were developed using a similar method: collagen membrane with a polyurethane membrane onlay, polyurethane-coated collagen membrane and collagen membrane on gauze.Results It was demonstrated that the use of frozen bovine tendon was stable, and that the prepared collagen sponge contained pores of 50-400 μm in diameter. Conclusions Collagen could be used as wound dressing.

  12. Preparation of polyol esters based on vegetable and animal fats.

    Science.gov (United States)

    Gryglewicz, S; Piechocki, W; Gryglewicz, G

    2003-03-01

    The possibility of using some natural fats: rapeseed oil, olive oil and lard, as starting material for the preparation of neopentyl glycol (NPG) and trimethylol propane (TMP) esters is reported. The syntheses of final products were performed by alcoholysis of fatty acid methyl esters, obtained from natural fats studied, with the appropriate polyhydric alcohol using calcium methoxide as a catalyst. The basic physicochemical properties of the NPG and TMP esters synthesized were the following: viscosity at 40 degrees C in the range of 13.5-37.6 cSt, pour point between -10.5 and -17.5 degrees C and very high viscosity indices, higher than 200. Generally, the esters of neopentyl alcohols were characterized by higher stability in thermo-oxidative conditions in comparison to native triglycerides. Due to the low content of polyunsaturated acids, the olive oil based esters showed the highest thermo-oxidative resistance. Also, methyl esters of fatty acids of lard would constitute a good raw material for the synthesis of lubricating oils, provided that their saturated acids content was lowered. This permits synthesis of NPG and TMP esters with a lower pour point (below -10 degrees C) than natural lard (+33 degrees C).

  13. Plasmid mediated antibiotic resistance in isolated bacteria from burned patients.

    Science.gov (United States)

    Beige, Fahimeh; Baseri Salehi, Majid; Bahador, Nima; Mobasherzadeh, Sina

    2015-01-01

    Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. The samples were collected from two hundred twenty hospitalized burned patients in Isfahan burn hospital during a three-month period (March 2012 to June 2012). The samples were isolated and the Gram-negative bacteria were identified using phenotypic method and API 20E System. Antibiotic susceptibility and plasmid profile were determined by standard Agar disc diffusion and plasmid spin column extraction methods. Totally 117 Gram-negative bacteria were isolated, the most common were Pseudomonas aerugionsa (37.6%), P. fluorescens (25.6%), Acinetobacter baumanii (20/5%) and Klebsiella pneumoniae (7.6%), respectively. The isolates showed high frequency of antibiotic resistance against ceftazidime and co-amoxiclave (100%) and low frequency of antibiotic resistance against amikacin with (70%).The results indicated that 60% of the isolates harboured plasmid. On the other hand, the patients infected with A. baumanii and P. aeruginosa were cured (with 60% frequency) whereas, those infected with P. fluorescens were not cured. Hence, probably antibiotic resistance markers of A. baumanii and P. aeruginosa are plasmid mediated; however, P. fluorescens is chromosomally mediated. Based on our findings, P. aerugionsa is a major causative agent of wound infections and amikacin could be considered as a more effective antibiotic for treatment of the burned patients.

  14. Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

    Directory of Open Access Journals (Sweden)

    Miranda Kirchner

    Full Text Available In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

  15. Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

    Science.gov (United States)

    Kirchner, Miranda; Abuoun, Manal; Mafura, Muriel; Bagnall, Mary; Hunt, Theresa; Thomas, Christopher; Weile, Jan; Anjum, Muna F

    2013-01-01

    In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

  16. Rapid plasmid library screening using RecA-coated biotinylated probes.

    Science.gov (United States)

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M

    1986-12-01

    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  17. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA.

    Science.gov (United States)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-15

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  18. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

    Science.gov (United States)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-01

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  19. Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant.

    Science.gov (United States)

    Schlüter, Andreas; Krause, Lutz; Szczepanowski, Rafael; Goesmann, Alexander; Pühler, Alfred

    2008-08-31

    Plasmid metagenome nucleotide sequence data were recently obtained from wastewater treatment plant (WWTP) bacteria with reduced susceptibility to selected antimicrobial drugs by applying the ultrafast 454-sequencing technology. The sequence dataset comprising 36,071,493 bases (346,427 reads with an average read length of 104 bases) was analysed for genetic diversity and composition by using a newly developed bioinformatic pipeline based on assignment of environmental gene tags (EGTs) to protein families stored in the Pfam database. Short amino acid sequences deduced from the plasmid metagenome sequence reads were compared to profile hidden Markov models underlying Pfam. Obtained matches evidenced that many reads represent genes having predicted functions in plasmid replication, stability and plasmid mobility which indicates that WWTP bacteria harbour genetically stabilised and mobile plasmids. Moreover, the data confirm a high diversity of plasmids residing in WWTP bacteria. The mobile organic peroxide resistance plasmid pMAC from Acinetobacter baumannii was identified as reference plasmid for the most abundant replication module type in the sequenced sample. Accessory plasmid modules encode different transposons, insertion sequences, integrons, resistance and virulence determinants. Most of the matches to Transposase protein families were identified for transposases similar to the one of the chromate resistance transposon Tn5719. Noticeable are hits to beta-lactamase protein families which suggests that plasmids from WWTP bacteria encode different enzymes possessing beta-lactam-hydrolysing activity. Some of the sequence reads correspond to antibiotic resistance genes that were only recently identified in clinical isolates of human pathogens. EGT analysis thus proofed to be a very valuable method to explore genetic diversity and composition of the present plasmid metagenome dataset.

  20. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

    Science.gov (United States)

    Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

    2017-01-01

    Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

  1. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

  2. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  3. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  4. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  5. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  6. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  7. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  8. University-Based Teacher Preparation and Middle Grades Teacher Effectiveness

    Science.gov (United States)

    Preston, Courtney

    2017-01-01

    For over two decades, there have been calls to assess the relationship of the features of teacher preparation programs to teacher effectiveness, to provide guidance for program improvement. At the middle grades level, theory suggests that coursework in educational psychology is particularly important for teacher effectiveness. Using 4 years of…

  9. University-Based Teacher Preparation and Middle Grades Teacher Effectiveness

    Science.gov (United States)

    Preston, Courtney

    2017-01-01

    For over two decades, there have been calls to assess the relationship of the features of teacher preparation programs to teacher effectiveness, to provide guidance for program improvement. At the middle grades level, theory suggests that coursework in educational psychology is particularly important for teacher effectiveness. Using 4 years of…

  10. Preparation of Crystallized Carbon Nitride Based on Microwave Plasma CVD

    National Research Council Canada - National Science Library

    Masatoshi INOUE; Yukihiro SAKAMOTO; Matsufumi TAKAYA

    2010-01-01

    ... on. To obtain this material, generally CH4 is used as a carbon source. Therefore, to make clear the effects of the reaction gas on the preparation of carbon nitride, we tried to use C2H4 as a carbon source instead of CH4...

  11. Considering the Future of University-Based Teacher Preparation

    Science.gov (United States)

    Fraser, James W.

    2014-01-01

    In this commentary, James W. Fraser, a noted historian of education, cites examples from several teacher education programs at more than 30 universities partnering with the Woodrow Wilson Teaching Fellowships in various ways. Additionally, Fraser reviews decades of challenges to traditional teacher preparation, looks at some current reforms, and…

  12. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    Science.gov (United States)

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

  13. IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

    Science.gov (United States)

    Chen, Wenyao; Fang, Tingzi; Zhou, Xiujuan; Zhang, Daofeng; Shi, Xianming; Shi, Chunlei

    2016-01-01

    The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid

  14. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  15. A Case-Based Curriculum Approach to Special Education Teacher Preparation.

    Science.gov (United States)

    Anderson, Peggy L.; Baker, Barbara K.

    1999-01-01

    Describes how Metropolitan State College of Denver, a large undergraduate institution, developed and implemented a case-based special-education curriculum approach to special-education teacher preparation. Of 20 students prepared using the case-based method, 19 have successfully passed the State of Colorado mandated teacher competency test.…

  16. Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA.

    Science.gov (United States)

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Lin, Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu, Haiying; Katsumura, Yosuke

    2015-01-01

    Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10(8) dm(3) mol(-1) s(-1) and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10-1000 μmol dm(-3)) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid.

  17. PREPARATION AND CHARACTERIZATION OF POLYMER-BASED SPHERICAL ACTIVATED CARBONS

    Institute of Scientific and Technical Information of China (English)

    Zhao-lian Zhu; Ai-min Li; Ming-fang Xia; Jin-nan Wan; Quan-xing Zhang

    2008-01-01

    A series of spherical activated carbons(SACs)with different pore structures were prepared from chloromethylated polydivinylbenzene by ZnCl2 activation.The effects of activation temperature and retention time on the yield and textural properties of the resulting SACs were studied.All the SACs are generated with high yield of above 65% and exhibit relatively high mesopore fraction(me%) of 35.7%-43.6% compared with conventional activated carbons.The sample zlc28 prepared at 800℃ for 2 h has the largest BET surface area of 891m2g-1 and pore volume of 0.489 cm3g-1,SEM and XRD analyses of zlc28 verify the presence of developed porous structure composed of disordered micrographite stacking with large amounts of interspaces in the order of nanometers.

  18. Preparation of Zirconia Based Packing Material and Its Evaluation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new reversed-phase packing (C18-PBD-ZrO2) was prepared by depositing and cross-linking 1-octadecene (ODE or C18) and polybutadiene (PBD) onto the surface of porous zirconia microspheres (5~10 mm in diameter) which were synthesized by a sol-gel process. These novel column packings possess high mechanical and chemical stability,wider usable pH range and can be used to separate basic compounds with no observable peak tailing.

  19. Preparation of Glass Ceramic Based on Granulated Slag and Cullet

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The glass-ceramic was prepared on the basis of materials of granulated slag containing high-calcium oxide and cullet.The content of granulated slag ranges from 50%-60%wt in the glass compositions. The samples were analyzed by DTA, SEM and XRD.The results show that the main crystal phase of the glass-ceramic is β-CaSiO3,Which is in scattering fiber or column form.The applying properties have also been measured.

  20. Single photon emission based on coherent state preparation

    Science.gov (United States)

    Ester, P.; Lackmann, L.; Michaelis de Vasconcellos, S.; Hübner, M. C.; Zrenner, A.; Bichler, M.

    2007-09-01

    The authors report here on deterministic single photon emission after coherent optical state preparation in the p-shell of a single InGaAs /GaAs quantum dot. In the approach, they use p-shell Rabi flopping followed by relaxation to the s-shell ground state with subsequent spontaneous single photon emission. Pulsed photon correlation experiments show complete suppression of the correlation peak at zero time delay and hence demonstrate clean single photon emission.

  1. Preparation and Characterization of Amino Acids-Based Trimethoprim Salts

    Directory of Open Access Journals (Sweden)

    Afzal R. Mohammed

    2012-02-01

    Full Text Available Trimethoprim (TMP is a dihydrofolate reductase (DHFR inhibitor which prevents the conversion of dihydrofolic acid into tetrahydrofolic acid, resulting in the depletion of the latter and leading to bacterial death. Oral bioavailability of TMP is hindered by both its low solubility and low permeability. This study aims to prepare novel salts of TMP using anionic amino acids; aspartic and glutamic acid as counter ions in order to improve solubility and dissolution. TMP salts were prepared by lyophilisation and characterized using FT-IR spectroscopy, proton nuclear magnetic resonance (1HNMR, Differential Scanning Calorimetry (DSC and Thermogravimetric analysis (TGA. Both the amino acids formed salts with TMP in a 1:1 molar ratio and showed a 280 fold improvement in solubility. Investigation of the microbiological activity of the prepared salts against TMP sensitive Escherichia coli showed that the new salts not only retained antibacterial activity but also exhibited higher zone of inhibition which was attributed to improved physicochemical characters such as higher solubility and dissolution. The results are an important finding that could potentially impact on faster onset of antibacterial activity and reduced therapeutic dose when administered to patients. Studies are underway investigating the effect of ion-pairing TMP with amino acids on the permeability profile of the drug.

  2. Geminiviruses: a tale of a plasmid becoming a virus

    Directory of Open Access Journals (Sweden)

    Krupovic Mart

    2009-05-01

    Full Text Available Abstract Background Geminiviruses (family Geminiviridae are small single-stranded (ss DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons. Results Phylogenetic and clustering analyses of various RCR Reps suggest that Rep proteins of geminiviruses share a most recent common ancestor with Reps encoded on plasmids of phytoplasmas, parasitic wall-less bacteria replicating both in plant and insect cells and therefore occupying a common ecological niche with geminiviruses. Capsid protein of Satellite tobacco necrosis virus was found to be the best template for homology-based structural modeling of the geminiviral capsid protein. Good stereochemical quality of the generated models indicates that the geminiviral capsid protein shares the same structural fold, the viral jelly-roll, with the vast majority of icosahedral plant-infecting ssRNA viruses. Conclusion We propose a plasmid-to-virus transition scenario, where a phytoplasmal plasmid acquired a capsid-coding gene from a plant RNA virus to give rise to the ancestor of geminiviruses.

  3. Do liquid-based preparations of urinary cytology perform differently than classically prepared cases? Observations from the College of American Pathologists Interlaboratory Comparison Program in Nongynecologic Cytology.

    Science.gov (United States)

    Laucirica, Rodolfo; Bentz, Joel S; Souers, Rhona J; Wasserman, Patricia G; Crothers, Barbara A; Clayton, Amy C; Henry, Michael R; Chmara, Beth Anne; Clary, Karen M; Fraig, Mostafa M; Moriarty, Ann T

    2010-01-01

    The cytomorphology of liquid-based preparations in urine cytology is different than classic slide preparations. To compare the performance of liquid-based preparation specimens to classically prepared urine specimens with a malignant diagnosis in the College of American Pathologists Interlaboratory Comparison Program in Nongynecologic Cytology. Participant responses between 2000 and 2007 for urine specimens with a reference diagnosis of high-grade urothelial carcinoma/carcinoma in situ/dysplasia (HGUCA), squamous cell carcinoma, or adenocarcinoma were evaluated. ThinPrep and SurePath challenges were compared with classic preparations (smears, cytospins) for discordant responses. There were 18 288 pathologist, 11 957 cytotechnologist, and 8086 "laboratory" responses available. Classic preparations comprised 90% (n = 34 551) of urine challenges; 9% (n = 3295) were ThinPrep and 1% (n = 485) were SurePath. Concordance to the general category of "positive-malignant" was seen in 92% of classic preparations, 96.5% of ThinPrep, and 94.6% of SurePath challenges (P Liquid-based preparations performed significantly better in urinary cytology challenges when evaluating malignant categories in the College of American Pathologists interlaboratory comparison program. The liquid-based preparation challenges also performed better for the exact reference interpretation of HGUCA, but no difference was observed for adenocarcinoma challenges. Cytotechnologists perform better than pathologists for all slide types, as well as those demonstrating HGUCA. These results suggest that liquid-based preparations facilitate a more accurate diagnosis than conventional preparations.

  4. Preparing Methods and Its Influencing Factors about Nanoparticles Based on Dendritic Polymer

    Directory of Open Access Journals (Sweden)

    Zhang Jianwei

    2017-01-01

    Full Text Available Based on the properties, structure and application of dendritic polymer, this paper analysed the methods of the preparation of nanoparticles using dendritic polymer, detailed preparation process, technical parameters and application effect about a single metal nanoparticles, bimetallic nanoparticles, sulfide and halide nanoparticles. The influencing factors of the preparation about nanoparticles were discussed, including the molecular algebra, the molar ratio of the metal ions to the dendritic polymer, and so on.

  5. Preparation and study of heterojunctions based on chalcogenide glassy semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Kornev, K.P. [Kaliningrad State Univ., Kaliningrad (Russian Federation); Korneva, I.P. [Baltic Fishing Fleet State Academy, Kaliningrad (Russian Federation)

    2005-07-01

    Four types of heterojunctions were prepared: SnO{sub 2}-As{sub 2}(Se{sub 0.9}Te{sub 0.1}){sub 3}, SnO{sub 2}-(As{sub 0.67}Sb{sub 0.33}){sub 2}Se{sub 3}, n-GaAs-As{sub 2}Se{sub 3} and n-GaAs-As{sub 2}S{sub 3}. For all samples I-V characteristics and photosensitivity spectra were obtained. These heterostructures can be used for manufacturing rectifying devices and photoreceivers. (orig.)

  6. In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

    OpenAIRE

    Lopes, Marta B.; Gonçalves, Geisa A. L.; Felício-Silva, Daniel; Prather, Kristala L. J.; Monteiro, Gabriel; Prazeres, Duarte M. F.; Calado, Cecília Ribeiro da Cruz

    2015-01-01

    BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables su...

  7. An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

    Science.gov (United States)

    Schründer, J; Meinhardt, F

    1995-03-01

    Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

  8. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

    Science.gov (United States)

    Chee, Mark K; Haase, Steven B

    2012-05-01

    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  9. Massive preparation of pitch-based organic microporous polymers for gas storage.

    Science.gov (United States)

    Li, Wenqing; Zhang, Aijuan; Gao, Hui; Chen, Mingjie; Liu, Anhua; Bai, Hua; Li, Lei

    2016-02-14

    A general challenge for preparing organic microporous polymers (MOPs) is to use cheap and sustainable building blocks while retaining the advanced functions. We demonstrate a strategy to massively prepare pitch-based MOPs, which are thermally and chemically stable. A maximum BET surface area of 758 m(2) g(-1) and high gas storage capacity were achieved.

  10. Plasmid Profiles and Prevalence of Intermediately Virulent Rhodococcus equi from Pigs in Nakhonpathom Province, Thailand: Identification of a New Variant of the 70-kb Virulence Plasmid, Type 18

    Directory of Open Access Journals (Sweden)

    Chaithep Poolkhet

    2010-01-01

    Full Text Available The prevalence of intermediately virulent Rhodococcus equi isolates from pig submaxillary lymph nodes from four slaughterhouses in Nakhonpathom province, Thailand, was investigated. The isolates were tested for the presence of virulence plasmids and the 20-kDa virulence-associated protein antigen (VapB gene by PCR. Of the 734 submaxillary lymph nodes tested, 19 (2.6% produced positive cultures of R. equi. All 19 isolates were positive for the VapB gene and contained virulence plasmids that were identified as type 1 (six isolates, type 6 (two isolates, type 7 (one isolate, type 16 (two isolates, and a new variant (eight isolates. Based on the restriction digestion patterns of the plasmid DNAs, we tentatively designated the variant as type 18. Investigation of the prevalence and plasmid profiles of VapB-positive R. equi in pigs should be extended throughout Thailand to evaluate potential sources of zoonotic infections.

  11. JADOPPT: java based AutoDock preparing and processing tool.

    Science.gov (United States)

    García-Pérez, Carlos; Peláez, Rafael; Therón, Roberto; Luis López-Pérez, José

    2017-02-15

    AutoDock is a very popular software package for docking and virtual screening. However, currently it is hard work to visualize more than one result from the virtual screening at a time. To overcome this limitation we have designed JADOPPT, a tool for automatically preparing and processing multiple ligand-protein docked poses obtained from AutoDock. It allows the simultaneous visual assessment and comparison of multiple poses through clustering methods. Moreover, it permits the representation of reference ligands with known binding modes, binding site residues, highly scoring regions for the ligand, and the calculated binding energy of the best ranked results. JADOPPT, supplementary material (Case Studies 1 and 2) and video tutorials are available at http://visualanalytics.land/cgarcia/JADOPPT.html. carlosgarcia@usal.es or pelaez@usal.es. Supplementary data are available at Bioinformatics online.

  12. New Plasmid Tools for Genetic Analysis of Actinobacilus pleuropneumoniae and Other Pasteurellaceae

    National Research Council Canada - National Science Library

    Janine T Bossé; Andrew L Durham; Andrew N Rycroft; J Simon Kroll; Paul R Langford

    2009-01-01

      We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae...

  13. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  14. Development of plasmids for quantitative detection of integrated lentiviral vectors and evaluation of culture time to perform vector titer by real-time quantitative polymerase chain reaction assay

    Directory of Open Access Journals (Sweden)

    Elena Baiamonte

    2014-09-01

    Full Text Available The accurate assessment of provirus copy number per cell (VCN/cell is a fundamental issue in transgenesis as well as in gene therapy studies based on stably integrated vectors. To this end, real-time quantitative polymerase chain reaction (qPCR is a powerful method but it is sensible to differences in quality or concentration of the two-plasmid preparations used for the construction of the standard curves. In order to minimize technical errors we included genome specific sequences (mouse or human and vector specific sequences in the same plasmid. We evaluated the specificity and sensitivity of these bivalent plasmids by qPCR analysis on mouse and human genomic DNA containing a known number of a reporter lentiviral vector and we found that the system is reliable to measure up to 0.1 VCN/cell. Here we have applied this assay to measure vector titer of virus stock preparations and to determine the optimal cell passages at which viral titration effectively reflects the number of integrated vectors.

  15. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  16. Research Progress on Preparation for Biomass-based SiC Ceramic

    Directory of Open Access Journals (Sweden)

    CUI He-shuai

    2017-08-01

    Full Text Available Silicon carbide (SiC ceramics prepared by the conventional process has excellent properties and wide application prospects, but the increased cost of high-temperature preparation process restricts its further development. In contrast, the abundant porous structure of biomass makes itself to be ideal replacement of SiC ceramic prepared at low temperature. This paper reviewed the structure characteristics, preparation methods, pyrolysis mechanism and influence parameters of biomass-based SiC ceramic, and eventually explored the current problems and development trends of the pretreatment of carbon source and silicon source, the pyrolysis process and the application research on the preparation for biomass-based SiC ceramic.

  17. Robotic, MEMS-based Multi Utility Sample Preparation Instrument for ISS Biological Workstation Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This project will develop a multi-functional, automated sample preparation instrument for biological wet-lab workstations on the ISS. The instrument is based on a...

  18. Preparation of poly(epsilon-caprolactone)-based tissue engineering scaffolds by stereolithography

    NARCIS (Netherlands)

    Elomaa, Laura; Teixeira, Sandra; Hakala, Risto; Korhonen, Harri; Grijpma, Dirk W.; Seppala, Jukka V.

    2011-01-01

    A photocrosslinkable poly(epsilon-caprolactone) (PCL)-based resin was developed and applied using stereolithography. No additional solvents were required during the structure preparation process. Three-armed PCL oligomers of varying molecular weights were synthesized, functionalized with methacrylic

  19. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids.

    Science.gov (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C

    1988-06-01

    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  20. Facile preparation of superhydrophobic surfaces based on metal oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Xue-Mei; Cui, Jin-Feng; Sun, Han-Xue; Liang, Wei-Dong; Zhu, Zhao-Qi; An, Jin; Yang, Bao-Ping; La, Pei-Qing; Li, An, E-mail: lian2010@lut.cn

    2014-06-01

    A novel method for fabrication of superhydrophobic surfaces was developed by facile coating various metal oxide nanoparticles, including ZnO, Al{sub 2}O{sub 3} and Fe{sub 3}O{sub 4}, on various substrates followed by treatment with polydimethylsiloxane (PDMS) via chemical vapor deposition (CVD) method. Using ZnO nanoparticles as a model, the changes in the surface chemical composition and crystalline structures of the metal oxide nanoparticles by PDMS treatment were investigated by X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD) and Fourier transform infrared (FTIR) analysis. The results show that the combination of the improved surface roughness generated from of the nanoparticles aggregation with the low surface-energy of silicon-coating originated from the thermal pyrolysis of PDMS would be responsible for the surface superhydrophobicity. By a simple dip-coating method, we show that the metal oxide nanoparticles can be easily coated onto the surfaces of various textural and dimensional substrates, including glass slide, paper, fabric or sponge, for preparation of superhydrophobic surfaces for different purpose. The present strategy may provide an inexpensive and new route to surperhydrophobic surfaces, which would be of technological significance for various practical applications especially for separation of oils or organic contaminates from water.

  1. Preparation of a PTE simulacrum based on surface molecular imprinting

    Institute of Scientific and Technical Information of China (English)

    Yong Guo; Ying Yang; Tian Ying Guo

    2011-01-01

    Firstly, we synthesized N-methacryloyl-histidine monomer and N-methacryIoyl-histidine-Cu2+ complex (MAH-Cu2+). Then the molecular imprinting polymers (MIP) has been prepared by surface grafting on uniform polystyrene (PS) core using reversible addition-fragmentation transfer polymerization (RAFT) with MAH-Cu2+ as the functional monomer, methyl paraoxon as the template to simulate phosphotriesterase (PTE). Finally, we have investigated the catalytic hydrolytic activities of MIP and non-imprinting polymers (NIP) to the template methyl paraoxon and the template analogue ethyl paraoxon respectively by UV spectrophotometry. The results showed that the catalytic hydrolytic activity of MIP to the template methyl paraoxon was highest and the value of k is 8.67 × 105 mmol L-1 min-1 3.89-fold higher than MIP to the template analogue ethyl paraoxon, 2.79-fold higher than NIP to the template methyl paraoxon. The KM, rm of MIP are also determined, and Km = 3.95 × 10-4 mol/L, rm = 2.12 μmol/ min. The MIP can be reused with only lose 7% of catalytic activity for four cycles.

  2. Facile preparation of superhydrophobic surfaces based on metal oxide nanoparticles

    Science.gov (United States)

    Bao, Xue-Mei; Cui, Jin-Feng; Sun, Han-Xue; Liang, Wei-Dong; Zhu, Zhao-Qi; An, Jin; Yang, Bao-Ping; La, Pei-Qing; Li, An

    2014-06-01

    A novel method for fabrication of superhydrophobic surfaces was developed by facile coating various metal oxide nanoparticles, including ZnO, Al2O3 and Fe3O4, on various substrates followed by treatment with polydimethylsiloxane (PDMS) via chemical vapor deposition (CVD) method. Using ZnO nanoparticles as a model, the changes in the surface chemical composition and crystalline structures of the metal oxide nanoparticles by PDMS treatment were investigated by X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD) and Fourier transform infrared (FTIR) analysis. The results show that the combination of the improved surface roughness generated from of the nanoparticles aggregation with the low surface-energy of silicon-coating originated from the thermal pyrolysis of PDMS would be responsible for the surface superhydrophobicity. By a simple dip-coating method, we show that the metal oxide nanoparticles can be easily coated onto the surfaces of various textural and dimensional substrates, including glass slide, paper, fabric or sponge, for preparation of superhydrophobic surfaces for different purpose. The present strategy may provide an inexpensive and new route to surperhydrophobic surfaces, which would be of technological significance for various practical applications especially for separation of oils or organic contaminates from water.

  3. Preparation of Astaxanthin Nanodispersions Using Gelatin-Based Stabilizer Systems

    Directory of Open Access Journals (Sweden)

    Navideh Anarjan

    2014-09-01

    Full Text Available The incorporation of lipophilic nutrients, such as astaxanthin (a fat soluble carotenoid in nanodispersion systems can either increase the water solubility, stability and bioavailability or widen their applications in aqueous food and pharmaceutical formulations. In this research, gelatin and its combinations with sucrose oleate as a small molecular emulsifier, sodium caseinate (SC as a protein and gum Arabic as a polysaccharide were used as stabilizer systems in the formation of astaxanthin nanodispersions via an emulsification-evaporation process. The results indicated that the addition of SC to gelatin in the stabilizer system could increase the chemical stability of astaxanthin nanodispersions significantly, while using a mixture of gelatin and sucrose oleate as a stabilizer led to production of nanodispersions with the smallest particle size (121.4 ± 8.6 nm. It was also shown that a combination of gelatin and gum Arabic could produce optimal astaxanthin nanodispersions in terms of physical stability (minimum polydispersity index (PDI and maximum zeta-potential. This study demonstrated that the mixture of surface active compounds showed higher emulsifying and stabilizing functionality compared to using them individually in the preparation of astaxanthin nanodispersions.

  4. Isolation of plasmid from the blue-green alga Spirulina platensis

    Science.gov (United States)

    Qin, Song; Tong, Shun; Zhang, Peijun; Tseng, C. K.

    1993-09-01

    CCC plasmid was isolated from an economically important blue-green alga — Spirulina platensis (1.7×106 dalton from the S6 strain and 1.2×106 dalton from the F3 strain) using a rapid method based on ultrasonic disruption of algal cells and alkaline removal of chromosomal DNA. The difference in the molecular weight of the CCC DNAs from the two strains differing in form suggests that plasmid may be related with the differentiation of algal form. This modified method, which does not use any lysozyme, is a quick and effective method of plasmid isolation, especially for filamentous blue-green algae.

  5. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    Science.gov (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  6. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  7. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  8. Current trends in separation of plasmid DNA vaccines: a review.

    Science.gov (United States)

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  9. Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ivanov Ivan

    2011-03-01

    Full Text Available Abstract Background Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ gene placed under the control of different regulatory elements (promoter and ribosome-binding sites were used as a model. Results Bacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985. The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites and segregational plasmid stability (determined by the above model was also observed. Conclusions Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy

  10. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  11. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  12. PREPARATION AND CHARACTERIZATION OF BIOCATALYSTS BASED ON IMMOBILIZED GLYCOSIDASES

    Directory of Open Access Journals (Sweden)

    O. L. Meshcheriakova

    2014-01-01

    Full Text Available Summary. Enzymes subclass glycosidases cleaving poly- and oligosaccharides to simple sugars, are of great practical importance for a variety of industries. Such enzymes include α-L-fucosidase and β-fructofuranosidase. α-L-fucosidase splits fucoidan kelp to fucose and fucooligosaccharides. Fucose has prebiotic, immunotropic action, and a wide spectrum of biological activity in vertebrates, fucooligosaccharides - antioxidant and prebiotic properties. In this regard, and fucose polymers may be demanded in the food, feed and pharmaceutical industry. β-fructofuranosidase sucrose hydrolysis with the formation of invert syrup high quality and biological value that is of interest to the sugar industry. In order to intensify the processes of hydrolysis of fucoidan and sucrose due to the higher stability and reusability of enzyme preparations carried immobilization α-L-fucosidase on chitosan and β-fructofuranosidase of ion exchange brand FIBAN A-6 adsorption method. Activity of the immobilized α-L-fucosidase and β-fructofuranosidase were 80 and 70% of the activity of the free enzyme, respectively. Found that immobilized β-fructofuranosidase exhibits maximal activity at pH 4,0-4,1, the immobilized α-L-fucosidase - at pH 7,0. The optimal pH of immobilized enzymes similar to those for the free enzyme. Optimal temperature hydrolysis substrates immobilized α-L-fucosidase and β-fructofuranosidase was 50 and 70 ° C respectively, 10 ° C and 20 ° C higher compared to free enzymes. Studies have shown sufficient stability of immobilized glycosidases, so at 4-fold using their enzymatic activity decreased by 1.5 times; Biocatalysts obtained in storage in the refrigerator for 4-6 months retained 80% of the catalytic activity of enzymes.

  13. Preparation and characterization of transparent PMMA-cellulose-based nanocomposites.

    Science.gov (United States)

    Kiziltas, Esra Erbas; Kiziltas, Alper; Bollin, Shannon C; Gardner, Douglas J

    2015-01-01

    Nanocomposites of polymethylmethacrylate (PMMA) and cellulose were made by a solution casting method using acetone as the solvent. The nanofiber networks were prepared using three different types of cellulose nanofibers: (i) nanofibrillated cellulose (NFC), (ii) cellulose nanocrystals (CNC) and (iii) bacterial cellulose from nata de coca (NDC). The loading of cellulose nanofibrils in the PMMA varied between 0.25 and 0.5 wt%. The mechanical properties of the composites were evaluated using a dynamic mechanical thermal analyzer (DMTA). The flexural modulus of the nanocomposites reinforced with NDC at the 0.5 wt% loading level increased 23% compared to that of pure PMMA. The NFC composite also exhibited a slightly increased flexural strength around 60 MPa while PMMA had a flexural strength of 57 MPa. The addition of NDC increased the storage modulus (11%) compared to neat PMMA at room temperature while the storage modulus of PPMA/CNC nanocomposite containing 0.25 and 0.5 wt% cellulose increased about 46% and 260% to that of the pure PMMA at the glass transition temperature, respectively. Thermogravimetric analysis (TGA) indicated that there was no significant change in thermal stability of the composites. The UV-vis transmittance of the CNF nanocomposites decreased by 9% and 27% with the addition of 0.25 wt% CNC and NDC, respectively. This work is intended to spur research and development activity for application of CNF reinforced PMMA nanocomposites in applications such as: packaging, flexible screens, optically transparent films and light-weight transparent materials for ballistic protection.

  14. Methods for preparing colloidal nanocrystal-based thin films

    Energy Technology Data Exchange (ETDEWEB)

    Kagan, Cherie R.; Fafarman, Aaron T.; Choi, Ji-Hyuk; Koh, Weon-kyu; Kim, David K.; Oh, Soong Ju; Lai, Yuming; Hong, Sung-Hoon; Saudari, Sangameshwar Rao; Murray, Christopher B.

    2016-05-10

    Methods of exchanging ligands to form colloidal nanocrystals (NCs) with chalcogenocyanate (xCN)-based ligands and apparatuses using the same are disclosed. The ligands may be exchanged by assembling NCs into a thin film and immersing the thin film in a solution containing xCN-based ligands. The ligands may also be exchanged by mixing a xCN-based solution with a dispersion of NCs, flocculating the mixture, centrifuging the mixture, discarding the supernatant, adding a solvent to the pellet, and dispersing the solvent and pellet to form dispersed NCs with exchanged xCN-ligands. The NCs with xCN-based ligands may be used to form thin film devices and/or other electronic, optoelectronic, and photonic devices. Devices comprising nanocrystal-based thin films and methods for forming such devices are also disclosed. These devices may be constructed by depositing NCs on to a substrate to form an NC thin film and then doping the thin film by evaporation and thermal diffusion.

  15. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  16. Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative.

    Science.gov (United States)

    Hughes, Meredith L; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A; McClane, Bruce A; Rood, Julian I

    2007-11-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

  17. Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative▿ †

    Science.gov (United States)

    Hughes, Meredith L.; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A.; McClane, Bruce A.; Rood, Julian I.

    2007-01-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ɛ-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ɛ-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative. PMID:17720791

  18. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  19. Preparation of stable ultrahydrophobic and superoleophobic silica-based coating.

    Science.gov (United States)

    Nimittrakoolchai, On-Uma; Supothina, Sitthisuntorn

    2012-06-01

    Silica-Based coatings having excellent water- and oil-repellent properties and good weathering stability have been deposited onto glass surface by a simple one-step dip coating technique. To achieve ultra water repellency and super oil repellency, the chemical composition of SiO2 nanoparticle employed as surface roughness enhancer and trichloro(1H,1H,2H,2H-perfluorooctyl)silane employed as surface-energy reducing substance was varied. At the optimum synthesis condition, the coating exhibited very high contact angles of 173.2, 146.7 and 147.6 degrees for water, ethylene glycol and seed oil, respectively. The achievement of excellent water- and oil-repellency is also described based on the presence of air trapped in micropore of the coating in addition to its high surface roughness and low surface free energy. The coatings have good weathering stability based on natural and accelerated weathering tests indicating feasibility for practical use.

  20. Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  1. Regular Cellular Distribution of Plasmids by Oscillating and Filament-forming ParA ATPase of Plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  2. Pressure-driven one-step solid phase-based on-chip sample preparation on a microfabricated plastic device and integration with flow-through polymerase chain reaction (PCR).

    Science.gov (United States)

    Tran, Hong Hanh; Trinh, Kieu The Loan; Lee, Nae Yoon

    2013-10-01

    In this study, we fabricate a monolithic poly(methylmethacrylate) (PMMA) microdevice on which solid phase-based DNA preparation and flow-through polymerase chain reaction (PCR) units were functionally integrated for one-step sample preparation and amplification operated by pressure. Chelex resin, which is used as a solid support for DNA preparation, can capture denatured proteins but releases DNA, and the purified DNA can then be used as a template in a subsequent amplification process. Using the PMMA microdevices, DNA was successfully purified from both Escherichia coli and human hair sample, and the plasmid vector inserted in E. coli and the D1S80 locus in human genomic DNA were successfully amplified from on-chip purified E. coli and human hair samples. Furthermore, the integration potential of the proposed sample preparation and flow-through PCR units was successfully demonstrate on a monolithic PMMA microdevice with a seamless flow, which could pave the way for a pressure-driven, simple one-step sample preparation and amplification with greatly decreased manufacture cost and enhanced device disposability.

  3. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Efficient encapsulation of plasmid DNA in anionic liposomes by a freeze/thaw extrusion procedure

    NARCIS (Netherlands)

    Schoen, P; Bijl, L; Wilschut, J

    1998-01-01

    In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity

  5. Preparation of microemulsions with soybean oil-based surfactants

    Science.gov (United States)

    Emulsions are widely applied in food, cosmeceutical and medicinal formulations. Smaller and highly stable droplets of emulsions are important for their application. This research reports that by using soybean oil-based surfactants, the higher stabilized oil-in-water emulsions were obtained via an ul...

  6. Preparing Instructional Designers for Game-Based Learning: Part 1

    Science.gov (United States)

    Hirumi, Atsusi; Appelman, Bob; Rieber, Lloyd; Van Eck, Richard

    2010-01-01

    Like many rapidly growing industries, advances in video game technology are far outpacing research on its design and effectiveness. Relatively little is understood about how to apply what we know about teaching and learning to optimize game-based learning. For the most part, instructional designers know little about game development and video game…

  7. Preparing Students for Flipped or Team-Based Learning Methods

    Science.gov (United States)

    Balan, Peter; Clark, Michele; Restall, Gregory

    2015-01-01

    Purpose: Teaching methods such as Flipped Learning and Team-Based Learning require students to pre-learn course materials before a teaching session, because classroom exercises rely on students using self-gained knowledge. This is the reverse to "traditional" teaching when course materials are presented during a lecture, and students are…

  8. Preparation of ultrafine Ti (C, N)-based cermet using oxygen-rich powders

    Institute of Scientific and Technical Information of China (English)

    FENG Ping; HE Yue-hui; XIONG Wei-hao; XIAO Yi-feng

    2005-01-01

    The availability using oxygen-rich powders to prepare ultrafine Ti(C,N)-based cermets was investigated. The deoxidation process, denitrification phenomenon and the effect of deoxidation on microstructure and mechanical properties of sintered samples were discussed, respectively. The results show that oxygen in the samples prepared even with high oxygen contained in starting powders can be almost completely cleaned away through suitable sintering process. The ultrafine oxygen-rich powders have a significant effect on microstructure, which promotes the formation of white core phase. A ultrafine Ti(C,N)-based cermet with mean particle size of 0. 30 μm, uniform microstructure and excellent mechanical properties is successfully prepared. It is also found that there exists severe denitrification phenomenon in the preparation process of ultrafine Ti(C,N)-based cermet.

  9. Mask process matching using a model based data preparation solution

    Science.gov (United States)

    Dillon, Brian; Saib, Mohamed; Figueiro, Thiago; Petroni, Paolo; Progler, Chris; Schiavone, Patrick

    2015-10-01

    Process matching is the ability to precisely reproduce the signature of a given fabrication process while using a different one. A process signature is typically described as systematic CD variation driven by feature geometry as a function of feature size, local density or distance to neighboring structures. The interest of performing process matching is usually to address differences in the mask fabrication process without altering the signature of the mask, which is already validated by OPC models and already used in production. The need for such process matching typically arises from the expansion of the production capacity within the same or different mask fabrication facilities, from the introduction of new, perhaps more advanced, equipment to deliver same process of record masks and/or from the re-alignment of processes which have altered over time. For state-of-the-art logic and memory mask processes, such matching requirements can be well below 2nm and are expected to reduce below 1nm in near future. In this paper, a data preparation solution for process matching is presented and discussed. Instead of adapting the physical process itself, a calibrated model is used to modify the data to be exposed by the source process in order to induce the results to match the one obtained while running the target process. This strategy consists in using the differences among measurements from the source and target processes, in the calibration of a single differential model. In this approach, no information other than the metrology results is required from either process. Experimental results were obtained by matching two different processes at Photronics. The standard deviation between both processes was of 2.4nm. After applying the process matching technique, the average absolute difference between the processes was reduced to 1.0nm with a standard deviation of 1.3nm. The methods used to achieve the result will be described along with implementation considerations, to

  10. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  11. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  12. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

    Institute of Scientific and Technical Information of China (English)

    Bogdan MUNTEANU; Mario BRAUN; Kajohn BOONROD

    2012-01-01

    QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid.It can be applied successfully to small plasmids (up to 10 kb).However,this method cannot efficiently mutate bigger plasmids.Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers,we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes.Moreover,only six polymerase chain reaction (PCR) cycles and 0.5 μl of polymerase (instead of 18 PCR cycles and 1.0 μl of enzyme In the standard protocol) were sufficient for the reaction.

  13. Usefulness of liquid-based preparation in urine cytology.

    Science.gov (United States)

    Hwang, Eu Chang; Park, Seong Hoon; Jung, Seung Il; Kwon, Dong Deuk; Park, Kwangsung; Ryu, Soo Bang; Park, Chang Soo

    2007-07-01

    ThinPrep (TP), a liquid-based cytological and non-invasive technique to confirm the diagnosis of bladder cancer, is reported to be a better screening test than the conventional cytospin method. This study compared the new MonoPrep2 (MP), a liquid-based cytological technique, with TP for diagnosing bladder cancer. Between January 2003 and June 2004, urine samples from 284 patients were processed using the TP and MP methods. The cytological diagnosis and the determination of specimen quality were performed separately. The cytological diagnoses were classified into four categories: unsatisfactory, benign, borderline, and malignant. A subsequent biopsy was performed in 73 patients. The cytological diagnoses were compared with the biopsy results to evaluate the sensitivity and specificity of the two methods. Considering all the features examined, the overall specimen quality was comparable between the MP and TP techniques in the majority of cases. The rate of satisfactory specimens was 100% for TP and 98.6% for MP. The diagnostic capacity was similar between MP and TP. The overall sensitivities with MP and TP were 58.6 and 62.0%, respectively, and the specificities were 100 and 97.7%; the differences were not significant (P > 0.05). MP and TP produced comparable results in diagnosing bladder cancer. As MP is less expensive than TP, we recommend MP as an alternative liquid-based cytology method for use in bladder cancer screening.

  14. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  15. Novel Epoxy Resin/SiO2 Nanocomposites Preparation Method Based on Diminutive Bubbles Explosion

    Institute of Scientific and Technical Information of China (English)

    NIE Peng; ZHAO Xue-zeng; CHEN Fang; WANG Wei-jie; BAI Yong-ping

    2006-01-01

    To obtain suspended dispersion of nano-particles in liquid without any dispersant, a novel epoxy resin/SiO2 nanocomposites preparation method based on diminutive bubbles explosion is presented. And, corresponding nanocomposites preparation system was designed. The preparation system applies compressed gas as transmission medium to carry nanomaterials into epoxy resin solution. The compressed gas with nanomaterials turns into diminutive bubbles distributing in epoxy resin/SiO2. The great pressure difference between inner and outer-bubbles led to bubbles inflation and explosion. During the bubble inflation, bubble oscillation may generate. The stretching rate may reach 106 s-1, which favors more homogeneous dispersion of nanoparticles. During the bubbles explosion the released energy and the explosion shock waves disperse the nanoparticles into epoxy resin solution. By using the preparation system, epoxy resin/SiO2 nanocomposites were prepared. The SiO2 dispersed into epoxy as the configuration of 15 nm - 30 nm particles.

  16. Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

    Science.gov (United States)

    Peng, Yun; Lu, Jun; Wong, Joyce J W; Edwards, Ross A; Frost, Laura S; Mark Glover, J N

    2014-11-11

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

  17. Enzymatic preparation and characterization of soybean lecithin-based emulsifiers

    Directory of Open Access Journals (Sweden)

    R. C. Reddy Jala

    2016-12-01

    Full Text Available Simple enzymatic methods were developed for the synthesis of lysolecithin, glycerolyzed lecithin and hydrolyzed lecithin. The products were characterized in terms of their acetone insoluble matter, hexane insoluble matter, moisture, phospholipid distribution and fatty acid composition. The HLB value ranges of different products with different acid values were detected. The efficiency of optimally hydrolyzed lecithin was examined at high calcium ion, low pH, and aqueous solutions and compared with commercially available standard lecithin-based emulsifiers. Overall, lysolecithin powder was proven to be the best emulsifier even at strong and medium acidic conditions.

  18. Silica base for dentifrice and process for its preparation

    Energy Technology Data Exchange (ETDEWEB)

    Shinpo, S.; Fushino, T.; Hachijo, A.; Ohtsu, S.

    1988-04-19

    A method for production of a silica base material for a dentifrice formulation, is described which comprises reacting an alkali metal silicate solution and hydrochloric or sulfuric acid in the presence of an electrolyte in two stages, namely a silica crystallization stage for which the pH of reaction mixture is brought to 10.0, and an acidification stage for which the pH is finally brought down to 3.5 or less, and wherein the ratio of the rate of addition of chloride or sulfate ion in the acidification stage to the rate of the addition in the silica crystallization stage is at least 3:2, and the acidification is carried out within 30 minutes; the silica base material having a specific surface area by the BET method of 270-500 m/sup 2//g-anhydride, and by the CTAB method of 5-60 m/sup 2//g-anhydride, giving amorphous X-ray diffraction patterns after firing at 1100/sup 0/C., and having a refractive index of 1.455-1.470.

  19. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor.

    Science.gov (United States)

    Samson, Jacopo; Piscopo, Irene; Yampolski, Alex; Nahirney, Patrick; Parpas, Andrea; Aggarwal, Amit; Saleh, Raihan; Drain, Charles Michael

    2011-10-21

    Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  20. Facilities for preparing actinide or fission product-based targets

    CERN Document Server

    Sors, M

    1999-01-01

    Research and development work is currently in progress in France on the feasibility of transmutation of very long-lived radionuclides such as americium, blended with an inert medium such as magnesium oxide and pelletized for irradiation in a fast neutron reactor. The process is primarily designed to produce ceramics for nuclear reactors, but could also be used to produce targets for accelerators. The Actinide Development Laboratory is part of the ATALANTE complex at Marcoule, where the CEA investigates reprocessing, liquid and solid waste treatment and vitrification processes. The laboratory produces radioactive sources; after use, their constituents are recycled, notably through R and D programs requiring such materials. Recovered americium is purified, characterized and transformed for an experiment known as ECRIX, designed to demonstrate the feasibility of fabricating americium-based ceramics and to determine the reactor transmutation coefficients.

  1. Copper base materials prepared by gel-casting process

    Institute of Scientific and Technical Information of China (English)

    LIU Weihua; JIA Chengchang; SHI Yantao; HAN Yuepeng

    2008-01-01

    Gel-casting process was developed as a new molding process in the field of copper base powder metallurgy to manufacture metal parts with excellent performance and complex shapes.Through changing the parameters of gel-casting process,such as dispersant and solid loading,the corresponding effects on the rheology of Cu slurries,molding and sintering behaviors were studied.The results show that the viscosity of Cu slurries was significantly reduced with an increase in dispersant.The most appropriate solid loading was found to be 61% and the sintering temperature was 910℃ in these experiments.After the optimization of parameters of gel-casting process,copper composite parts with relatively high density and better properties were obtained.

  2. CO responses of sensors based on cerium oxide thick films prepared from clustered spherical nanoparticles.

    Science.gov (United States)

    Izu, Noriya; Matsubara, Ichiro; Itoh, Toshio; Akamatsu, Takafumi; Shin, Woosuck

    2013-03-08

    Various types of CO sensors based on cerium oxide (ceria) have been reported recently. It has also been reported that the response speed of CO sensors fabricated from porous ceria thick films comprising nanoparticles is extremely high. However, the response value of such sensors is not suitably high. In this study, we investigated methods of improving the response values of CO sensors based on ceria and prepared gas sensors from core-shell ceria polymer hybrid nanoparticles. These hybrid nanoparticles have been reported to have a unique structure: The core consists of a cluster of ceria crystallites several nanometers in size. We compared the characteristics of the sensors based on thick films prepared from core-shell nanoparticles with those of sensors based on thick films prepared from conventionally used precipitated nanoparticles. The sensors prepared from the core-shell nanoparticles exhibited a resistance that was ten times greater than that of the sensors prepared from the precipitated nanoparticles. The response values of the gas sensors based on the core-shell nanoparticles also was higher than that of the sensors based on the precipitated nanoparticles. Finally, improvements in sensor response were also noticed after the addition of Au nanoparticles to the thick films used to fabricate the two types of sensors.

  3. Chemical Preparation of Supported Bimetallic Catalysts. Gold-Based Bimetallic, a Case Study

    OpenAIRE

    Louis, Catherine

    2016-01-01

    International audience; This review focuses on the chemical methods used to prepare supported bimetallic heterogeneous catalysts, i.e., bimetallic nanoparticles deposited on a support. The review is limited to the preparation of gold-based bimetallic catalysts and moreover to bimetallic nanoparticles supported on powder inorganic supports, i.e., on the surface or in the porosity, and not on model supports such as single crystals.

  4. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

    OpenAIRE

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Jaana K.H. Bamford; Buckling, Angus

    2011-01-01

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple ant...

  5. Impact of bowel preparation type on the quality of colonoscopy: a multicenter community-based study

    Directory of Open Access Journals (Sweden)

    Daniel Martin

    2016-04-01

    Full Text Available Background: High-quality bowel preparation is crucial for achieving the goals of colonoscopy. However, choosing a bowel preparation in clinical practice can be challenging because of the many formulations. This study aims to assess the impact the type of bowel preparation on the quality of colonoscopy in a community hospital setting. Methods: A retrospective, observational study was conducted utilizing a colonoscopy screening/surveillance database in central Illinois during the period of January 1, 2010, to March 31, 2014. Patients without bowel preparation assessment were excluded from this study. Controlling for the confounders, generalized linear models were used to estimate the adjusted impact [odds ratio (OR] of bowel preparation type on the quality of preparation (excellent, good, fair, and poor, and on the detection of advanced adenoma. The association between the time of withdrawal after insertion and the quality of preparation was also examined using a linear model. Results: A total of 28,368 colonoscopies; half the patients were male, and the average age was 61±9 years. Polyethylene glycol (PEG was used in the majority (70.2% of bowel preparations, followed by sodium sulfate (21.4%, sodium phosphate (2.5%, magnesium sulfate (0.4%, and others. Compared with PEG, magnesium sulfate had a poorer quality of bowel preparations (OR=0.6, 95% CI 0.4–0.9; p<0.05, whereas the quality of bowel preparation was significantly improved by using sodium sulfate (OR=5.7, 95% CI 5.4–6.1; p<0.001 and sodium phosphate (OR=2.1, 95% CI 1.8–2.5; p<0.001. For those who had adequate bowel preparation, the better quality of preparation significantly increased the detection rate of advanced adenoma (5.0, 3.6, and 2.9% for excellent, good, and fair, respectively. Conclusion: When possible, sodium sulfate–based preparations should be recommended in the community setting for colonoscopy because of their high quality of bowel preparation.

  6. Construction of the plasmid-free strain for human growth hormone production.

    Science.gov (United States)

    Schulga, A A; Mechev, P V; Kirpichnikov, M P; Skryabin, K G; Deyev, S M

    2016-01-01

    The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

  7. PREPARATION AND THE CULTURE OF LO2 CELLS ON PVA-BASED MICROCARRIERS

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong; YU Yaoting; PAN Jilun; WANG lianyong

    2001-01-01

    Using polyvinyl alcohol (PVA) as raw material and vacuum pump oil as oil phase medium,PVA-based microcarriers were prepared by suspension method The diameters of the beads were 100-180 μ m. LO2 cells were cultured on PVA-based microcarriers and cytodexⅢ microcarriers.Morphology, attachment and growth rate of LO2 cells were studied.

  8. PREPARATION AND THE CULTURE OF LO2 CELLS ON PVA—BASED MICROCARRIERS

    Institute of Scientific and Technical Information of China (English)

    ZHANGHong; YUYaoting; 等

    2001-01-01

    Using polyvinyl alcohol(PVA) as raw material and vacuum pump oil as oil phase medium,PVA-based microcarriers were prepared by suspension method,The diameters of the beads were 100-180um,LO2 cells were cultured on PVA-based microcarriers and cytodexⅢ microcarriers.Morphology,attachment and growth rate of LO2 cells were studied.

  9. PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501-and pHT beta-related replicons associated with glycopeptide resistance and stabilizing toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Rosvoll, T.C.S.; Pedersen, T.; Sletvold, H.;

    2010-01-01

    A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHT beta (n=14) were observed in 83% of the strains, while pS86, pCF10, p......RUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage...... of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments....

  10. Development of a gene transfer system for curing of plasmids in the marine fish pathogen Vibrio salmonicida.

    Science.gov (United States)

    Valla, S; Frydenlund, K; Coucheron, D H; Haugan, K; Johansen, B; Jørgensen, T; Knudsen, G; Strøm, A

    1992-01-01

    All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence. Images PMID:1622274

  11. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  12. Therapeutic low-intensity red laser for herpes labialis on plasmid survival and bacterial transformation.

    Science.gov (United States)

    Sergio, Luiz Philippe da Silva; Marciano, Roberta da Silva; Teixeira, Gleica Rocha; Canuto, Keila da Silva; Polignano, Giovanni Augusto Castanheira; Guimarães, Oscar Roberto; Geller, Mauro; de Paoli, Flavia; da Fonseca, Adenilson de Souza

    2013-05-01

    A low-intensity laser is used in treating herpes labialis based on the biostimulative effect, albeit the photobiological basis is not well understood. In this work experimental models based on Escherichia coli cultures and plasmids were used to evaluate effects of low-intensity red laser on DNA at fluences for treatment of herpes labialis. To this end, survival and transformation efficiency of plasmids in E. coli AB1157 (wild type), BH20 (fpg/mutM(-)) and BW9091 (xthA(-)), content of the supercoiled form of plasmid DNA, as well as nucleic acids and protein content from bacterial cultures exposed to the laser, were evaluated. The data indicate low-intensity red laser: (i) alters the survival of plasmids in wild type, fpg/mutM(-) and xthA(-)E. coli cultures depending of growth phase, (ii) alters the content of the supercoiled form of plasmids in the wild type and fpg/mutM(-)E. coli cells, (iii) alters the content of nucleic acids and proteins in wild type E. coli cells, (iv) alters the transformation efficiency of plasmids in wild type and fpg/mutM(-)E. coli competent cells. These data could be used to understand positive effects of low-intensity lasers on herpes labialis treatment.

  13. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  14. Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae.

    Directory of Open Access Journals (Sweden)

    Shukriti Sharma

    Full Text Available Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.

  15. Preparation of tamarind gum based soft ion gels having thixotropic properties.

    Science.gov (United States)

    Sharma, Mukesh; Mondal, Dibyendu; Mukesh, Chandrakant; Prasad, Kamalesh

    2014-02-15

    Tamarind gum was used to prepare ion gels using both synthetic ionic liquids (ILs) namely 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium bromide and bio-based ionic liquids (Bio-ILs) namely choline acrylate, choline caproate and choline caprylate by heating cooling process. The gels were found to have good thermal stability and exhibited thixotropic behaviour. Upon relaxation after applied breaking strain, the recovery of gel structures after ten consecutive cycles was observed. The hydrogel of the gum prepared using ethanol aqueous solution had much inferior quality in terms of viscosity, viscoelasticity, thermal stability and thixotropicity when compared with the ion gels. The ion gels also showed very good adherence to human finger muscles and skin. The ion gels thus prepared may find application in electrochemistry, sensors, actuators and the gels prepared with Bio-ILs could even be useful in biomedical applications.

  16. Replicon typing of plasmids carrying blaCTX-M-15 among Enterobacteriaceae isolated at the environment, livestock and human interface.

    Science.gov (United States)

    Zurfluh, Katrin; Glier, Melinda; Hächler, Herbert; Stephan, Roger

    2015-07-15

    One of the currently most important antibiotic resistance mechanisms in Enterobacteriaceae is based on the production of ESBL enzymes that inactivate β-lactam antibiotics including cephalosporins and monobactams by hydrolyzing their β-lactam ring. In humans, the most prevalent ESBL enzyme type is encoded by blaCTX-M-15. CTX-M-15 producing enterobacterial strains were also frequently isolated from environmental samples including surface water and freshwater fish. Plasmids, which can be grouped in different plasmid incompatibility types, play a key role in the horizontal spread of these multidrug resistance genes. The purpose of this study was to investigate the diversity of plasmids that carry blaCTX-M-15 genes among Enterobacteriaceae isolated at the environment, livestock and human interface. In total, 81 blaCTX-M-15-harboring isolates collected between 2009 and 2014 were tested for its ability to transfer blaCTX-M-15 by conjugation. These plasmids were further typed. Transfer of a single blaCTX-M-15-harboring plasmid was observed in 32 (39.5%) of the isolates. The most frequent replicon types detected among these plasmids are IncF-type plasmids (n=12) (mostly multi replicon plasmids with a combination of following replicons: IncFII, IncFIA and IncFIB), followed by IncI1 (n=8), IncK (n=3) and IncR (n=1). A noticeable number of plasmids (n=8) could not be assigned to any of the tested replicon types. Knowledge about the plasmid types circulating in bacterial populations is indispensable for understanding epidemiological dynamics and for establishing intervention strategies to stop further dissemination of particular plasmids.

  17. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  18. Development and application of a general plasmid reference material for GMO screening.

    Science.gov (United States)

    Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

    The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Evaluation of roll compaction as a preparation method for hydroxypropyl cellulose-based matrix tablets

    Directory of Open Access Journals (Sweden)

    Imjak Jeon

    2011-01-01

    Full Text Available Roll compaction was applied for the preparation of hydroxypropyl cellulose (HPC-based sustained-release matrix tablets. Matrix tablets made via roll compaction exhibited higher dosage uniformity and faster drug release than direct-compacted tablets. HPC viscosity grade, roll pressure, and milling speed affected tablet properties significantly. Roll compaction seems to be an adequate granulation method for the preparation of HPC-based matrix tablets due to the simplicity of the process, less handling difficulty from HPC tackiness as well as easier particle size targeting. Selecting the optimum ratio of plastic excipients and the particle size of starting materials can however be critical issues in this method.

  20. Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain

    Directory of Open Access Journals (Sweden)

    Skilton Rachel J

    2009-05-01

    suggest that the plasmid is not a highly mobile genetic element and does not transfer readily between isolates. Comparative analysis of the plasmid sequences has revealed the most conserved regions that should be used to design future plasmid based nucleic acid amplification tests, to avoid diagnostic failures.

  1. Characterization of dicalcium phosphate dihydrate cements prepared using a novel hydroxyapatite-based formulation

    Energy Technology Data Exchange (ETDEWEB)

    Alge, Daniel L [Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47908 (United States); Cruz, Grace Santa; Chu, Tien-Min Gabriel [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Goebel, W Scott, E-mail: tgchu@iupui.ed [Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States)

    2009-04-15

    Dicalcium phosphate dihydrate (DCPD) cements are typically prepared using beta-tricalcium phosphate (beta-TCP) as the base component. However, hydroxyapatite (HA) is an interesting alternative because of its potential for reducing cement acidity, as well as modulating cement properties via ionic substitutions. In the present study, we have characterized DCPD cements prepared with a novel formulation based on monocalcium phosphate monohydrate (MCPM) and HA. Cements were prepared using a 4:1 MCPM:HA molar ratio. The reactivity of HA in this system was verified by showing DCPD formation using poorly crystalline HA, as well as highly crystalline HA. Evaluation of cements prepared with poorly crystalline HA revealed that setting occurs rapidly in the MCPM/HA system, and that the use of a setting regulator is necessary to maintain workability of the cement paste. Compressive testing showed that MCPM/HA cements have strengths comparable to what has previously been published for DCPD cements. However, preliminary in vitro analysis of cement degradation revealed that conversion of DCPD to HA may occur much more rapidly in the MCPM/HA system compared to cements prepared with beta-TCP. Future studies should investigate this property further, as it could have important implications for the use of HA-based DCPD cement formulations.

  2. Preparation and optimization of glyceryl behenate-based highly porous pellets containing cilostazol.

    Science.gov (United States)

    Hwang, Kyu-Mok; Byun, Woojin; Cho, Cheol-Hee; Park, Eun-Seok

    2016-11-03

    The aim of this study was to prepare a highly porous multiparticulate dosage form containing cilostazol for gastroretentive drug delivery. The floating pellets were prepared with glyceryl behenate as a matrix former and camphor as a sublimating agent by extrusion/spheronization and sublimation under vacuum. Granules prepared with sublimation at 60 °C displayed a slower dissolution rate and smoother surface morphology than those prepared at lower temperatures. This was unexpected as the reported melting point of glyceryl behenate is higher than 69 °C. The DSC study revealed that melting began at a lower temperature owing to the multicomponent property of glyceryl behenate, which led to a sintering effect. The prepared pellets were spherical with unimodal size distribution. They also had porous structures with increased porosity, which led to immediate buoyancy. As cilostazol is a hydrophobic drug that has an erosion-based release mechanism, drug release profile was highly correlated with the percentage of disintegrated pellets. Various excipients were added to the glyceryl behenate-based formulation to increase the floating duration. When hydroxyethyl cellulose was added to the glyceryl behenate-based pellets, acceptable dissolution rate and buoyancy were acquired. This system could potentially be used for gastroretentive delivery of various hydrophobic drugs, which was generally considered difficult.

  3. Study on preparation and regeneration condition of protoplasts of Cephalosporium acremonium and transformation of plasmid DNA with vgb gene%顶头孢霉原生质体制备、再生及带vgb基因的质粒DNA转化条件的研究

    Institute of Scientific and Technical Information of China (English)

    戴秀君; 马文婵; 李术娜; 王全; 李红亚; 朱宝成

    2009-01-01

    The formation and regeneration conditions of Cephalosporium acremonium protoplast were studied. The optimum conditions of protoplast preparation were as follow: After Cephalosporium acremonium mycelium has been cultivated for 110 -120 h on peptone medium, using enzyme 1%snailase+1% cellulase("Onozoka R - 10") to digest the cell wall for 3. 5 h at 30 ℃ , the number of protoplast reaches about 2. 377 × 10~7 per milliliter. Meanwhile the regeneration was tested on solid media containing KC (0. 6 mol/L KCl+25 mmol/L CaCL_2 · 2H_2O) as osmotic stabilizer, the regeneration ratio is about 40%. Then the constructed shuttle plasmid pMK4 - LV was transformed into protoplasts by using electroporation method. The recombi-nant was obtained. Successfully. The transfer ratio is about 10 recombinants/μg DNA. The vgb gene was testified to have been recombined into the Cephalosporium acremonium genome through the PCR amplification.%对头孢菌素C的工业生产菌种顶头孢霉(Cephalosporium acremonium)原生质体形成和再生条件进行了研究.优化后的制备及再生条件为:在蛋白胨固体培养基上培养110~120 h的菌丝体,30 ℃条件下经1%蜗牛酶和1%纤维素酶混合液酶解3.5 h,原生质体得率最高可达2.377×10~7 个/mL;在以KC(0 6 mol/L KCl+25 mmol/L CaCl_2·2H_2O)为渗透压稳定剂的再生培养基上进行培养,再生率可达40%.用pMK4-LV质粒通过电击法对原生质体进行转化,成功得到了重组子,转化率约为10 个转化子/μg质粒DNA.转化子的 PCR鉴定结果表明,外源基因已转入顶头孢霉基因组.

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

    Science.gov (United States)

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

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

  5. Abundance and diversity of plasmid-associated genes among clinical isolates of Enterococcus faecalis.

    Science.gov (United States)

    Wardal, Ewa; Gawryszewska, Iwona; Hryniewicz, Waleria; Sadowy, Ewa

    2013-11-01

    Enterococcus faecalis, a normal compound of the human intestinal microbiome, plays an important role in hospital-acquired infections. Plasmids make a significant contribution to the acquisition of the novel traits such as antimicrobial resistance and virulence by this pathogen. The study investigated the plasmid content and the diversity of plasmid-associated genes in a group of 152 hospital isolates of E. faecalis. The majority of plasmids visualized by pulsed-field gel electrophoresis of S1 nuclease-digested DNA fell into the range of 50-100 kb. PCR-based screening allowed detection of genes of the rep1(pIP501), rep2(pRE25), rep4(pMBB1), rep6(pS86), rep7(pT181), rep8(pAM373), rep9(pAD1/pTEF2/pCF10), rep10(pIM13) and rep13(pC194) families in 29 different combinations. The par and ω-ε-ζ plasmid stabilization systems were ubiquitous (45 isolates, 29.6% and 88 isolates, 57.9%, respectively), while the axe-txe system was not found. The asa1 gene homologues encoding aggregation substance characteristic for the pAD1 and related group of pheromone-responsive plasmids were present in 106 isolates. A variety of sequence variants, including novel ones, of genes associated with pheromone-responsive plasmids, such as rep8(pAM373), rep9(pAD1/pTEF2/pCF10), par, and asa1 were observed. In conclusion, there is a big and only partially characterized pool of diverse plasmids in clinical E. faecalis.

  6. Serious overestimation in quantitative PCR by circular (supercoiled plasmid standard: microalgal pcna as the model gene.

    Directory of Open Access Journals (Sweden)

    Yubo Hou

    Full Text Available Quantitative real-time PCR (qPCR has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form and linear DNA standards (linearized plasmid DNA or PCR amplicons, using proliferating cell nuclear gene (pcna, the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.

  7. Altered murine tissue colonization by Borrelia burgdorferi following targeted deletion of linear plasmid 17-carried genes.

    Science.gov (United States)

    Casselli, Timothy; Tourand, Yvonne; Bankhead, Troy

    2012-05-01

    The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined both in vitro and in vivo. A mutant strain lacking the genes bbd16 to bbd25 showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein C in vitro, which was correlated with the absence of the gene bbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone of B. burgdorferi and reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

  8. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density.

    Science.gov (United States)

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan

    2014-06-01

    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Preparation and testing of plant seed meal-based wood adhesives.

    Science.gov (United States)

    He, Zhongqi; Chapital, Dorselyn C

    2015-01-01

    Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable and environment-friendly. These natural products may serve as alternatives to petroleum-based adhesives to ease environmental and sustainability concerns. This work demonstrates the preparation and testing of the plant seed-based wood adhesives using cottonseed and soy meal as raw materials. In addition to untreated meals, water washed meals and protein isolates are prepared and tested. Adhesive slurries are prepared by mixing a freeze-dried meal product with deionized water (3:25 w/w) for 2 hr. Each adhesive preparation is applied to one end of 2 wood veneer strips using a brush. The tacky adhesive coated areas of the wood veneer strips are lapped and glued by hot-pressing. Adhesive strength is reported as the shear strength of the bonded wood specimen at break. Water resistance of the adhesives is measured by the change in shear strength of the bonded wood specimens at break after water soaking. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for substitution of synthetic-based wood adhesives. Adjustments to the adhesive formulation with or without additives and bonding conditions could optimize their adhesive properties for various practical applications.

  10. Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

    2015-01-01

    The gut epithelium formed between an organism and the environment plays an essential role in host–microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins...... are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were...... of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved....

  11. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  12. Preparation and characterization of zirconium based colloidal suspensions; Preparacao e caracterizacao de suspensoes coloidais a base de zirconio

    Energy Technology Data Exchange (ETDEWEB)

    Chiavacci, Leila Aparecida; Pulcinelli, Sandra Helena; Santilli, Celso Valentim [UNESP, Araraquara, SP (Brazil). Inst. de Quimica

    1995-12-31

    Attention has been paid to zirconia based ceramic materials due to its technological applications, like catalysts, ionic changers, sensors, etc. Colloidal suspensions based on zirconium, containing different cation/anion molar ratio, have been prepared aiming the improvement of its extrinsical properties. The systems have been characterized by turbidimetry and the xerogels have been analysed by scanning electronic microscopy, infrared spectroscopy and x-ray diffraction. Results make evident the viability of preparation of transparent colloidal suspensions, gel and powders, in which the particles size and morphology could be controlled. (author) 3 figs.

  13. Differentiating between benign follicular nodules and follicular neoplasms in thyroid liquid-based cytology preparations.

    Science.gov (United States)

    Suzuki, Ayana; Hirokawa, Mitsuyoshi; Higuchi, Miyoko; Takada, Nami; Yamao, Naoki; Kuma, Seiji; Miyauchi, Akira

    2016-08-01

    The cytological morphology observed in liquid-based cytology (LBC) preparations is dissimilar to that of conventional preparations. The aim of this report is to clarify the cytological differences between benign follicular nodules (BFNs) and follicular neoplasms (FNs) in LBC preparations and identify novel diagnostic criteria for LBC preparations. A retrospective review of LBC preparations from 38 BFN and 74 FN (57 follicular adenomas and 17 follicular carcinomas) cases confirmed by histological examination was conducted. LBC samples were obtained from the washout fluid in aspiration needles, fixed with CytoRich-RED(TM) , and prepared using the SurePath(TM) method. Fibrin was observed in 43.2% of FNs and in 23.7% of BFNs. The incidences of intercellular spaces, distinct outer margin, and cytoplasmic process were significantly higher in BFNs (P < 0.01, P < 0.01, and P < 0.05, respectively). Elongated microfollicles were seen in 55.4% of FNs and 10.5% of BFNs (P < 0.01). Membranous materials encircling the outer edge of the follicles were observed only in BFN cases (5.8%). There were no significant differences between follicular adenoma and carcinoma. Intercellular spaces, distinct outer margins of the follicular clusters, cytoplasmic process, and membranous materials constitute indicators of BFN in LBC preparations. Fibrin and elongated microfollicles point to FN. We believe that these findings will improve the diagnostic accuracy of thyroid LBC preparations. Diagn. Cytopathol. 2016;44:659-664. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Amplification of full-length hepatitis C virus genome based on plasmid pJFH-1%基于pJFH-1的HCV全基因组扩增方法的建立

    Institute of Scientific and Technical Information of China (English)

    郭艳; 兰林; 何长龙; 洪国祜; 程玲; 毛青

    2011-01-01

    目的 基于pJFH-1建立能稳定扩增HCV全基因组的长链PCR方法.方法 以pJFH-1为测试模板,通过优化PCR扩增中各个重要环节,包括引物的选择、甘油和/或DMSO最适浓度的筛选、循环条件的摸索等,建立能稳定扩增HCV全基因组的长链PCR方案.结果 高Tm值(>65 ℃)的引物更有利于HCV全基因组的扩增;5%、10%甘油或5% DMSO可显著提高PCR扩增的特异性和扩增效率,且甘油的促进作用优于DMSO;双温法较三温法能获得更高产量的PCR产物.结论 通过优化长链PCR反应体系及条件,成功实现HCV基因全长的扩增.%Objective To optimize the protocols of long-PCR for amplifying full-length HCV genome based on plasmid pJFH-1. Methods Optimization of long-PCR strategies was performed by testing a series of primers, adding various concentrations of glycerol, DMSO or both, using different cycle systems to select the optimal long PCR conditions. Results Primers that have higher melting temperatures ( > 65 C )could improve the efficiency of amplification. Glycerol of 5% and 10% or DMSO of 5% improved the specificity and efficiency of PCR amplification of full-length HCV genome. The promoting effect of glycerol was better than that of DMSO. Compared to three-stage temperature method, two-step temperature produced more PCR product. Conclusion Through the optimization of long-PCR protocol, full-length HCV genome is successfully achieved.

  15. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  16. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  17. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  18. A Competency-Based Approach to the Master's Degree Preparation of Higher Education Professionals

    Science.gov (United States)

    Ott, Molly; Baca, Evelyn; Cisneros, Jesus; Bates, Evan

    2015-01-01

    The purpose of this manuscript is to describe a competency-based approach to designing and assessing master's level professional preparation programs in the field of higher education administration. Given the absence of a universal set of competencies defined for HEA master's degree programs, the authors draw from the CAHEP (2010) and Wright…

  19. Preparing Biology Teachers to Teach Evolution in a Project-Based Approach

    Science.gov (United States)

    Cook, Kristin; Buck, Gayle; Park Rogers, Meredith

    2012-01-01

    This study investigates a project-based learning (PBL) approach to teaching evolution to inform efforts in teacher preparation. Data analysis of a secondary biology educator teaching evolution through a PBL approach illuminated: (1) active student voice, which allowed students to reflect on their positioning on evolution and consider multiple…

  20. Succinate-based preparation alleviates manifestations of the climacteric syndrome in women.

    Science.gov (United States)

    Peskov, A B; Maevskii, E I; Uchitel', M L; Sakharova, N Yu; Vize-Khripunova, M A

    2005-09-01

    Clinical placebo-controlled study of Enerlit-Clima (bioactive succinate-based food additive) a showed positive effect of the preparation on general clinical and psychoemotional manifestations of the climacteric syndrome. A trend to an increase in estradiol level in early pathological climacteric and normalization of the endometrial status were observed.

  1. Carbon-based Composite Electrodes: Preparation, Characterization and Application in Electroanalysis

    NARCIS (Netherlands)

    Corb, I.; Manea, F.; Radovan, C.; Pop, A.; Burtica, G.; Malchev, P.G.; Picken, S.J.; Schoonman, J.

    2007-01-01

    Electrodes based on carbon, i.e., expanded graphite (20%, wt.)-epoxy composite (20EG-Epoxy) and expanded graphite (20%, wt.)-polystyrene composite (20EG-PS) have been prepared, characterized using scanning electron microscopy (SEM) and cyclic voltammetry (CV), and tested as anodic sensors. The elect

  2. Preparing Instructional Designers for Game-Based Learning: Part III. Game Design as a Collaborative Process

    Science.gov (United States)

    Hirumi, Atsusi; Appelman, Bob; Rieber, Lloyd; Van Eck, Richard

    2010-01-01

    In this three part series, four professors who teach graduate level courses on the design of instructional video games discuss their perspectives on preparing instructional designers to optimize game-based learning. Part I set the context for the series and one of four panelists discussed what he believes instructional designers should know about…

  3. Preparation and characterization of gas separation hollow fiber membranes based on polyethersulfone-polyimide miscible blends

    NARCIS (Netherlands)

    Kapantaidakis, G.C.; Koops, G.H.; Wessling, M.

    2002-01-01

    In this work the preparation and characterization of gas separation hollow fibers based on polyethersulfone Sumikaexcel (PES) and polyimide Matrimid 5218 (PI) blends are reported. Scanning Electron Microscopy (SEM) was used to investigate the morphological characteristics and structure of the asymme

  4. Flexible Pedagogies: Employer Engagement and Work-Based Learning. Flexible Pedagogies: Preparing for the Future Series

    Science.gov (United States)

    Kettle, Jane

    2013-01-01

    This publication focuses on national and international policy initiatives to develop a better understanding of work-based learners and the types of flexibility that may well enhance their study especially pedagogically. As part of our five-strand research project "Flexible Pedagogies: preparing for the future" it: (1) highlights the…

  5. Inventing Motivates and Prepares Student Teachers for Computer-Based Learning

    Science.gov (United States)

    Glogger-Frey, I.; Kappich, J.; Schwonke, R.; Holzäpfel, L.; Nückles, M.; Renkl, A.

    2015-01-01

    A brief, problem-oriented phase such as an inventing activity is one potential instructional method for preparing learners not only cognitively but also motivationally for learning. Student teachers often need to overcome motivational barriers in order to use computer-based learning opportunities. In a preliminary experiment, we found that student…

  6. Embedding Evidence-Based Practice in Pre-Service Teacher Preparation

    Science.gov (United States)

    Bain, Alan; Lancaster, Julie; Zundans, Lucie; Parkes, Robert John

    2009-01-01

    In this study, the authors sought to establish the differential effects on achievement of embedding evidence-based practice in the design of an inclusive education teacher preparation course. Embedded design involves creating self-repeating patterns in the instructional design of a course by expressing essential design features at multiple levels…

  7. Bismuth oxide based ceramics with improved electrical and mechanical properties: Part I. Preparation and characterisation

    NARCIS (Netherlands)

    Kruidhof, H.; Seshan, K.; Lippens Jr., B.C.; Gellings, P.J.; Burggraaf, A.J.

    1987-01-01

    A study of the preparative variables in the synthesis of ceramics based on Bi2O3-Er2O3 solid solutions has shown that the best results are obtained with a coprecipitation method. Critical parameters in the synthesis are found to be i) wet milling of the precalcined powder with an appropriate liquid

  8. Unfolding Case-Based Practicum Curriculum Infusing Crisis, Trauma, and Disaster Preparation

    Science.gov (United States)

    Greene, Catie A.; Williams, Amy E.; Harris, Pamela N.; Travis, Sterling P.; Kim, Sharon Y.

    2016-01-01

    The authors evaluated an unfolding case-based approach to a practicum in counseling course infusing crisis, trauma, and disaster preparation for changes in students' crisis self-efficacy across a semester. The course, informed by constructivist-developmental pedagogy and centered on the unfolding case, resulted in significant increases in…

  9. Plasmid pORF-hTRAIL targeting to glioma using transferrin-modified polyamidoamine dendrimer

    Directory of Open Access Journals (Sweden)

    Gao S

    2015-12-01

    Full Text Available Song Gao,1,* Jianfeng Li,2 Chen Jiang,2 Bo Hong,3 Bing Hao4,* 1Department of Clinical Laboratory, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 2Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 3Department of Pathology, The Second Affiliated Hospital, 4Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: A gene drug delivery system for glioma therapy based on transferrin (Tf-modified polyamidoamine dendrimer (PAMAM was prepared. Gene drug, tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL-encoding plasmid open reading frame (pORF-hTRAIL, Trail, was condensed by Tf-modified PAMAM to form nanoparticles (NPs. PAMAM-PEG-Tf/DNA NPs showed higher cellular uptake, in vitro gene expression, and cytotoxicity than PAMAM-PEG/DNA NPs in C6 cells. The in vivo targeting efficacy of NPs was visualized by ex vivo fluorescence imaging. Tf-modified NPs showed obvious glioma-targeting trend. Plasmid encoding green fluorescence protein (GFP was also condensed by modified or unmodified PAMAM to evaluate the in vivo gene expression level. The PAMAM-PEG-Tf/plasmid encoding enhanced green fluorescence protein (pEGFP NPs exhibited higher GFP expression level than PAMAM-PEG/pEGFP NPs. TUNEL assay revealed that Tf-modified NPs could induce much more tumor apoptosis. The median survival time of PAMAM-PEG-Tf/Trail-treated rats (28.5 days was longer than that of rats treated with PAMAM-PEG/Trail (25.5 days, temozolomide (24.5 days, PAMAM-PEG-Tf/pEGFP (19 days, or saline (17 days. The therapeutic effect was further confirmed by magnetic resonance imaging. This study demonstrated that targeting gene delivery system had potential application for the

  10. Plasmids spread very fast in heterogeneous bacterial communities.

    Science.gov (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  11. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  12. Preparation and Adsorption Properties of PAM Based Adsorbents for Plasma Lipoproteins

    Institute of Scientific and Technical Information of China (English)

    Hai Tao LI; Zhi YUAN; Xin Fu CHEN; Bin LIU; Bin SHEN; Bing Lin HE

    2004-01-01

    Crosslinked macroporous polyacrylamide(PAM)was prepared with inverse phase suspension polymerization technique.After treatment with hydrazine,the polymer was functionalized with chloroacetic acid,trifluoroacetic acid diethylenetriaminepentaacetic acid (DEPAA), and maleic acid, respectively,and PAM based adsorbents bearing carboxyl functional groups for low density lipoprotein(LDL)apheresis use were obtained.The blood compatibility and the adsorption properties for plasma lipoproteins of PAM based adsorbents were investigated.

  13. CO Responses of Sensors Based on Cerium Oxide Thick Films Prepared from Clustered Spherical Nanoparticles

    OpenAIRE

    Woosuck Shin; Takafumi Akamatsu; Toshio Itoh; Ichiro Matsubara; Noriya Izu

    2013-01-01

    Various types of CO sensors based on cerium oxide (ceria) have been reported recently. It has also been reported that the response speed of CO sensors fabricated from porous ceria thick films comprising nanoparticles is extremely high. However, the response value of such sensors is not suitably high. In this study, we investigated methods of improving the response values of CO sensors based on ceria and prepared gas sensors from core-shell ceria polymer hybrid nanoparticles. These hybrid nano...

  14. Preparation of electrodes on cfrp composites with low contact resistance comprising laser-based surface pre-treatment

    KAUST Repository

    Almuhammadi, Khaled Hamdan

    2016-12-29

    Various examples are provided related to the preparation of electrodes on carbon fiber reinforced polymer (CFRP) composites with low contact resistance. Laser-based surface preparation can be used for bonding to CFRP composites. In one example, a method includes preparing a pretreated target area on a CFRP composite surface using laser pulsed irradiation and bonding an electrode to exposed fibers in the pretreated target area. The surface preparation can allow the electrode to have a low contact resistance with the CFRP composite.

  15. The influence of the base material surface preparation on the properties of thermally sprayed coatings

    Directory of Open Access Journals (Sweden)

    V. Marušić

    2010-01-01

    Full Text Available Using specimens, a research was conducted to determine the influence of the base material surface preparation for 42CrMo4 on the final coating, prior to actual thermal spraying. During thermal spraying, an Al-Ni-alloy was used as an interlayer before the actual coating with Cr-Mo-Ni. The surface hardness and the hardness distribution across the thickness of the sprayed coating were measured and the structure of respective sprayed coatings was photographed. A comparison of experimental results enabled the identification of the particular material preparation method with an optimal ratio of the satisfactory coating thickness and its hardness.

  16. Room temperature ethanol sensor based on ZnO prepared via laser ablation in water

    Science.gov (United States)

    Kondo, Takahiro; Sato, Yoshihiro; Kinoshita, Masahiro; Shankar, Prabakaran; Mintcheva, Neli N.; Honda, Mitsuhiro; Iwamori, Satoru; Kulinich, Sergei A.

    2017-08-01

    The present work reports on room-temperature ethanol sensing performance of ZnO nanospheres and nanorods prepared using pulsed laser ablation in water. Nanosecond and millisecond lasers were used to prepare ZnO nanomaterials with hexagonal wurtzite crystal structure. The two contrasting nanostructures were tested as gas sensors towards volatile compounds such as ethanol, ammonia, and acetone. At room temperature, devices based on both ZnO nanomaterials demonstrated selectivity for ethanol vapor. The sensitivity of nanospheres was somewhat higher compared to that of nanorods, with response values of ∼19 and ∼14, respectively, towards 250 ppm. Concentrations as low as 50 ppm could be easily detected.

  17. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

    OpenAIRE

    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G

    1984-01-01

    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  18. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  19. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  20. Immunization responses against equine influenza virus (EIV) based on recombinant Alphavirus replicon plasmid expressing EIV hemagglutinin gene%马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

    Institute of Scientific and Technical Information of China (English)

    仇铮; 郭巍; 孙元; 戚亭; 仇华吉; 相文华

    2012-01-01

    To evaluate DNA vaccination against equine influenza virus (EIV), the hemagglutinin (HA) genes of American and European H3N8 subtype EIV were amplified by RT-PCR and cloned into a Semliki forest virus (SFV) replicon based plasmid of pSFV to construct the recombinant plasmid pSFV1CS-EIV-HA, respectively. The HA expressions were identified in the 293T cells transfected with the recombinant plasmid by indirect immunofluorescence assay. Furthermore, the specific HI antibody, IFN-γ or IL-4 level were detected in BALB/c mice immunized with either single or both of the recombinant plasmids. However, the virus was failed to detect in immunized BALB/c mice by the RT-PCR detection or the virus isolation. The results indicated that the recombinant plasmids possessed a capability for inducing effective immune responses in animals, which was a promising candidate DNA vaccine against EIV infection.%为评价马流感病毒(EIV) HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒.并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高.攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR 均未检测到病毒.上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力.

  1. Preparation and evaluation of cationic bolaform surfactants for water-based drilling

    Directory of Open Access Journals (Sweden)

    M.M. Dardir

    2017-03-01

    Full Text Available Three cationic bolaform surfactants with different spacer lengths were prepared from the reaction of two moles of triisopropanolamine with one mole of each of the following 1,4-dibromobutane, 1,5-dibromopentane and 1,6-dibromohexane. The chemical structures of the prepared compounds were confirmed via: FTIR spectroscopy, 1H NMR and elemental microanalysis. The surface activity of these bolaform surfactants was studied. The prepared cationic bolaform surfactants were evaluated as viscosifier additives for water-based drilling fluids. The evaluation includes the study of rheological properties of the formulated mud (apparent viscosity, plastic viscosity and yield point, gel strength and thixotropy, effect of temperature on the rheological properties and also, the study of mineralogical properties of the water-based before and after treatment with the prepared surfactants using: X-ray diffraction (XRD, scanning electron microscope (SEM and transmission electron microscope (TEM. The results of the evaluation were compared to the water-based mud formulated from commercial grade bentonite.

  2. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  3. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  4. Preparation and characterization of amidated pectin based hydrogels for drug delivery system.

    Science.gov (United States)

    Mishra, R K; Datt, M; Pal, K; Banthia, A K

    2008-06-01

    In the current studies attempts were made to prepare hydrogels by chemical modification of pectin with ethanolamine (EA) in different proportions. Chemically modified pectin products were crosslinked with glutaraldehyde reagent for preparing hydrogels. The hydrogels were characterized by Fourier transform infrared spectroscopy (FTIR), organic elemental analysis, X-ray diffraction studies (XRD), swelling studies, biocompatibility and hemocompatibility studies. Mechanical properties of the prepared hydrogels were evaluated by tensile test. The hydrogels were loaded with salicylic acid (used as a model drug) and drug release studies were done in a modified Franz's diffusion cell. FTIR spectroscopy indicated the presence of primary and secondary amide absorption bands. XRD studies indicated increase in crystallinity in the hydrogels as compared to unmodified pectin. The degree of amidation (DA) and molar and mass reaction yields (YM and YN) was calculated based on the results of organic elemental analysis. The hydrogels showed good water holding properties and were found to be compatible with B-16 melanoma cells & human blood.

  5. Repeating cytological preparations on liquid-based cytology samples: A methodological advantage?

    Science.gov (United States)

    Pinto, Alvaro P; Maia, Henrique Felde; di Loretto, Celso; Krunn, Patrícia; Túlio, Siumara; Collaço, Luis Martins

    2007-10-01

    This study investigates the rule that repeating cytological preparations on liquid-based cytology improves sample adequacy, diagnosis, microbiological, and hormonal evaluations. We reviewed 156 cases of pap-stained preparations of exfoliated cervical cells in two slides processed by DNA-Cytoliq System. After sample repeat/dilution, limiting factors affecting sample adequacy were removed in nine cases and three unsatisfactory cases were reclassified as satisfactory. Diagnosis was altered in 24 cases. Of these, the original diagnosis in 15 was atypical squamous cells of undetermined significance; after the second slide examination, diagnosis in 5 of the 15 cases changed to low-grade squamous intraepithelial lesion, 3 to high-grade squamous intraepithelial lesion, and 7 to absence of lesion. Microbiological evaluation was altered, with Candida sp. detected in two repeated slides. Repeat slide preparation or dilution of residual samples enhances cytological diagnosis and decreases effects of limiting factors in manually processed DIGENE DCS LBC.

  6. Preparation and characterization of hybrid nanoparticles based on chitosan and poly(methacryloylglycylglycine)

    Science.gov (United States)

    Ferri, Marcella; Dash, Mamoni; Cometa, Stefania; De Giglio, Elvira; Sabbatini, Luigia; Chiellini, Federica

    2014-05-01

    The present work investigated the possibility of preparing nanoparticles based on methacryloylglycylglycine (MAGG) and chitosan (CS) by in situ polymerization. The study revealed that nanoparticle formation was strictly dependent on ionic interactions between NH3 + groups from CS and COO- groups arising from the anionic monomer MAGG. The subsequent in situ polymerizations of MAGG in the presence of CS led to the formation of nanoparticles with homogeneous morphology, a uniform particle size distribution, and a good spherical shape as confirmed by laser diffraction granulometry and scanning electron microscopy analyses. Nanoparticle formulations with different amounts of CS and MAGG were prepared, and their chemical compositions were investigated by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The obtained results showed that the polymerization of MAGG in the presence of CS appears to be a very promising approach in the preparation of nanoparticles for drug delivery applications.

  7. Sintering of anorthite based ceramics prepared from kaolin DD2 and calcite

    Energy Technology Data Exchange (ETDEWEB)

    Zaiou, S.; Harabi, A.; Harabi, E.; Guechi, A.; Karboua, N.; Benhassine, M.-T.; Zouai, S.; Guerfa, F., E-mail: Zaiou_21@yahoo.fr, E-mail: harabi52@gmail.com, E-mail: semouni84@gmail.com, E-mail: guechia@yahoo.fr, E-mail: kanour17@yahoo.fr, E-mail: mtb25dz@gmail.com, E-mail: zouaisouheila@yahoo.fr, E-mail: guerfatiha@gmail.com [Ceramics Lab., Faculty of Exact Science, Physics Department, Mentouri University of Constantine (Algeria)

    2016-10-15

    In this work, the preparation of anorthite based ceramics using a modified milling system and 80 wt% kaolin (DD2 type) and 20 wt% calcium oxide extracted from CaCO{sub 3} is shown. The choice of these raw materials was dictated by their natural abundance. Previous studies have shown that a simple and vibratory multidirectional milling system using a bimodal distribution of highly resistant ceramics can be successfully used for obtaining fine powders. The prepared samples were sintered at different temperatures ranging between 800 and 1100 °C. It has been found that the relative density of samples sintered at 900 °C for 1 h with a heating rate of 5 °C/min was about 96% of the theoretical density of anorthite (2.75 g/cm{sup 3} ). Finally, the prepared samples were also characterized by scanning electron microscopy, X-ray diffraction and Raman spectroscopy. (author)

  8. Preparation of New Type Ni-P Micro/Nano Metal Material Based on Bacteria Shape

    Institute of Scientific and Technical Information of China (English)

    Xin Liang; Jianhua Liu; Songmei Li

    2009-01-01

    A new type of Ni-P alloy with rod-shape was prepared by electroless deposition method based on the shape of Nocadia, a kind of bacteria. The material was characterized by microbiological method, scanning elec-tron microscope, energy dispersion spectroscopy, transmission electron microscopy, fourier transform infrared spectroscopy, X-ray diffraction and vibrant sample magnetometer. It was found that Ni-P alloy deposited on Nocadia surface was amorphous when pH=8.0. The amount of Ni crystalline increased with pH of plating solution. Ni-P nano-particles deposited on active locations on the surface at the initial stage, and then ho-mogeneous Ni-P film formed with time. Nocadia remained their original rod shape after Ni-P nano-particles deposition. The new type metal material formed of Ni-P alloy with nano-particles was prepared. The mag-netization of the material prepared at pH=9.7 is greater than that prepared at pH=8.0. The magnetic loss of the material prepared at pH=9.7 is less than 0.1. The dielectric loss exceeds 0.3 when frequency is higher than 14 GHz, which is 1.5 at 18 GHz. The new type Ni-P metal material with Nocadia shape has dielectric loss property.

  9. Scalable preparation, characterization, and application of alkali-treated starch as a new organic base catalyst.

    Science.gov (United States)

    Tamaddon, Fatemeh; KazemiVarnamkhasti, MohammadTaghi

    2017-01-02

    Preparation, characterization, and application of alkali starch (AS) given by dry co-grinding of starch and alkali is described in this work. Grinding using a mortar (agate) and pestle or, more conveniently, a ball mill has been found to be satisfactory for the preparation of the AS. The AS products were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) and x-ray fluorescence (XRF) analyses. The base capacities of ASs were 4.25-4.45 mmol/g, respectively. AS is a low cost and easy to handle base catalyst that showed promising catalytic performance in the synthesis of a dihydroquinazoline-based antibacterial drug that involves tandem hydration or decarboxylative amidation, imination, and Aza-Michael reactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Preparation of six quinazoline schiff bases and their inhibitory effect on HHCC and Bcap-37 cells

    Institute of Scientific and Technical Information of China (English)

    陈惠; 孙晓莉; 刘志红; 张生勇; 药立波

    2003-01-01

    Objective: To prepare six quinazoline schiff bases by six steps of chemistry organic synthesis and test their inhibitory effect on hepatomacellular carcinoma cells HHCC and mammary cancer cell Bcap-37, furthmore,to compare their antitumor activities on these two kinds of cells. Methods: 2-Amino-5-nitro-benzylcarbonitrile was the initial material, and it was under the reaction of hydrolysis, ring-closing, halogenation, addition, reduction and substitution in turn to get the six quinazoline schiff bases, MTT method was adopted to compare their anticancer activities against the two cancer cells. Result and Conclusion: Six 6-imine-4-halo substituted anilinoquinozolines were prepared. The anticancer activities against both HHCC and Bcap-37were found, furthermore, they have more potency that on HHCC than on Bcap-37. In the six compounds, the schiff base Ⅵ is the most potent compound.

  11. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    Science.gov (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  12. Development and stability of semisolid preparations based on a supercritical CO2 Arnica extract.

    Science.gov (United States)

    Bilia, Anna Rita; Bergonzi, Maria Camilla; Mazzi, Giovanni; Vincieri, Franco Francesco

    2006-05-03

    Conventional herbal drug preparations (HDP) based on Arnica montana L. have a low content of the active principles, sesquiterpene lactones, which show poor stability and low physical compatibility in semisolid formulations. Recently, an innovative supercritical carbon dioxide (CO2) extract with high sesquiterpene content has been marketed. Development of six semisolid preparations (cetomacrogol, polysorbate 60, polawax, anphyphil, natrosol and sepigel) based on this innovative CO2 extract is discussed. Stability of these preparations was investigated according to ICH guidelines. The evaluation of in vitro release of active constituents was performed using the cell method reported in the European Pharmacopoeia. Preliminary data on in vivo permeation of three selected formulations is demonstrated using the "skin stripping" test, according to the FDA, in healthy subjects. Analysis of sesquiterpene lactones within the extract and in vitro and in vivo studies was performed by RP-HPLC-DAD-MS method. The cetomacrogol showed the best release profile in the in vitro test, while in the in vivo test the best preparation resulted polysorbate 60 and polawax.

  13. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

    Directory of Open Access Journals (Sweden)

    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  14. A facile method to prepare a high performance solid-state flexible paper-based supercapacitor

    Energy Technology Data Exchange (ETDEWEB)

    Shieh, Jen-Yu; Zhang, Sheng-Hui; Wu, Cheng-Hung [Institute of Electro-Optical and Materials Science, National Formosa University, 64 Wenhua Road, Huwei, Yunlin 63208, Taiwan (China); Yu, Hsin Her, E-mail: hhyu@nfu.edu.tw [Department of Biotechnology, National Formosa University, 64 Wenhua Road, Huwei, Yunlin 63208, Taiwan (China)

    2014-09-15

    Graphical abstract: A flexible paper-based supercapacitor was assembled into a sandwich structure, which exhibits well-retained triangular-shaped curves. The cycle life stability of this device still retains about 96% of the initial capacitance after 2000 cycles at a scan rate of 400 mV/s. An as-fabricated paper-based supercapacitor could light a red LED well after charging at constant potential of 3 V. - Highlights: • A facile approach is proposed to fabricate paper-based supercapacitors. • Apple pectin is an excellent dispersant for MWCNTs. • Paper provides a strong binding and flexible characteristic for electrode. • A paper-based supercapacitor could light a red LED after charging. • This device shows excellent electrochemical performance and cycling stability. - Abstract: We propose a low cost and simple method to prepare a paper-based supercapacitor in this study. Multi-walled carbon nanotubes (MWCNTs) were dispersed with a pectin solution under an ultrasonic homogenizer. Carbon nanotube suspension was prepared using a centrifuge to eliminate impurities. The dispersed MWCNTs suspension was dropped and dried onto the shallow surface of commercial copy paper. A paper-based conductive paper was formed as the electrodes. The electrical conductivity and dispersed morphology of the paper-based conductive paper were examined by four probes, atomic force microscope (AFM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The solid-state electrolyte was prepared by casting a solution of phosphoric acid and polyvinyl alcohol onto a glass plate. The paper-based supercapacitor was constructed with one solid-state electrolyte inserted between two electrodes, which were assembled into a sandwich structure by hot press. The specific capacitance and cycle-life stability of the paper-based supercapacitor was investigated by cyclic voltammetry analysis.

  15. Preparation and characterization of lignin based macromonomer and its copolymers with butyl methacrylate.

    Science.gov (United States)

    Liu, Xiaohuan; Wang, Jifu; Yu, Juan; Zhang, Mingming; Wang, Chunpeng; Xu, Yuzhi; Chu, Fuxiang

    2013-09-01

    Copolymerization of butyl methacrylate (BMA) with biobutanol lignin (BBL) was achieved by free-radical polymerization (FRP) using a lignin-based macromonomer. The lignin-based macromonomer containing acrylic groups was prepared by reacting acryloyl chloride with biobutanol lignin using triethylamine (TEA) as absorb acid agentin. From the results of elemental analysis and GPC, the average degree of polymerization (DP) of BBL was estimated to be five. A detailed molecular characterization has been performed, including techniques such as (1)H NMR, (13)C NMR and UV-vis spectroscopies, which provided quantitative information about the composition of the copolymers. The changes in the solubility of lignin-g-poly(BMA) copolymers in ethyl ether were dependent on the length of poly(BMA) side chain. TGA analysis indicated that the lignin-containing poly(BMA) graft copolymers exhibited high thermal stability. The bulky aromatic group of lignin increased the glass-transition temperature of poly(BMA). In order to confirm the main structure of copolymer, (AC-g-BBL)-co-BMA copolymer was also synthesized by atom transfer radical polymerization (ATRP), and the results revealed that the copolymer prepared by ATRP had the same solution behavior as that prepared by FRP, and the lignin-based macromonomer showed no homopolymerizability due to the steric hindrance. In addition, the lignin-co-BMA copolymer had a surprisingly higher molecular weight than poly(BMA) under the same reaction condition, suggesting that a branched lignin based polymer could be formed.

  16. Preparation and research on poisoning resistant Zr-Co based hydrogen storage alloys

    Institute of Scientific and Technical Information of China (English)

    LI Hualing; WANG Shumao; JIANG Lijun; ZHANG Lidong; LIU Xiaopeng; LI Zhinian

    2008-01-01

    At present,all hydrogen storage alloys are poisoned by hydrogen mixed with CO,CO2,etc,which decreases the hydrogen storage property sharply.Zr-Co based hydrogen storage alloys with good poisoning resistance were prepared by alloying,fluorinating,and electroless plating.The experiment results show that the poisoning resistance of the Zr-Co based alloy was improved remarkably after the treatments.The poisoning resistance mechanism of the Zr-Co based hydrogen storage alloys was analyzed.

  17. Comparison of Preparation Methods of Copper Based PGMFree Diesel-Soot Oxidation Catalysts

    Directory of Open Access Journals (Sweden)

    R. Prasad

    2011-05-01

    Full Text Available CuO-CeO2 systems have been proposed as a promising catalyst for low temperature diesel-soot oxidation. CuO-CeO2 catalysts prepared by various methods were examined for air oxidation of the soot in a semi batch tubular flow reactor. The air oxidation of soot was carried out under tight contact with soot/catalyst ratio of 1/10. Air flow rate was 150 ml/min, soot-catalyst mixture was 110 mg, heating rate was 5 0C/min. Prepared catalysts were calcined at 500 0C and their stability was examined by further heating to 800 0C for 4 hours. It was found that the selectivity of all the catalysts was nearly 100% to CO2 production. It was observed that the activity and stability of the catalysts greatly influenced by the preparation methods. The strong interaction between CuO and CeO2 is closely related to the preparation route that plays a crucial role in the soot oxidation over the CuO-CeO2 catalysts. The ranking order of the preparation methods of the catalysts in the soot oxidation performance is as follows: sol-gel > urea nitrate combustion > Urea gelation method > thermal decomposition > co-precipitation. Copyright © 2011 BCREC UNDIP. All rights reserved.(Received: 27th June 2010, Revised: 7th August 2010; Accepted: 13rd October 2010[How to Cite: R. Prasad, V.R. Bella. (2011. Comparison of Preparation Methods of Copper Based PGMFree Diesel-Soot Oxidation Catalysts. Bulletin of Chemical Reaction Engineering and Catalysis, 6(1: 15-21. doi:10.9767/bcrec.6.1.822.15-21][How to Link / DOI: http://dx.doi.org/10.9767/bcrec.6.1.822.15-21 || or local: http://ejournal.undip.ac.id/index.php/bcrec/article/view/822 | View in 

  18. Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis.

    Science.gov (United States)

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2007-08-01

    Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

  19. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    at high frequencies from diverse donors, I showed plasmid or donor dependence of plasmid transfer to other species. Additionally, environmental factors like stress also impact the permissiveness of phylogenetic groups towards plasmids. The developed method and results increase our ability to predict......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways...... and the extent of bacterial phyla permissive towards plasmid receipt are largely unknown. Historically, methods exploring the underlying genetic and environmental factors of plasmid transfer have been heavily reliant on cultivation and expression of plasmid encoded phenotypes. This has provided an incomplete...

  20. 78 FR 29120 - Notice of Intent To Prepare an Environmental Impact Statement for the Main Operating Base 2 (MOB...

    Science.gov (United States)

    2013-05-17

    ... of Intent To Prepare an Environmental Impact Statement for the Main Operating Base 2 (MOB-2) for the... prepare an Environmental Impact Statement (EIS) for the Main Operating Base 2 (MOB-2) for the beddown of... the MOB-2 at existing Air National Guard (ANG) installations within the continental United States....

  1. Modeling sRNA-Regulated Plasmid Maintenance

    Science.gov (United States)

    Klumpp, Stefan

    2017-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  2. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  3. Preparation of PVdF-based electrospun membranes and their application as separators

    OpenAIRE

    Yanhuai Ding et al

    2008-01-01

    A one-step method preparing of poly(vinylidene fluoride)-based electrospun membranes (PEMs) containing TiO2 has been developed. The effect of TiO2 on the morphology, degree of crystallization and electrochemical behavior of PEMs was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC) and electrochemical measurements. The PEMs containing TiO2 show improved ionic conductivity and cycling performance compared with pure PEMs.

  4. Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production.

    Science.gov (United States)

    Mo, Solveig Sølverød; Slettemeås, Jannice Schau; Berg, Einar Sverre; Norström, Madelaine; Sunde, Marianne

    2016-01-01

    Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid

  5. Improvement of in vivo transfer of plasmid DNA in muscle : Comparison of electroporation versus ultrasound

    NARCIS (Netherlands)

    Kusumanto, Yoka H.; Mulder, Nanno H.; Dam, Wendy A.; Losen, Mario H.; Meijer, Coby; Hospers, Geke A. P.

    2007-01-01

    Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods-electroporation and ultrasound-that facilitate DNA uptake into cel

  6. Chitosan-based delivery systems for diclofenac delivery: preparation and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Dreve, Simina; Kacso, Irina; Bratu, Ioan; Indrea, Emil, E-mail: simina.dreve@itim-cj.r [National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath, 400293 Cluj-Napoca (Romania)

    2009-08-01

    The preparation and characterization of novel materials for drug delivery has rapidly gained importance in development of innovative medicine. The paper concerns the uses of chitosan as an excipient in oral formulations and as a drug delivery vehicle for burnt painful injuries. The use of chitosan (CTS) as base in polyelectrolyte complex systems, to prepare liquid release systems as hydrogels and solid release systems as sponges is presented. In this paper the preparation of CTS hydrogels and sponges carrying diclofenac (DCF), as anti-inflammatory drug is reported. The immobilization of DCF in CTS is done by mixing the CTS hydrogel with the anti-inflammatory drug solutions. The concentration of anti-inflammatory drug in the CTS hydrogel generating the sponges was of 57 mg/l, 72 mg/l and 114 mg/l. The CTS sponges with anti-inflammatory drugs were prepared by freeze-drying at -610{sup 0}C and 0,09 atm. The characterization of the hydrogels and sponges was done by infrared spectra (FTIR) and ultraviolet-visible spectroscopy (UV-VIS). The results indicated the formation of CTS-DCF intermediates. The DCF molecules are forming temporary chelates in CTS hydrogels and sponges and they are compatible with skin or some of biological fluids with satisfactory results.

  7. Nanosized cobalt-based catalyst prepared by supercritical phase condition for Fischer-Tropsch synthesis

    Institute of Scientific and Technical Information of China (English)

    Jingmiao Li; Jingchang Zhang; Runduo Zhang; Weiliang Cao

    2009-01-01

    A series of nanosized Co/Zn/Mn/K composite catalysts for Fischer-Tropsch synthesis (FTS) were prepared by supercritical fluid drying (SCFD) method and common drying (CD) method. The nanosized cobalt-based catalysts were characterized by XRD,TEM and BET techniques. Their catalytic performances were tested in a slurry-bed reactor under FTS reaction conditions. The drying and crystallization were carded out simultaneously during SCFD,therefore,the catalysts prepared by SCFD method have ideal structure and show the FTS performance superior to the others prepared by CD method. The FTS activity and selectivity were improved via adding Zn,Mn and K promoters,and less CH_4 and CO_2 as well as higher yield of C~(5+) products were achieved. The optimal performance of a 92% CO conversion and a 65% C~(5+) product yield was obtained over a catalyst with the component of Co/Zn/Mn/K = 100/50/10/7. Furthermore,the catalytic performance was studied under the conditions of liquid-phase and supercriticai phase slurry-bed,and C~(5+) product yield were 57.4% and 65.4%,respectively. In summary,better catalytic performance was obtained using the nanosized catalyst prepared by SCFD method under supercritical reaction conditions,resulting in higher conversion of CO,less CO_2 byproduct,and higher yield of C~(5+) products,

  8. Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

    Science.gov (United States)

    Wooddell, Christine I; Subbotin, Vladimir M; Sebestyén, Magdolna G; Griffin, Jacob B; Zhang, Guofeng; Schleef, Martin; Braun, Serge; Huss, Thierry; Wolff, Jon A

    2011-02-01

    Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

  9. A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.

    Science.gov (United States)

    Short, Francesca L; Monson, Rita E; Salmond, George P C

    2015-01-01

    Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.

  10. Overview of nongynecological samples prepared with liquid-based cytology medium.

    Science.gov (United States)

    Ren, Shuyue; Solomides, Charalambos; Draganova-Tacheva, Rossitza; Bibbo, Marluce

    2014-01-01

    Liquid-based cytology of nongynecological specimens is commonly used in cytology laboratories throughout the world and various processing methods, such as ThinPrep and SurePath, have been reported. The cytological features and performance of liquid-based cytology for various cytology specimens, including body cavity fluids, urine, brushing specimens and fine-needle aspiration of various lesions, were reviewed and compared with the experience of our laboratory and the literature published in PubMed. The parameters for the evaluation of liquid-based cytology and conventional smears were described in the various types of specimens. Criteria for the interpretation of nongynecological liquid-based cytology were highlighted to show differences in cell morphology, background and artifacts. The interpretation requires familiarity with the appearance of liquid-based cytology in the various types of preparations to avoid misdiagnosis. Cell blocks can be prepared with specimens preserved in a liquid-based cytology medium and immunocytochemical stains and molecular testing can be successfully performed. These are important adjuncts in order to reach a definitive diagnosis.

  11. Preparing students to practice evidence-based dentistry: a mixed methods conceptual framework for curriculum enhancement.

    Science.gov (United States)

    Palcanis, Kent G; Geiger, Brian F; O'Neal, Marcia R; Ivankova, Nataliya V; Evans, Retta R; Kennedy, Lasonja B; Carera, Karen W

    2012-12-01

    This article describes a mixed methods conceptual framework for evidence-based dentistry to enhance the curriculum at the University of Alabama at Birmingham School of Dentistry. A focus of recent curriculum reform has been to prepare students to integrate evidence-based dentistry into clinical practice. The authors developed a framework consisting of four conceptual phases to introduce curriculum innovation: 1) exploration of the phenomenon; 2) development of two new instruments; 3) data collection, analysis, outcomes, and evaluation; and 4) application to curricular reform. Eight sequential procedural steps (literature review; focus group discussions; development of themes; survey design; internal review; data collection, analysis, and evaluation; development of recommendations with external review; and implementation of recommendations for curricular enhancement) guided the curricular enhancement. Faculty members supported the concept of teaching evidence-based dentistry to facilitate major curriculum reform, and course directors incorporated evidence-based teaching to prepare scientist-practitioners who meet dental performance standards. The new curriculum implemented following completion of the study is in its third year. Much of its structure is based on evidence-based teaching methodologies, and approximately one-third of the content consists of small groups researching clinical problems with applied science and discussing the findings. The framework described in this article proved useful to guide revision of predoctoral clinical education at one dental school and may be useful in other settings.

  12. Plasmid selection in Escherichia coli using an endogenous essential gene marker

    Directory of Open Access Journals (Sweden)

    Good Liam

    2008-08-01

    Full Text Available Abstract Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.

  13. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    Directory of Open Access Journals (Sweden)

    Kazuhiro Iiyama

    2015-06-01

    Full Text Available A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

  14. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  15. A corrosion resistant cerium oxide based coating on aluminum alloy 2024 prepared by brush plating

    Energy Technology Data Exchange (ETDEWEB)

    Tang Junlei; Han Zhongzhi [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Zuo Yu, E-mail: zuoy@mail.buct.edu.cn [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Tang Yuming [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China)

    2011-01-15

    Cerium oxide based coatings were prepared on AA2024 Al alloy by brush plating. The characteristic of this technology is that hydrogen peroxide, which usually causes the plating solution to be unstable, is not necessary in the plating electrolyte. The coating showed laminated structures and good adhesive strength with the substrate. X-ray diffraction and X-ray photoelectron spectroscopy analysis showed that the coatings were composed of Ce(III) and Ce(IV) oxides. The brush plated coatings on Al alloys improved corrosion resistance. The influence of plating parameters on structure and corrosion resistance of the cerium oxide based coating was studied.

  16. CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

    Directory of Open Access Journals (Sweden)

    F BENSALAH

    2003-06-01

    Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

  17. IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes.

    Science.gov (United States)

    Heuer, Holger; Binh, Chu T T; Jechalke, Sven; Kopmann, Christoph; Zimmerling, Ute; Krögerrecklenfort, Ellen; Ledger, Thomas; González, Bernardo; Top, Eva; Smalla, Kornelia

    2012-01-01

    The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  18. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  19. Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A.

    Directory of Open Access Journals (Sweden)

    Päivi Lahti

    Full Text Available Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

  20. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit

    1997-01-01

    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

  1. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

    OpenAIRE

    Hill, K E; A. J. Weightman; Fry, J C

    1992-01-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobil...

  2. Preparation of GaN-based cross-sectional TEM specimens by laser lift-off.

    Science.gov (United States)

    Zilan, Li; Xiaodong, Hu; Ke, Chen; Ruijuan, Nie; Xuhui, Luo; Xiaoping, Zhang; Tongjun, Yu; Bei, Zhang; Song, Chen; Zhijian, Yang; Zhizhong, Chen; Guoyi, Zhang

    2005-01-01

    Laser lift-off (LLO) technology is successfully used to prepare GaN-based TEM cross-sectional specimens. Detailed procedures of the method to prepare the specimens are demonstrated. Large thin areas suitable for TEM analysis were obtained. TEM images of the resulting GaN interface are studied, and the changes in structural quality are confined to approximately the first 250 nm of the epilayer. Clear TEM images of the whole epilayer and the InGaN quantum wells and the HRTEM images of the superlattice layer are demonstrated, showing that LLO is a quick and ideal method to study the crystal structure of the epilayer, especially if only the upper layers are of interest.

  3. Preparation of Graphene Oxide-Based Hydrogels as Efficient Dye Adsorbents for Wastewater Treatment

    Science.gov (United States)

    Guo, Haiying; Jiao, Tifeng; Zhang, Qingrui; Guo, Wenfeng; Peng, Qiuming; Yan, Xuehai

    2015-06-01

    Graphene oxide (GO) sheets exhibit superior adsorption capacity for removing organic dye pollutants from an aqueous environment. In this paper, the facile preparation of GO/polyethylenimine (PEI) hydrogels as efficient dye adsorbents has been reported. The GO/PEI hydrogels were achieved through both hydrogen bonding and electrostatic interactions between amine-rich PEI and GO sheets. For both methylene blue (MB) and rhodamine B (RhB), the as-prepared hydrogels exhibit removal rates within about 4 h in accordance with the pseudo-second-order model. The dye adsorption capacity of the hydrogel is mainly attributed to the GO sheets, whereas the PEI was incorporated to facilitate the gelation process of GO sheets. More importantly, the dye-adsorbed hydrogels can be conveniently separated from an aqueous environment, suggesting potential large-scale applications of the GO-based hydrogels for organic dye removal and wastewater treatment.

  4. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.

    Science.gov (United States)

    Urich, Mark A; Nery, Joseph R; Lister, Ryan; Schmitz, Robert J; Ecker, Joseph R

    2015-03-01

    Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein-DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.

  5. Preparation and characterization of plasticized high molecular weight PVC-based polymer electrolytes

    Indian Academy of Sciences (India)

    S Ramesh; Geok Bee Teh; Rong-Fuh Louh; Yong Kong Hou; Pung Yen Sin; Lim Jing Yi

    2010-02-01

    Poly(vinyl chloride) (PVC)-based polymer electrolytes films consisting of lithium trifluromethanesulfonate (LiCF3SO3)-ethylene carbonate (EC) were prepared by the solution-casting method. Ionic conductivities of the electrolytes have been determined by an impedance studies in the temperature range of 298–373 K. Complexation of the prepared electrolytes is studied by X-ray diffraction (XRD) analysis. Thermogravimetric analysis (TGA) was used to confirm the thermal stability of the polymer electrolytes. The conductivity–temperature plots were found to follow an Arrhenius nature. All these films are found to be thermally stable until 132–167°C.

  6. Zinc-oxide-based sorbents and processes for preparing and using same

    Science.gov (United States)

    Gangwal, Santosh Kumar; Turk, Brian Scott; Gupta, Raghubir Prasael

    2010-03-23

    Zinc oxide-based sorbents, and processes for preparing and using them are provided. The sorbents are preferably used to remove one or more reduced sulfur species from gas streams. The sorbents comprise an active zinc component, optionally in combination with one or more promoter components and/or one or more substantially inert components. The active zinc component is a two phase material, consisting essentially of a zinc oxide (ZnO) phase and a zinc aluminate (ZnAl.sub.2O.sub.4) phase. Each of the two phases is characterized by a relatively small crystallite size of typically less than about 500 Angstroms. Preferably the sorbents are prepared by converting a precursor mixture, comprising a precipitated zinc oxide precursor and a precipitated aluminum oxide precursor, to the two-phase, active zinc oxide containing component.

  7. Progress in the preparation and application of three-dimensional graphene-based porous nanocomposites

    Science.gov (United States)

    Yan, Zhengquan; Yao, Wenli; Hu, Lei; Liu, Dandan; Wang, Chundong; Lee, Chun-Sing

    2015-03-01

    Due to high specific surface area, excellent conductivity, low mass density, good compatibility and elegant flexibility, three-dimensional graphene composites with interconnected porous structures possess unusual and novel physical and electronic properties, unsurpassed chemical functionalities and other attractive features. Therefore, different three-dimensional graphene-based nanoporous scaffolds have been extensively designed, prepared and investigated for practical applications in lithium-ion batteries, super-capacitors, solar cells, catalysis, thermal management, environment pollution enrichment and separation, and chemical sensors with high performance from both fundamental and technological viewpoints. To present readers with a better understanding of this kind of important porous material, in this feature article, we will highlight the main achievements made in the preparation of 3D graphene micro- and/or nano-architectures and their potential applications in the aforementioned fields.

  8. Preparation and characterization of novel super-artificial hair fiber based on biomass materials.

    Science.gov (United States)

    Yang, Lijun; Guo, Jing; Zhang, Sen; Gong, Yumei

    2017-06-01

    A novel super-artificial hair fiber basing on sodium alginate (SA) and Antarctic Krill protein (AKP) was prepared by wet spinning successfully. Such SA/AKP fiber did not only have similar crystalline structure with human hair, but also had super flame resistance and mechanical performance. It should be noted that the whole preparation process was green without any incorporation of non-toxic solution. Moreover, comparing with human hair, the SA/AKP fiber had a lot of unique groove upon the fiber surface, which contributed a lot to excellent hygroscopicity. Meanwhile, the dyeing performance could be improved notably due to incorporation of protein into the matrix. Herein, the SA/AKP fiber with superior mechanical and functional performance had practical value for application in the field of synthetic wig. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Polylactide-based microspheres prepared using solid-state copolymerized chitosan and d,l-lactide.

    Science.gov (United States)

    Demina, T S; Akopova, T A; Vladimirov, L V; Zelenetskii, A N; Markvicheva, E A; Grandfils, Ch

    2016-02-01

    Amphiphilic chitosan-g-poly(d,l-lactide) copolymers have been manufactured via solid-state mechanochemical copolymerization and tailored to design polyester-based microspheres for tissue engineering. A single-step solid-state reactive blending (SSRB) using low-temperature co-extrusion has been used to prepare these copolymers. These materials have been valorized to stabilize microspheres processed by an oil/water emulsion evaporation technique. Introduction of the copolymers either in water or in the oil phase of the emulsion allowed to replace a non-degradable emulsifier typically used for microparticle preparation. To enhance cell adhesion, these copolymers were also tailored to bring amino-saccharide positively charged segments to the microbead surface. Size distribution, surface morphology, and total microparticle yield have been studied and optimized as a function of the copolymer composition. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Preparations, properties and applications of chitosan based nanofibers fabricated by electrospinning

    Directory of Open Access Journals (Sweden)

    2011-04-01

    Full Text Available Chitosan is soluble in most acids. The protonation of the amino groups on the chitosan backbone inhibits the electrospinnability of pure chitosan. Recently, electrospinning of nanofibers based on chitosan has been widely researched and numerous nanofibers containing chitosan have been prepared by decreasing the number of the free amino groups of chitosan as the nanofibiers have enormous possibilities for better utilization in various areas. This article reviews the preparations and properties of the nanofibers which were electrospun from pure chitosan, blends of chitosan and synthetic polymers, blends of chitosan and protein, chitosan derivatives, as well as blends of chitosan and inorganic nanoparticles, respectively. The applications of the nanofibers containing chitosan such as enzyme immobilization, filtration, wound dressing, tissue engineering, drug delivery and catalysis are also summarized in detail.

  11. Optimization of Preparation Program for Biomass Based Porous Active Carbon by Response Surface Methodology Based on Adsorptive Property

    Directory of Open Access Journals (Sweden)

    ZHANG Hao

    2017-06-01

    Full Text Available With waste walnut shell as raw material, biomass based porous active carbon was made by microwave oven method. The effects of microwave power, activation time and mass fraction of phosphoric acid on adsorptive property of biomass based porous active carbon in the process of physical activation of active carbon precursor were studied by response surface method and numerical simulation method, the preparation plan of biomass based porous active carbon was optimized, and the optimal biomass based porous active carbon property was characterized. The results show that three factors affect the adsorptive property of biomass based porous active carbon, but the effect of microwave power is obviously more significant than that of mass fraction of phosphoric acid, and the effect of mass fraction of phosphoric acid is more significant than that of activation time. The optimized preparation conditions are:microwave power is 746W, activation time is 11.2min and mass fraction of phosphoric acid is 85.9% in the process of physical activation of activated carbon precursor by microwave heating method. For the optimal biomass based porous active carbon, the adsorption value of iodine is 1074.57mg/g, adsorption value of methylene blue is 294.4mL/g and gain rate is 52.1%.

  12. Education of the impellent-plastic expressiveness at a stage of specialized base preparation in art gymnastics.

    Directory of Open Access Journals (Sweden)

    Kravchuk T.N.

    2010-12-01

    Full Text Available In the given article is proved and opened the technique of education of the impellent-plastic expressiveness at a stage of specialized base preparation in art gymnastics. Criteria and modern requirements to the impellent-plastic expressiveness of gymnasts are analyzed. Its basic components are defined: musical-rhythmic preparation; mastering by skills of expressive movement; choreographic preparation; studying of elements of national and ball dances.

  13. Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.

    Directory of Open Access Journals (Sweden)

    Xabier Osteikoetxea

    Full Text Available In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody and ganglioside GM1 (cholera toxin subunit B. We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition, may prove useful for quality control of extracellular vesicle related basic and clinical studies.

  14. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor

    Directory of Open Access Journals (Sweden)

    Charles Michael Drain

    2011-10-01

    Full Text Available Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  15. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  16. Superiority of needle-free transdermal plasmid delivery for the induction of antigen-specific IFNgamma T cell responses in the dog.

    Science.gov (United States)

    Goubier, A; Fuhrmann, L; Forest, L; Cachet, N; Evrad-Blanchard, M; Juillard, V; Fischer, L

    2008-04-24

    Although successful needle-free DNA vaccination has been described on several occasions, the true benefit of this delivery technology over needle-based injections for DNA vaccination of dogs has not yet been documented. We conducted a side-by-side comparison of needle-free transdermal plasmid delivery vs. intramuscular vs. intradermal needle-based delivery of the same plasmid in dogs. Our data confirmed the importance of the route of plasmid delivery and further established the unique potential of needle-free transdermal plasmid delivery to elicit strong antigen-specific, hTyr-specific IFNgamma T in the dog. Further, this study demonstrated that properly enabled DNA vaccination has the potential to trigger very significant cell-based immune responses in dogs, establishing needle-free transdermal plasmid delivery as a critical technology for successful immunotherapy of cancer and/or chronic infectious diseases in companion animal medicine.

  17. Evidence-based public health education as preparation for medical school.

    Science.gov (United States)

    Riegelman, Richard K; Garr, David R

    2008-04-01

    The Institute of Medicine has recommended that all undergraduates have access to public health education. An evidence-based public health framework including curricula such as "Public Health 101" and "Epidemiology 101" was recommended for all colleges and universities by arts and sciences, public health, and clinical health professions educators as part of the Consensus Conference on Undergraduate Public Health Education. These courses should foster critical thinking whereby students learn to broadly frame options, critically analyze data, and understand the uncertainties that remain. College-level competencies or learning outcomes in research literature reading, determinants of health, basic understanding of health care systems, and the synergies between health care and public health can provide preparation for medical education. Formally tested competencies could substitute for a growing list of prerequisite courses. Grounded in principles similar to those of evidence-based medicine, evidence-based public health includes problem description, causation, evidence-based recommendations for intervention, and implementation considering key issues of when, who, and how to intervene. Curriculum frameworks for structuring "Public Health 101" and "Epidemiology 101" are provided by the Consensus Conference that lay the foundation for teaching evidence-based public health as well as evidence-based medicine. Medical school preparation based on this foundation should enable the Clinical Prevention and Population Health Curriculum Framework, including the evidence base for practice and health systems and health policy, to be fully integrated into the four years of medical school. A faculty development program, curriculum guide, interest group, and clear student interest are facilitating rapid acceptance of the need for these curricula.

  18. Preparation of ultrafine Ce-based oxide nanoparticles and their catalytic performances for diesel soot combustion

    Institute of Scientific and Technical Information of China (English)

    韦岳长; 赵震; 焦金庆; 刘坚; 段爱军; 姜桂元

    2014-01-01

    The ultrafine Ce-based oxide nanoparticles with different element dopings (Zr, Y) were synthesized by the method of mi-cropores-diffused coprecipitation (MDC) using ammonia solution as the precipitation agent. The activities of the catalysts for soot oxidation were evaluated by the temperature-programmed oxidation (TPO) reaction. Ce-based oxides prepared in this study exhibited high catalytic activity for soot oxidation under the condition of loose contact between soot particles and catalysts, and the catalytic ac-tivity of ultrafine Ce0.9Zr0.1O2 nanoparticle for soot combustion was the highest, whose T10, T50 and SCO2m was 364, 442 ºC and 98.3%, respectively. All catalysts were systematically characterized by means of X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brumauer-Emett-Teller (BET), Fourier transform infrared spectroscopy (FT-IR) and UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS). It was indicated that the MDC method could prepare the ultrafine Ce-based oxide nanoparticles whose the crystal lattice were perfect, and the BET surface area and average crystal size of the ultrafine nanoparticles changed with the different element dopings (Zr, Y). The H2-TPR measurements showed that the ultrafine Ce-based ox-ide nanoparticles with the doping-Zr cation could be favorable for improving the redox property of the catalysts.

  19. Evaluation of sustained release suppositories prepared with fatty base including solid fats with high melting points.

    Science.gov (United States)

    Takatori, Toshihito; Shimono, Norihito; Higaki, Kazutaka; Kimura, Toshikiro

    2004-07-08

    To prepare the sustained release suppositories, solid fats such as polyglycerol ester of fatty acids (PGEFs) or beeswax were utilized with a fatty suppository base, Witepsol H15. PGEFs such as decaglycerol heptabehenate (HB750) and hexaglycerol pentastearate (PS500), and beeswax have relatively high melting points. The addition of PGEFs or beeswax to Witepsol H15 increased the apparent viscosity of suppository bases at 37 degrees C without any large change in the melting point of Witepsol H15. Moreover, the apparent viscosity of a mixed base with HB750, PS500 or beeswax at 37 degrees C was significantly correlated with the amount of each solid fat in a mixed base. The release of acetaminophen (AAP), a model drug, from suppositories was delayed by HB750, PS500 or beeswax, and an excellent correlation was observed between the apparent viscosity of these mixed bases and Higuchi's rate constants in each mixed base suppository, suggesting that these solid fats could regulate the drug release from the mixed base suppositories by changing their viscosity. In the in vivo absorption study in rats, several suppositories made from Witepsol H15-HB750 or Witepsol H15-beeswax mixed bases prolonged the rectal absorption of AAP without reducing AUC. In conclusion, by using solid fats such as HB750 and beeswax with relatively high melting points, it is possible to control the rate of drug release from fatty base suppositories for maintaining the plasma concentration of drugs for longer time periods.

  20. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids.

    Science.gov (United States)

    Ranjbar, R; Owlia, P; Saderi, H; Bameri, Z; Izadi, M; Jonaidi, N; Morovvati, S

    2007-09-01

    Aim of this study was to investigate the presence of plasmids among the strains of P. aeruginosa isolated from clinically diagnosed cases in Tehran in 2006. A total of 38 strains of P. aeruginosa were isolated. With the exception of one isolate, all P. aeruginosa strains harbored at least one plasmid band. The electrophoretic analysis of plasmid DNAs showed different number of plasmid bands among the strains tested. The DNA band of 1.4 kbp was evident in 84.2% of the strains. Approximately 71 and 21% of the isolates harbored concomitantly two and three plasmids, respectively. Isolation of strains with diverse types of plasmids suggests the different cluster of P. aeruginosa might be disseminated during the current study period.

  1. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

    Notani, N.K.; Setlow, J.K.; McCarthy, D.; Clayton, N.L.

    1981-12-01

    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  2. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    Science.gov (United States)

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains. Images PMID:8265367

  3. Process to prepare stable trifluorostyrene containing compounds grafted to base polymers using a solvent/water mixture

    Science.gov (United States)

    Roelofs, Mark Gerrit; Yang, Zhen-Yu; Han, Amy Qi

    2010-06-15

    A fluorinated ion exchange polymer is prepared by grafting at least one grafting monomer derived from trifluorostyrene on to at least one base polymer in a organic solvent/water mixture. These ion exchange polymers are useful in preparing catalyst coated membranes and membrane electrode assemblies used in fuel cells.

  4. Building Energy Storage Panel Based on Paraffin/Expanded Perlite: Preparation and Thermal Performance Study

    Directory of Open Access Journals (Sweden)

    Xiangfei Kong

    2016-01-01

    Full Text Available This study is focused on the preparation and performance of a building energy storage panel (BESP. The BESP was fabricated through a mold pressing method based on phase change material particle (PCMP, which was prepared in two steps: vacuum absorption and surface film coating. Firstly, phase change material (PCM was incorporated into expanded perlite (EP through a vacuum absorption method to obtain composite PCM; secondly, the composite PCM was immersed into the mixture of colloidal silica and organic acrylate, and then it was taken out and dried naturally. A series of experiments, including differential scanning calorimeter (DSC, scanning electron microscope (SEM, best matching test, and durability test, have been conducted to characterize and analyze the thermophysical property and reliability of PCMP. Additionally, the thermal performance of BESP was studied through a dynamic thermal property test. The results have showed that: (1 the surface film coating procedure can effectively solve the leakage problem of composite phase change material prepared by vacuum impregnation; (2 the optimum adsorption ratio for paraffin and EP was 52.5:47.5 in mass fraction, and the PCMP has good thermal properties, stability, and durability; and (3 in the process of dynamic thermal performance test, BESP have low temperature variation, significant temperature lagging, and large heat storage ability, which indicated the potential of BESP in the application of building energy efficiency.

  5. Preparation, Properties, and Self-Assembly Behavior of PTFE-Based Core-Shell Nanospheres

    Directory of Open Access Journals (Sweden)

    Katia Sparnacci

    2012-01-01

    Full Text Available Nanosized PTFE-based core-shell particles can be prepared by emulsifier-free seed emulsion polymerization technique starting from spherical or rod-like PTFE seeds of different size. The shell can be constituted by the relatively high Tg polystyrene and polymethylmethacrylate as well as by low Tg polyacrylic copolymers. Peculiar thermal behavior of the PTFE component is observed due to the high degree of PTFE compartmentalization. A very precise control over the particle size can be exerted by properly adjusting the ratio between the monomers and the PTFE seed. In addition, the particle size distribution self-sharpens as the ratio monomer/PTFE increases. Samples with uniformity ratios suited to build 2D and 3D colloidal crystals are easily prepared. In particular, 2D colloidal crystal of spheres leads to very small 2D nanostructuration, useful for the preparation of masks with a combination of nanosphere lithography and reactive ion etching. 3D colloidal crystals were also obtained featuring excellent opal quality, which is a direct consequence of the monodispersity of colloids used for their growth.

  6. Building Energy Storage Panel Based on Paraffin/Expanded Perlite: Preparation and Thermal Performance Study.

    Science.gov (United States)

    Kong, Xiangfei; Zhong, Yuliang; Rong, Xian; Min, Chunhua; Qi, Chengying

    2016-01-25

    This study is focused on the preparation and performance of a building energy storage panel (BESP). The BESP was fabricated through a mold pressing method based on phase change material particle (PCMP), which was prepared in two steps: vacuum absorption and surface film coating. Firstly, phase change material (PCM) was incorporated into expanded perlite (EP) through a vacuum absorption method to obtain composite PCM; secondly, the composite PCM was immersed into the mixture of colloidal silica and organic acrylate, and then it was taken out and dried naturally. A series of experiments, including differential scanning calorimeter (DSC), scanning electron microscope (SEM), best matching test, and durability test, have been conducted to characterize and analyze the thermophysical property and reliability of PCMP. Additionally, the thermal performance of BESP was studied through a dynamic thermal property test. The results have showed that: (1) the surface film coating procedure can effectively solve the leakage problem of composite phase change material prepared by vacuum impregnation; (2) the optimum adsorption ratio for paraffin and EP was 52.5:47.5 in mass fraction, and the PCMP has good thermal properties, stability, and durability; and (3) in the process of dynamic thermal performance test, BESP have low temperature variation, significant temperature lagging, and large heat storage ability, which indicated the potential of BESP in the application of building energy efficiency.

  7. Characterization of AZO and Ag based films prepared by RF magnetron sputtering

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Dagang [Institute of Textiles and Clothing, The Hong Kong Polytechnic University, Hong Kong (China); Jiang, Shouxiang, E-mail: kinor.j@polyu.edu.hk [Institute of Textiles and Clothing, The Hong Kong Polytechnic University, Hong Kong (China); Zhao, Hongmei [Department of Prosthodontics, The Affiliated Hospital of Qingdao University, Qingdao (China); Shang, Songmin; Chen, Zhuoming [Institute of Textiles and Clothing, The Hong Kong Polytechnic University, Hong Kong (China)

    2014-12-15

    Highlights: • Highly infrared reflective AZO and Ag based films were prepared. • Ag showed better crystallization on AZO film than on glass substrate. • Infrared reflection rate was inversely proportional to the film sheet resistance. • Film with infrared reflection of 97% in FIR region was acquired. - Abstract: Ag, AZO/Ag, Ag/AZO and AZO/Ag/AZO films were prepared on glass substrates by radio frequency (RF) magnetron sputtering technology. The prepared films were systematically investigated by X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), UV–visible spectrophotometer, a four-point probe system and Fourier Transform Infrared Spectroscopy. The results indicated that Ag inner layer starts forming a continuous film at the thickness of 10 nm and Ag layer presents superior crystallization on AZO substrate than that on glass substrate. The continuous Ag inner layer film provided the highest average visible transmittance of 85.4% (AZO/Ag/AZO). The lowest sheet resistance of 3.21 Ω/sq and the highest infrared reflection rate of 97% in FIR region can be obtained on AZO/Ag (15 nm)/AZO film. The high infrared reflection property of the AZO/Ag/AZO coating makes it a promising candidate for solar control films.

  8. Modern Focused-Ion-Beam-Based Site-Specific Specimen Preparation for Atom Probe Tomography.

    Science.gov (United States)

    Prosa, Ty J; Larson, David J

    2017-02-06

    Approximately 30 years after the first use of focused ion beam (FIB) instruments to prepare atom probe tomography specimens, this technique has grown to be used by hundreds of researchers around the world. This past decade has seen tremendous advances in atom probe applications, enabled by the continued development of FIB-based specimen preparation methodologies. In this work, we provide a short review of the origin of the FIB method and the standard methods used today for lift-out and sharpening, using the annular milling method as applied to atom probe tomography specimens. Key steps for enabling correlative analysis with transmission electron-beam backscatter diffraction, transmission electron microscopy, and atom probe tomography are presented, and strategies for preparing specimens for modern microelectronic device structures are reviewed and discussed in detail. Examples are used for discussion of the steps for each of these methods. We conclude with examples of the challenges presented by complex topologies such as nanowires, nanoparticles, and organic materials.

  9. Preparation and characterization of novel antibacterial castor oil-based polyurethane membranes for wound dressing application.

    Science.gov (United States)

    Yari, Abbas; Yeganeh, Hamid; Bakhshi, Hadi; Gharibi, Reza

    2014-01-01

    Preparation of novel antibacterial and cytocompatible polyurethane membranes as occlusive dressing, which can provide moist and sterile environment over mild exudative wounds is considered in this work. In this regard, an epoxy-terminated polyurethane (EPU) prepolymer based on castor oil and glycidyltriethylammonium chloride (GTEAC) as a reactive bactericidal agent were synthesized. Polyurethane membranes were prepared through cocuring of EPU and different content of GTEAC with 1,4-butane diamine. The physical and mechanical properties, as well as cytocompatibility and antibacterial performance of prepared membranes were studied. Depending on their chemical formulations, the equilibrium water absorption and water vapor transmission rate values of the membranes were in ranges of 3-85% and 53-154g m(-2) day(-1), respectively. Therefore, these transparent membranes can maintain for a long period the moist environment over the wounds with low exudates. Detailed cytotoxicity analysis of samples against mouse L929 fibroblast and MCA-3D keratinocyte cells showed good level of cytocompatibility of membranes after purification via extraction of residual unreacted GTEAC moieties. The antibacterial activity of the membranes against Escherichia coli and Staphylococcus aureus bacteria was also studied. The membrane containing 50% GTEAC exhibited an effective antibacterial activity, while showed acceptable cytocompatibility and therefore, can be applied as an antibacterial occlusive wound dressing.

  10. Preparation of liquefied wood-based resins and their application in molding material

    Institute of Scientific and Technical Information of China (English)

    Zhang Qiu-hui; Zhao Guang-jie; Yu Li-li; Jie Shu-jun

    2007-01-01

    To investigate value in use of liquefied wood-based resin applications in molding material, Chinese fir (Cunninghamia lanceolata) and poplar (Populus tomentosa) wood meal were liquefied in phenol. The reactant was co-condensed with formaldehyde to obtain liquefied wood-based resin. For this paper, we investigated the characterization of the resin and its application in molding material. The result shows that the basic properties of liquefied wood-based resin were satisfactory; the bonding strength of plywood prepared with liquefied Chinese fir and liquefied poplar resin can reach 1.54 and 1.00 MPa, respectively. The compression strengths of the molding material prepared with two kinds of liquefied wood resin were 73.01 and 73.58 MPa, almost the same as that of PF resin molding material. The limiting volume swelling of molding material made with liquefied Chinese resin and liquefied poplar resin were 8.5% and 8.3%, thickness swelling rates of water absorption were 3.3% and 4.2%, and the maximum weight ratios of water absorption were 25.9% and 26.2%, respectively. The soil burial test result shows that the weight loss rate of the molding materials made with liquefied Chinese resin and liquefied poplar resin were 8.3% and 9.1% and that of the PF resin molding material was 7.9%. After the soil internment test, the reduction ratio of compression strength of the two kinds of molding material achieved 16.9%and 17.7%, while that of the PF resin molding material was 15.4%. The test results of wood fungi inoculation on the three surfaces of the molding material indicate the breeding rate of molding material prepared with liquefied Chinese resin and liquefied poplar resin were at level 4 and that of PF resin molding material was at level 1 of the ISO standard.

  11. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

    Directory of Open Access Journals (Sweden)

    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  12. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  13. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

    DEFF Research Database (Denmark)

    Shuyu, Wu; Dalsgaard, A.; Hammerum, A. M.

    2010-01-01

    Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids...... involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli...... isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids...

  14. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    Science.gov (United States)

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

  15. Preparation and Catalytic Properties of Polymer-Bound Schiff Base Ternary Complexes

    Institute of Scientific and Technical Information of China (English)

    HAO Cheng-jun; WANG Rong-min; HE Yu-feng; WANG Yun-pu; XIA Chun-gu

    2004-01-01

    The polymer-bound Schiff base ternary manganese complexes [PS-SalPhe-Mn-L (L = Phen, Bipy and 8HQ)-] have been prepared from the polymer-bound Schiff base ligand, a manganese salt and the second ligand, such as 1,10-phenanthroline(phen), 2,2′-bipyridyl(bipy) and 8-quinolinol(8HQ). The polymer-bound Schiff base ternary manganese complexes were characterized by means of infrared spectrometry and ICPAES. The catalytic activities of the complexes have been studied in the aerobic epoxidation of long-chain linear a[iphatic olefins. It is shown that 1-octene or 1-decene can be directly oxidized by molecular oxygen when catalyzed by PS-SalPhe-Mn-L(L=Phen, Bipy and 8HQ), and 1,2-epoxy alkane can be afforded in these reactions.

  16. Preparation and Characterization of Er2O3 Nanocrystals by Schiff Base Salt Combustion Method

    Institute of Scientific and Technical Information of China (English)

    刘建华; 孙杰; 李松梅

    2003-01-01

    The nanocrystals Er2O3 were prepared by using a combustion method with Schiff base as a chelating agent. The Er(Ⅲ) coordanation compound of Schiff base, obtained from erbium nitrate and retinal Schiff base, underwent a combustion process and voluminous ashes formed when calcimining the complex in air. Pure cubic Er2O3 nanocrystals with a diameter of 13 nm were produced. The nanocrystals were homogeneous and rigid coacervation was not observed. The photoluminescence emission spectrum of the erbium(Ⅲ) oxide nanocrystals shows that it has a characteristic peak at 1.54 μm, and some other shoulder peaks appear on both sides of the main peak.

  17. Synthesis and characterization of nanocomposites based on PANI and carbon nanostructures prepared by electropolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Petrovski, Aleksandar; Paunović, Perica [Faculty of Technology and Metallurgy, SS Cyril and Methodius University, Rudjer Bošković, 16, 1000, Skopje (Macedonia, The Former Yugoslav Republic of); Avolio, Roberto; Errico, Maria E.; Cocca, Mariacristina; Gentile, Gennaro [Institute for Polymers, Composites and Biomaterials, National Research Council, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli (Italy); Grozdanov, Anita, E-mail: anita.grozdanov@yahoo.com [Faculty of Technology and Metallurgy, SS Cyril and Methodius University, Rudjer Bošković, 16, 1000, Skopje (Macedonia, The Former Yugoslav Republic of); Avella, Maurizio [Institute for Polymers, Composites and Biomaterials, National Research Council, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli (Italy); Barton, John [Tyndall National Institute, University College Cork, Dyke Parade, T12 R5CP, Cork (Ireland); Dimitrov, Aleksandar [Faculty of Technology and Metallurgy, SS Cyril and Methodius University, Rudjer Bošković, 16, 1000, Skopje (Macedonia, The Former Yugoslav Republic of)

    2017-01-01

    Nanocomposites based on polyaniline (PANI) and carbon nanostructures (CNSs) (graphene (G) and multiwall carbon nanotubes (MWCNTs)) were prepared by in situ electrochemical polymerization. CNSs were inserted into the PANI matrix by dispersing them into the electrolyte before the electropolymerization. Electrochemical characterization by means of cyclic voltammetry and steady state polarization were performed in order to determine conditions for electro-polymerization. Electro-polymerization of the PANI based nanocomposites was carried out at 0.75 V vs. saturated calomel electrode (SCE) for 40 and 60 min. The morphology and structural characteristics of the obtained nanocomposites were studied by scanning electron microscopy (SEM) and Raman spectroscopy, while thermal stability was determined using thermal gravimetric analysis (TGA). According to the morphological and structural study, fibrous and porous structure of PANI based nanocomposites was detected well embedding both G and MWCNTs. Also, strong interaction between quinoidal structure of PANI with carbon nanostructures via π–π stacking was detected by Raman spectroscopy. TGA showed the increased thermal stability of composites reinforced with CNSs, especially those reinforced with graphene. - Highlights: • Nanocomposites of PANI with carbon nanostructures were prepared for sensing application. • By cyclic voltammetry, conductive form of PANI (green colored emeraldine phase) is obtained 0.75 V • Using 4 Probe method, nanocomposite PANI/CNS tablet was tested for sensing application. • Micro-structural properties of nanocomposites were studied by SEM, TGA and Raman analysis.

  18. PREPARATION AND CHARACTERIZATION OF MODIFIED STARCH-BASED BIODEGRADABLE MATERIALS EINFORCED WITH PULP FIBER

    Institute of Scientific and Technical Information of China (English)

    Lingfang Ji; Shucai Li

    2004-01-01

    Native com starch and hydroxypropyl starch (HPS)based plastic films were prepared using the short pulp fiber as reinforcement and the glycerol as the plasticizer. The results of tensile test showed that the tensile strength and the elongation at break increased with the pulp contents. With the glycerol contents,the elongation at break increased considerably, but the tensile strength decreased. The water uptake of the films decreased with the pulp contents and hydroxypropylation, but increased with the glycerol contents. So it is concluded that the films was reinforced by pulp fiber and hydroxypropylation.

  19. A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer

    Institute of Scientific and Technical Information of China (English)

    Qing Quan Wei; Tian Xin Wei

    2011-01-01

    A novel method to prepare surface plasmon resonance (SPR) sensor chips based on grafted imprinted polymer is explored. Benzophenone photografting system is used to grow molecularly imprinted polymer (MIP) films from the modified surface of gold substrate. The surface morphology and thickness of MIP films were investigated by scanning electronic microscope (SEM). The adsorption properties of sensor chip were studied by SPR spectroscopy. The results demonstrate that nano-MIP films can be constructed on the surface of gold substrate with the good adsorption of template molecules.

  20. Fast preparation and thermal transport property of TiCoSb-based half-Heusler compounds

    Institute of Scientific and Technical Information of China (English)

    Xie Wen-Jie; Tang Xin-Feng; Zhang Qing-Jie

    2007-01-01

    TiCoSb-based half-Heusler compounds with the substitution of Zr for Ti have been prepared quickly by combining high-energy ball milling method with spark plasma sintering technique, and their thermal transport properties have been investigated. With the increase of the concentration of Zr, the thermal conductivity of Ti1-xZrxCoSb compounds decreases significantly. Compared with the thermal conductivity of TiCoSb compound, that of Ti0.5Zr0.5CoSb decreases by 200% at 1000 K.

  1. Thermo-optically driven adaptive mirror based on thermal expansion: preparation and resolution

    Science.gov (United States)

    Reinert, Felix; Lüthy, W.

    2005-12-01

    A thermo-optically driven adaptive mirror is presented. It is based on the thermal expansion of a thin film heated with a light pattern. We describe a procedure for the preparation of a silicon elastomer with a high-quality optical surface. This material, Sylgard 184, has a high linear thermal expansion coefficient of 3.1μ10-4 K-1. Surface modulations are recorded in an interferometer. Modulations of 350 nm result at an intensity of 370 mW/cm2. The resolution is measured with a line pattern. The contrast drops to 30 % at 1.6 line pairs per millimeter (lp/mm).

  2. PREPARATION AND CHARACTERIZATION OF MODIFIED STARCH-BASED BIODEGRADABLE MATERIALS REINFORCED WITH PULP FIBER

    Institute of Scientific and Technical Information of China (English)

    LingfangJi; ShucaiLi

    2004-01-01

    Native corn starch and hydroxypropyl starch (HPS) based plastic films were prepared using the short pulp fiber as reinforcement and the glycerol as the plasticizer. The results of tensile test showed that the tensile strength and the elongation at break increased with the pulp contents. With the glycerol contents, the elongation at break increased considerably, but the tensile strength decreased. The water uptake of the films decreased with the pulp contents and hydroxypropylation, but increased with the glycerol contents. So it is concluded that the films was reinforced by pulp fiber and hydroxypropylation.

  3. Preparation and Properties of Biocomposite Based on Natural Rubber and Bagasse Nanocellulose

    Directory of Open Access Journals (Sweden)

    Jarnthong Methakarn

    2015-01-01

    Full Text Available Biocomposite based on natural rubber (NR and bagasse nanocellulose (BNC was prepared in latex state. The mechanical, morphological and thermal properties of NR/BNC biocomposite were investigated. It was found that the addition of 3 wt% of BNC in NR film caused significant increase in modulus at 100% and 300% elongations and improved thermal stability of NR/BNC biocomposite. However, the strength at break and elongation at break of the biocomposite were not enhanced correlating to the morphological result obtained from scanning electron microscope (SEM.

  4. Python-based geometry preparation and simulation visualization toolkits for STEPS.

    Science.gov (United States)

    Chen, Weiliang; De Schutter, Erik

    2014-01-01

    STEPS is a stochastic reaction-diffusion simulation engine that implements a spatial extension of Gillespie's Stochastic Simulation Algorithm (SSA) in complex tetrahedral geometries. An extensive Python-based interface is provided to STEPS so that it can interact with the large number of scientific packages in Python. However, a gap existed between the interfaces of these packages and the STEPS user interface, where supporting toolkits could reduce the amount of scripting required for research projects. This paper introduces two new supporting toolkits that support geometry preparation and visualization for STEPS simulations.

  5. Python-Based Geometry Preparation and Simulation Visualization Toolkits for STEPS

    Directory of Open Access Journals (Sweden)

    Weiliang eChen

    2014-04-01

    Full Text Available STEPS is a stochastic reaction-diffusion simulation engine that implements a spatial extension of Gillespie’s Stochastic Simulation Algorithm (SSA in complex tetrahedral geometries. An extensive Python-based interface is provided to STEPS so that it can interact with the large number of scientific packages in Python. However, a gap existed between the interfaces of these packages and the STEPS user interface, where supporting toolkits could reduce the amount of scripting required for research projects. This paper introduces two new supporting toolkits that support geometry preparation and visualization for STEPS simulations.

  6. Preparation of Polyacrylate-based Conductive Coatings and Its PTC Effect

    Institute of Scientific and Technical Information of China (English)

    XIONG Chuan-xi; HU Hui-rong; ZHOU Zhi-yong; ZHANG Yi; DONG Li-jie

    2004-01-01

    Polyacrylate- based conductive coatings were prepared from polyacrylate emulsion as matrix andcarbon black (CB) whose surface was treated with titanate coupling agent as conducting particles. One kind oforganic crystal was added to study its effects on the electrical conductivity and PTC ( positive temperature coeffi-cient ) effect of the conductive coatings. Experimental results show that the coatings containing only polyacrylateemulsion and CB exhibit an excellent electrical conductivity but bad PTC effect, and when organic crystal is added,PTC effect is characterized and can increase by 2 orders of magtitude. The critical transformation temperature ofpolyacrylate emulsion/CB PTC composites is decided by melting point of organic crystals.

  7. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  8. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  9. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains.

    Science.gov (United States)

    Leverstein-van Hall, M A; Dierikx, C M; Cohen Stuart, J; Voets, G M; van den Munckhof, M P; van Essen-Zandbergen, A; Platteel, T; Fluit, A C; van de Sande-Bruinsma, N; Scharinga, J; Bonten, M J M; Mevius, D J

    2011-06-01

    Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

  10. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

    Science.gov (United States)

    Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

    2004-04-01

    Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

  11. Factors that affect transfer of the IncI1 β-lactam resistance plasmid pESBL-283 between E. coli strains.

    Directory of Open Access Journals (Sweden)

    Nadine Händel

    Full Text Available The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates

  12. Factors that affect transfer of the IncI1 β-lactam resistance plasmid pESBL-283 between E. coli strains.

    Science.gov (United States)

    Händel, Nadine; Otte, Sarah; Jonker, Martijs; Brul, Stanley; ter Kuile, Benno H

    2015-01-01

    The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these

  13. Preparation and Mechanism of a New Enhanced Flocculant Based on Bentonite for Drinking Water

    Directory of Open Access Journals (Sweden)

    Feng-shan Zhou

    2015-01-01

    Full Text Available Bentonite is characterized by the large specific surface, good adsorption, ion exchange ability, and nontoxicity. An enhanced bentonite base composite flocculant (BTA can be prepared from treating the calcium base bentonite and compositing various functional additives. Bentonite was firstly treated by citric acid, then the talc and activated carbon turned to be acid part and simultaneously the part that was treated by sodium bicarbonate and calcium hydroxide turned to be alkaline part, and finally the acid bentonite part and alkaline bentonite part were mixed up with preground powder of polymeric chloride aluminium (PAC, cationic polyacrylamide (CPAM, ferrous sulfate, and aluminum sulfate, and after all of the processing flocculant BTA was obtained. The optimum preparation process of flocculant BTA has shown 29.5% acid bentonite part, 29.5% alkaline bentonite part, 15% PAC, 1% CPAM, 5% ferrous sulfate, and 20% aluminum sulfate. BTA was used to treat drinking water with high turbidity and metal ion in Karamay City, Xinjiang. The treated water was surely up to the drinking water standard of China in decolorization rate, deodorization rate, heavy metal ion removal rate, and so forth, and contents of residual aluminum ions and acrylamide monomer in drinking water were considerably decreased.

  14. Effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pembroke, J.T.; Stevens, E. (University Coll., Galway (Ireland))

    1984-07-01

    The presence of the IncJ plasmids R391, R997, R705, R706, R748, and R749 was shown to sensitize Escherichia coli AB1157 and both its uvr A and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA/sup +/ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

  15. Development and validation of a SYBR-Green I real-time PCR protocol to detect hazelnut (Corylus avellana L.) in foods through calibration via plasmid reference standard.

    Science.gov (United States)

    D'Andrea, Matteo; Coïsson, Jean Daniel; Travaglia, Fabiano; Garino, Cristiano; Arlorio, Marco

    2009-12-09

    Many tree nuts are considered to be a serious problem in food safety, because of the presence of causative factors in IgE-mediated food allergies. Among these, hazelnut (Corylus avellana L.) seeds are largely used in a range of confectionery products and contain many well-characterized allergens. DNA-based methods and ELISA tests may prove to be useful to assess the presence of hidden ingredients in foods. The aim of this work was the development and validation of a species-specific SYBR Green I real-time PCR protocol for the detection of hazelnut in foods. A novel efficient primer pair on the Cor a 8 genomic coding region was designed by preparing a plasmid vector-based internal reference standard to calibrate the PCR. A good sensitivity, down to 20 (genomic) and 15 (plasmid) DNA copies, was established. All of the commercial samples considered in our study (containing hazelnut as ingredient or as a potential trace cross-contamination) were effectively amplified by PCR, showing a perfect correspondence with an ELISA commercial test, employed as a reference standard method.

  16. Preparation and evaluation of a novel therapeutic dairy-based drink for phenylketonuria

    Directory of Open Access Journals (Sweden)

    Ahmed M. Abdel-Salam

    2010-02-01

    Full Text Available Background: People with phenylketonuria need to eat a special diet which contains a low level of phenylalanine. Most of these special diets have high protein levels which contain phenylalanine. Control of phenylalanine levels in the early years of life is crucial and remains important throughout childhood, especially for cognitive function and behavior. Aims: The current study evaluated the biological and sensory properties of a novel dairy-based drink for patients with phenylketonuria (PKU. Methods and Materials: The novel dairy-based drink was prepared by emulsifying corn germ oil with casein glycomacropeptide (GMP solution in milk permeates. The chemical composition and sensory properties of the dairy-based drink were determined. In addition, the dairy-based drink was nutritionally evaluated using patient volunteers. These patients followed a strict diet limiting phenylalanine in their food. Phenylalanine levels were measured before and after three days of consuming the dairy-based drink. Results: The results of the sensory evaluation showed that the product was ranked that there were decreases in “good” and was acceptable by all test panels and volunteers. Serum phenylalanine levels in all volunteers decreased between 30% - 80%. Conclusions: The data obtained from the sensory evaluation and the decreases in serum phenylalanine levels encourage us to utilize this formulated dairy-based drink for therapeutic feeding of PKU patients.

  17. Preparation and evaluation of a novel therapeutic dairy-based drink for phenylketonuria

    Directory of Open Access Journals (Sweden)

    Ahmed M Abdel-Salam

    2010-01-01

    Full Text Available Background: People with phenylketonuria need to eat a special diet which contains a low level of phenylalanine. Most of these special diets have high protein levels which contain phenylalanine. Control of phenylalanine levels in the early years of life is crucial and remains important throughout childhood, especially for cognitive function and behavior. Aims: The current study evaluated the biological and sensory properties of a novel dairy-based drink for patients with phenylketonuria (PKU. Methods and Materials: The novel dairy-based drink was prepared by emulsifying corn germ oil with casein glycomacropeptide (GMP solution in milk permeates. The chemical composition and sensory properties of the dairy-based drink were determined. In addition, the dairy-based drink was nutritionally evaluated using patient volunteers. These patients followed a strict diet limiting phenylalanine in their food. Phenylalanine levels were measured before and after three days of consuming the dairy-based drink. Results: The results of the sensory evaluation showed that the product was ranked that there were decreases in "good" and was acceptable by all test panels and volunteers. Serum phenylalanine levels in all volunteers decreased between 30% - 80%. Conclusions : The data obtained from the sensory evaluation and the decreases in serum phenylalanine levels encourage us to utilize this formulated dairy-based drink for therapeutic feeding of PKU patients.

  18. Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles.

    Science.gov (United States)

    Tse, Man Tsuey; Blatchford, Chris; Oya Alpar, H

    2009-03-31

    Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.1M NaHCO(3) and 0.07M Na(2)HPO(4) and the effect these have on the encapsulation efficiency and other critical parameters associated with these encapsulated DNA materials. Both buffers preserved the conformation of the original plasmid DNA during the homogenization process, but those made with 0.07M Na(2)HPO(4) had higher encapsulation efficiencies, as well as smaller diameters, compared with those made with 0.1M NaHCO(3) (encapsulation efficiencies of 40.72-45.65%, and mean volume diameters of 2.96-4.45microm). Buffers with a range of pH from 5 to 12 were investigated, and it was demonstrated that pH 9 was the point at which the highest amount of supercoiled DNA was balanced with the highest encapsulation efficiency. To simulate in vitro release, it was shown that microparticles made with 0.07M Na(2)HPO(4) had lower DNA release rates than those made with 0.1M NaHCO(3). These results demonstrate that the use of different buffers can aid in retaining the conformation of plasmid DNA, and can also modulate the amount of DNA encapsulated and the release profiles of microparticles.

  19. Microevolutionary events involving narrow host plasmids influences local fixation of vancomycin-resistance in Enterococcus populations.

    Directory of Open Access Journals (Sweden)

    Ana R Freitas

    Full Text Available Vancomycin-resistance in enterococci (VRE is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm and ST6 Enterococcus faecalis (Efs human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs from Portuguese hospitals and aquatic surroundings (1996-2008 was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1 and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30-150 kb with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1, and narrow host RepA_N plasmids (pRUM, pAD1-like. TAs of Inc18 (ω-ε-ζ and pRUM (Axe-Txe plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  20. Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations

    Science.gov (United States)

    Freitas, Ana R.; Novais, Carla; Tedim, Ana P.; Francia, María Victoria; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.

    2013-01-01

    Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  1. Preparation and characterization of manganese ferrite-based magnetic liposomes for hyperthermia treatment of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Pradhan, Pallab [School of Biosciences and Bioengineering, Mumbai (India); Giri, Jyotsnendu [Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology, Mumbai 400076 (India); Banerjee, Rinti [School of Biosciences and Bioengineering, Mumbai (India); Bellare, Jayesh [School of Biosciences and Bioengineering, Mumbai (India); Bahadur, Dhirendra [Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology, Mumbai 400076 (India)]. E-mail: dhirenb@iitb.ac.in

    2007-04-15

    Comparative evaluation of two different methods of magnetic liposomes preparation, namely thin film hydration (TFH) and double emulsion (DE) with different molar ratios of egg-phosphatidyl choline (egg-PC) and cholesterol using lauric acid coated manganese ferrite-based aqueous magnetic fluid, is reported. TFH was found to be a better method of encapsulation and TFH 2:1 (egg-PC: cholesterol) magnetic liposomes showed the highest encapsulation efficiency and comparable heating ability to that of magnetic fluids. Stealth TFH 2:1 magnetic liposomes containing DSPE-PEG{sub 2000} were three-fold more cytocompatible as compared to the magnetic fluid. Stealth TFH 2:1 manganese ferrite-based magnetic liposomes might be useful for hyperthermia treatment of cancer.

  2. Preparation and characterization of manganese ferrite-based magnetic liposomes for hyperthermia treatment of cancer

    Science.gov (United States)

    Pradhan, Pallab; Giri, Jyotsnendu; Banerjee, Rinti; Bellare, Jayesh; Bahadur, Dhirendra

    2007-04-01

    Comparative evaluation of two different methods of magnetic liposomes preparation, namely thin film hydration (TFH) and double emulsion (DE) with different molar ratios of egg-phosphatidyl choline (egg-PC) and cholesterol using lauric acid coated manganese ferrite-based aqueous magnetic fluid, is reported. TFH was found to be a better method of encapsulation and TFH 2:1 (egg-PC: cholesterol) magnetic liposomes showed the highest encapsulation efficiency and comparable heating ability to that of magnetic fluids. Stealth TFH 2:1 magnetic liposomes containing DSPE-PEG 2000 were three-fold more cytocompatible as compared to the magnetic fluid. Stealth TFH 2:1 manganese ferrite-based magnetic liposomes might be useful for hyperthermia treatment of cancer.

  3. An innovative monolithic column preparation for the isolation of 25 kilo base pairs DNA.

    Science.gov (United States)

    Ongkudon, Clarence M; Pan, Sharadwata; Danquah, Michael K

    2013-11-29

    The use of large DNAs in preparing multivalent vaccines that will eventually give protective immunity against multiple pathogenic microbes is becoming a major debate nowadays. One of the important issues in ensuring the successful implementation of the new vaccine technology is the development of a chromatographic technique that can handle larger DNAs. This paper reports the development of a novel conical monolithic column format with pore and surface characteristics engineered for the isolation of 25 kbp DNA in a single step fashion. An effective method of eliminating wall channelling, a defect of most conventional monolithic chromatography systems which has caused significant loss of product, was applied to maximise DNA recovery. This method was based on a systematic reduction of wall channel size based on a predetermined correlation between column’s back pressure and wall channel size of a particular monolith pore size.

  4. Preparation, Characterization, and Analytical Application of Ramipril Membrane-Based Ion-Selective Electrode

    Directory of Open Access Journals (Sweden)

    Hassan Arida

    2009-01-01

    Full Text Available The fabrication and electrochemical evaluation of two PVC membrane-based Ion-Selective electrodes responsive for ramipril drug have been proposed. The sensitive membranes were prepared using ramipril-phosphomolibdate and ramipril-tetraphenylborate ion-pair complexes as electroactive sensing materials in plasticized PVC support. The electrodes based on these materials provide near-Nernestian response (sensitivity of 53±0.5–54±0.5 mV/concentration decade covering the concentration range of 1.0×10-2–1.0×10-5 mol L−1 with a detection limit of 3.0×10-6–4.0×10-6 mol L−1. The suggested electrodes have been successfully used in the determination of ramipril drug in some pharmaceutical formulations using direct potentiometry with average recovery of >96% and mean standard deviation of <3% (n=5.

  5. Preparation and Properties of Silver Nanowire-Based Transparent Conductive Composite Films

    Science.gov (United States)

    Tian, Ji-Li; Zhang, Hua-Yu; Wang, Hai-Jun

    2016-06-01

    Silver nanowire-based transparent conductive composite films with different structures were successfully prepared using various methods, including liquid polyol, magnetron sputtering and spin coating. The experimental results revealed that the optical transmittance of all different structural composite films decreased slightly (1-3%) compared to pure films. However, the electrical conductivity of all composite films had a great improvement. Under the condition that the optical transmittance was greater than 78% over the wavelength range of 400-800 nm, the AgNW/PVA/AgNW film became a conductor, while the AZO/AgNW/AZO film and the ITO/AgNW/ITO film showed 88.9% and 94% reductions, respectively, for the sheet resistance compared with pure films. In addition, applying a suitable mechanical pressure can improve the conductivity of AgNW-based composite films.

  6. Design and preparation of Zn-based materials possessing both high damping and good mechanical properties

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new idea of design and manufacture of metal-based materials possessing both high damping and good mechanical properties was proposed. The key of the idea is the combination of fining restriction-damping structures, using all mechanisms and taking advantages of different materials. Based upon this idea a foam ZA27 was prepared by the technology of prefabricated salt-mass centrifugal seeping foundry, its tensile strength and compressive strength are 83~119 MPa and 100~189 MPa, respectively. The damping properties of the foam ZA27 increase remarkably after the carpenter pastern or rosin (the damping-increased materials) was immerged into it, which approaches to the level of viscous-elastic polymer materials (Q-1≥20×10-3).

  7. Cellulose-based graft copolymers prepared by simplified electrochemically mediated ATRP

    Directory of Open Access Journals (Sweden)

    P. Chmielarz

    2017-02-01

    Full Text Available Brush-shaped block copolymer with a dual hydrophilic poly(acrylic acid-block-poly(oligo(ethylene glycol acrylate (PAA-b-POEGA arms was synthesized for the first time via a simplified electrochemically mediated ATRP (seATRP under both constant potential electrolysis and constant current electrolysis conditions, utilizing only 30 ppm of catalyst complex. The polymerization conditions were optimized to provide fast reactions while employing low catalyst concentrations and preparation of cellulose-based brush-like copolymers with narrow molecular weight distributions. The results from proton nuclear magnetic resonance (1H NMR spectral studies support the formation of cellulose-based graft (copolymers. It is expected that these new polymer brushes may find application as pH- and thermo-sensitive drug delivery systems.

  8. Advances in host and vector development for the production of plasmid DNA vaccines.

    Science.gov (United States)

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  9. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  10. Destabilization of IncA and IncC plasmids by SGI1 and SGI2 type Salmonella genomic islands.

    Science.gov (United States)

    Harmer, Christopher J; Hamidian, Mohammad; Ambrose, Stephanie J; Hall, Ruth M

    Both the Salmonella genomic islands (SGI) and the conjugative IncC plasmids are known to contribute substantially to the acquisition of resistance to multiple antibiotics, and plasmids in the A/C group are known to mobilize the Salmonella genomic island SGI1, which also carries multiple antibiotic resistance genes. Plasmid pRMH760 (IncC; A/C2) was shown to mobilize SGI1 variants SGI1-I, SGI1-F, SGI1-K and SGI2 from Salmonella enterica to Escherichia coli where it was integrated at the preferred location, at the end of the trmE (thdF) gene. The plasmid was transferred at a similar frequency. However, we observed that co-transfer of the SGI and the plasmid was rarer. In E. coli to E. coli transfer, the frequency of transfer of the IncC plasmid pRMH760 was at least 1000-fold lower when the donor carried SGI1-I or SGI1-K, indicating that the SGI suppresses transfer of the plasmid. In addition, pRMH760 was rapidly lost from both E. coli and S. enterica strains that also carried SGI1-I, SGI1-F or SGI2. However, plasmid loss was not seen when the SGI1 variant was SGI1-K, which lacks two segments of the SGI1 backbone. The complete sequence of the SGI1-I and SGI1-F were determined and SGI1-K also carries two single base substitutions relative to SGI1-I. The IncA (A/C1) plasmid RA1 was also shown to mobilize SGI2-A and though there are significant differences between the backbones of IncA and IncC plasmids, RA1 was also rapidly lost when SGI2-A was present in the same cell. We conclude that there are multiple interactions, both cooperative and antagonistic, between an IncA or IncC plasmid and the SGI1 and SGI2 family genomic islands. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Sequence analysis and characterization of rolling-circle replicating plasmid pVCM01 from Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Penido, A. F. B.

    2013-12-01

    Full Text Available Aims: Characterization of cryptic plasmid pVCM01 (accession number JX133088 isolated from Salmonella enterica Enteritidis. Methodology and results: The complete sequence of pVCM01 was obtained. This plasmid possesses 1981 bp, with G+C content of 57% in agreement of the range of Salmonella genomic DNA. pVCM01 has a high degree of similarity to pB and pJ plasmids. It possesses six main open reading frames, only one have a very high degree of amino acid identity with protein involved in the rolling-circle-like replication (RCR. Based on the sequence similarities, pVCM01 plasmid belonged to the pC194/pUB110 rolling-circle replicating plasmid family. The Rep pVCM01 possesses the motifs: FLTLTVRN, HPHFHTL, SGDGYVKHERW, which were present in all Rep proteins. Conclusion, significance and impact of study: The small size of pVCM01 plasmid and its stability in E. coli cells, make it an attractive candidate to develop new vectors, such as cloning and/or expression vector.

  12. Transformation of serum-susceptible Escherichia coli O111 with p16Slux plasmid to allow for real-time monitoring of complement-based inactivation of bacterial growth in bovine milk.

    Science.gov (United States)

    Maye, S; Stanton, C; Fitzgerald, G F; Kelly, P M

    2016-01-01

    Complement activity has only recently been characterized in raw bovine milk. However, the activity of this component of the innate immune system was found to diminish as milk was subjected to heat or partitioning during cream separation. Detection of complement in milk relies on a bactericidal assay. This assay exploits the specific growth susceptibility of Escherichia coli O111 to the presence of complement. Practical application of the assay was demonstrated when a reduction in complement activity was recorded in the case of pasteurized and reduced-fat milks. This presented an opportunity to improve the functionality of the bactericidal assay by incorporating bioluminescence capability into the target organism. Following some adaptation, the strain was transformed by correctly integrating the p16Slux plasmid. Growth properties of the transformed strain of E. coli O111 were unaffected by the modification. The efficacy of the strain adaptation was correlated using the LINEST function analysis [r=0.966; standard error of prediction (SEy)=0.957] bioluminescence with that of bactericidal assay total plate counts within the range of 7.5 to 9.2 log cfu/mL using a combination of raw and processed milk samples. Importantly, the transformed E. coli O111 p16Slux strain could be identified in milk and broth samples using bioluminescence measurement, thus enabling the bactericidal assay-viability test to be monitored in real time throughout incubation.

  13. Preparation and Photocatalytic Property of TiO2/Diatomite-Based Porous Ceramics Composite Materials

    Directory of Open Access Journals (Sweden)

    Shuilin Zheng

    2012-01-01

    Full Text Available The diatomite-based porous ceramics was made by low-temperature sintering. Then the nano-TiO2/diatomite-based porous ceramics composite materials were prepared by hydrolysis deposition method with titanium tetrachloride as the precursor of TiO2 and diatomite-based porous as the supporting body of the nano-TiO2. The structure and microscopic appearance of nano-TiO2/diatomite-based porous ceramics composite materials was characterized by XRD and SEM. The photocatalytic property of the composite was investigated by the degradation of malachite green. Results showed that, after calcination at 550°C, TiO2 thin film loaded on the diatomite-based porous ceramics is anatase TiO2 and average grain size of TiO2 is about 10 nm. The degradation ratio of the composite for 5 mg/L malachite green solution reached 86.2% after irradiation for 6 h under ultraviolet.

  14. Evidence-based recommendations for natural bodybuilding contest preparation: nutrition and supplementation

    Science.gov (United States)

    2014-01-01

    The popularity of natural bodybuilding is increasing; however, evidence-based recommendations for it are lacking. This paper reviewed the scientific literature relevant to competition preparation on nutrition and supplementation, resulting in the following recommendations. Caloric intake should be set at a level that results in bodyweight losses of approximately 0.5 to 1%/wk to maximize muscle retention. Within this caloric intake, most but not all bodybuilders will respond best to consuming 2.3-3.1 g/kg of lean body mass per day of protein, 15-30% of calories from fat, and the reminder of calories from carbohydrate. Eating three to six meals per day with a meal containing 0.4-0.5 g/kg bodyweight of protein prior and subsequent to resistance training likely maximizes any theoretical benefits of nutrient timing and frequency. However, alterations in nutrient timing and frequency appear to have little effect on fat loss or lean mass retention. Among popular supplements, creatine monohydrate, caffeine and beta-alanine appear to have beneficial effects relevant to contest preparation, however others do not or warrant further study. The practice of dehydration and electrolyte manipulation in the final days and hours prior to competition can be dangerous, and may not improve appearance. Increasing carbohydrate intake at the end of preparation has a theoretical rationale to improve appearance, however it is understudied. Thus, if carbohydrate loading is pursued it should be practiced prior to competition and its benefit assessed individually. Finally, competitors should be aware of the increased risk of developing eating and body image disorders in aesthetic sport and therefore should have access to the appropriate mental health professionals. PMID:24864135

  15. Nuclear characteristics of the endometrial cytology: liquid-based versus conventional preparation.

    Science.gov (United States)

    Norimatsu, Yoshiaki; Shigematsu, Yumie; Sakamoto, Shingo; Ohsaki, Hiroyuki; Yanoh, Kenji; Kawanishi, Namiki; Kobayashi, Tadao K

    2013-02-01

    The aim of this study was to assess the utility of liquid-based cytologic preparation (LP) compared with conventional preparation (CP) for the assessment of nuclear findings in endometrial glandular and stromal breakdown (EGBD) which may be misdiagnosed as carcinoma in EGBD cases. The material consists of cytologic smears including 20 cases of proliferative endometrium (PE), 20 cases of EGBD, and 20 cases of endometrioid adenocarcinoma grade1 (G1) for which histopathological diagnosis was obtained by endometrial curettage at the JA Suzuka General Hospital. Nuclear findings were examined in PE cells, EGBD-stromal cells, EGBD-metaplastic cells, and G1 cells, respectively. It was examined about the following items; (1) nuclear shape; (2) A long/minor axis ratio in cell nuclei; (3) an area of cell nuclei; (4) overlapping nuclei. Results are as follows: (1) nuclear shape; as for the reniform shape of EGBD-stromal cells and spindle shape of EGBD-metaplastic cells, the ratio of the LP method was a higher value than the CP method. (2) The long axis and area of cell nuclei; LP in all groups was a recognizable tendency for nuclear shrinkage. (3) The long/minor axis ratio in cell nuclei; only EGBD-metaplastic cells recognize a significant difference between CP and LP. (4) Overlapping nuclei; LP was a higher value in comparison with CP in the other groups except PE cells, and the degree of overlapping nuclei was enhanced about three times. Therefore, although a cell of LP has a shrinking tendency, (1) it is excellent that LP preserves a characteristic of nuclear shape than CP; (2) a cellular characteristic becomes clearer, because three-dimensional architecture of LP is preserved of than CP. As for the standard preparation method for endometrial cytology samples, we considered that a concrete introduction of the LP method poses no problems.

  16. Lyapunov-Based Feedback Preparation of GHZ Entanglement of N-Qubit Systems.

    Science.gov (United States)

    Liu, Yanan; Kuang, Sen; Cong, Shuang

    2016-07-09

    The Greenberger-Horne-Zeilinger (GHZ) entangled states are a typical class of entangled states in multiparticle systems and play an important role in the applications of quantum communication and quantum computation. For a general quantum system of N qubits, degenerate measurement operators are often met, which cause the convergence obstacle in the state preparation or stabilization problem. This paper first generalizes the traditional quantum state continuous reduction theory to the case of a degenerate measurement operator and chooses a measurement operator for an arbitrarily given target GHZ entangled state, then presents a state stabilization control strategy based on the Lyapunov method and achieves the feedback preparation of the target GHZ state. In our stabilization strategy, we separate the target GHZ state and all the other GHZ states that often form the equilibrium points of the closed-loop system by dividing the state space into several different regions; and formally design a switching control law between the regions, which contains the control Hamiltonians to be constructed. By analyzing the stability of the closed-loop system in the different regions, we propose a systematic method for constructing the control Hamiltonians and solve the convergence problem caused by the degenerate measurement operator. The global stability of the whole closed-loop stochastic system is strictly proved. Also, we perform some simulation experiments on a three-qubit system and prepare a three-qubit GHZ entangled state. At the same time, the simulation results show the effectiveness of the switching control law and the construction method for the control Hamiltonians proposed in this paper.

  17. Water-based preparation of spider silk films as drug delivery matrices.

    Science.gov (United States)

    Agostini, Elisa; Winter, Gerhard; Engert, Julia

    2015-09-10

    The main focus of this work was to obtain a drug delivery matrix characterized by biocompatibility, water insolubility and good mechanical properties. Moreover the preparation process has to be compatible with protein encapsulation and the obtained matrix should be able to sustain release a model protein. Spider silk proteins represent exceptional natural polymers due to their mechanical properties in combination with biocompatibility. As both hydrophobic and slowly biodegrading biopolymers, recombinant spider silk proteins fulfill the required properties for a drug delivery system. In this work, we present the preparation of eADF4(C16) films as drug delivery matrices without the use of any organic solvent. Water-based spider silk films were characterized in terms of protein secondary structure, thermal stability, zeta-potential, solubility, mechanical properties, and water absorption and desorption. Additionally, this study includes an evaluation of their application as a drug delivery system for both small molecular weight drugs and high molecular weight molecules such as proteins. Our investigation focused on possible improvements in the film's mechanical properties including plasticizers in the film matrix. Furthermore, different film designs were prepared, such as: monolayer, coated monolayer, multilayer (sandwich), and coated multilayer. The release of the model protein BSA from these new systems was studied. Results indicated that spider silk films are a promising protein drug delivery matrix, capable of releasing the model protein over 90 days with a release profile close to zero order kinetic. Such films could be used for several pharmaceutical and medical purposes, especially when mechanical strength of a drug eluting matrix is of high importance. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Evidence-based recommendations for natural bodybuilding contest preparation: nutrition and supplementation.

    Science.gov (United States)

    Helms, Eric R; Aragon, Alan A; Fitschen, Peter J

    2014-01-01

    The popularity of natural bodybuilding is increasing; however, evidence-based recommendations for it are lacking. This paper reviewed the scientific literature relevant to competition preparation on nutrition and supplementation, resulting in the following recommendations. Caloric intake should be set at a level that results in bodyweight losses of approximately 0.5 to 1%/wk to maximize muscle retention. Within this caloric intake, most but not all bodybuilders will respond best to consuming 2.3-3.1 g/kg of lean body mass per day of protein, 15-30% of calories from fat, and the reminder of calories from carbohydrate. Eating three to six meals per day with a meal containing 0.4-0.5 g/kg bodyweight of protein prior and subsequent to resistance training likely maximizes any theoretical benefits of nutrient timing and frequency. However, alterations in nutrient timing and frequency appear to have little effect on fat loss or lean mass retention. Among popular supplements, creatine monohydrate, caffeine and beta-alanine appear to have beneficial effects relevant to contest preparation, however others do not or warrant further study. The practice of dehydration and electrolyte manipulation in the final days and hours prior to competition can be dangerous, and may not improve appearance. Increasing carbohydrate intake at the end of preparation has a theoretical rationale to improve appearance, however it is understudied. Thus, if carbohydrate loading is pursued it should be practiced prior to competition and its benefit assessed individually. Finally, competitors should be aware of the increased risk of developing eating and body image disorders in aesthetic sport and therefore should have access to the appropriate mental health professionals.

  19. Synthesis and Study of Guest-Rebinding of MIP Based on MAA Prepared using Theophylline Template

    Science.gov (United States)

    Nurhayati, T.; Yanti; Royani, I.; Widayani; Khairurrijal

    2016-08-01

    A molecularly imprinted polymer (MIP) based on methacrylic acid (MAA) monomer and theophylline template has been synthesized using a modified bulk polymerization method. Theophylline was employed as a template and it formed a complex with MAA through hydrogen bonding. Self-assembly of template-monomer was followed by cross-linking process using ethylene glycol dimethacrylate (EGDMA) cross-linker. The polymerization process was initiated by thermal decomposition of benzoyl peroxide (BPO) as the initiator at 60oC after cooling treatment at -5oC. After 7 hours, a rigid polymer was obtained and followed by grinding the polymer and removing the template. As a reference, a nonimprinted polymer (NIP) has also been synthesized using similar procedure by excluding the template. FTIR study was carried out to investigate the presence of theophylline in the as- prepared polymer, MIP, and NIP. The spectra indicated that theophylline was successfully incorporated in the as-prepared polymer. This result was also confirmed by EDS analysis showing that N atoms of the as-prepared polymer were derived from amino group of theophylline. Furthermore, the polymer particles of MIP were irregular in shape and size as shown by its SEM image. The capability of guest-rebinding of the MIP was analyzed through Batchwise guest-binding experiment. The results showed that for initial concentration of theophylline in methanol/chloroform (1/1, v/v) of 0.333 mM, the binding capacity of the MIP was 23.22 /mol/g. Compared to the MIP, the adsorption capacity of the NIP was only 3.73 /mol/g. This result shows that MIP has higher affinity than NIP.

  20. Are general surgery residents adequately prepared for hepatopancreatobiliary fellowships? A questionnaire-based study

    Science.gov (United States)

    Osman, Houssam; Parikh, Janak; Patel, Shirali; Jeyarajah, D Rohan

    2015-01-01

    Background The present study was conducted to assess the preparedness of hepatopancreatobiliary (HPB) fellows upon entering fellowship, identify challenges encountered by HPB fellows during the initial part of their HPB training, and identify potential solutions to these challenges that can be applied during residency training. Methods A questionnaire was distributed to all HPB fellows in accredited HPB fellowship programmes in two consecutive academic years (n = 42). Reponses were then analysed. Results A total of 19 (45%) fellows responded. Prior to their fellowship, 10 (53%) were in surgical residency and the rest were in other surgical fellowships or surgical practice. Thirteen (68%) were graduates of university-based residency programmes. All fellows felt comfortable in performing basic laparoscopic procedures independently at the completion of residency and less comfortable in performing advanced laparoscopy. Eight (42%) fellows cited a combination of inadequate case volume and lack of autonomy during residency as the reasons for this lack of comfort. Thirteen (68%) identified inadequate preoperative workup and management as their biggest fear upon entering practice after general surgery training. A total of 17 (89%) fellows felt they were adequately prepared to enter HPB fellowship. Extra rotations in transplant, vascular or minimally invasive surgery were believed to be most helpful in preparing general surgery residents pursing HPB fellowships. Conclusions Overall, HPB fellows felt themselves to be adequately prepared for fellowship. Advanced laparoscopic procedures and the perioperative management of complex patients are two of the challenges facing HPB fellows. General surgery residents who plan to pursue an HPB fellowship may benefit from spending extra rotations on certain subspecialties. Focus on perioperative workup and management should be an integral part of residency and fellowship training. PMID:25387852

  1. Preparation and characterization of Fe-based bulk metallic glasses in plate form

    Energy Technology Data Exchange (ETDEWEB)

    Lavorato, G.C. [INTECIN (FIUBA-CONICET), Paseo Colon 850, Capital Federal (Argentina); Dipartimento di Chimica IFM and NIS, Universita di Torino, Torino (Italy); Fiore, G.; Castellero, A.; Baricco, M. [Dipartimento di Chimica IFM and NIS, Universita di Torino, Torino (Italy); Moya, J.A., E-mail: jmoya.fi.uba@gmail.com [IESIING, Facultad de Ingenieria e Informatica, UCASAL, Salta (Argentina); CONICET (Argentina)

    2012-08-15

    Amorphous alloys with composition (at%) Fe{sub 48}Cr{sub 15}Mo{sub 14}C{sub 15}B{sub 6}Gd{sub 2} (alloy A) and Fe{sub 48}Cr{sub 15}Mo{sub 14}C{sub 15}B{sub 6}Y{sub 2} (alloy B) were prepared either using pure elements (A and B1) and a commercial AISI430 steel as a base material (B2). When prepared from pure elements both alloys (A and B1) could be cast in plate form with a fixed thickness of 2 mm and variable lengths between 10 and 20 mm by means of copper-mold injection in air atmosphere. In the case of alloy B2, prepared using commercial grade raw materials, rods of 2 mm diameter were obtained. X-ray diffraction and scanning electron microscopy observations confirmed that an amorphous structure was obtained in all the as-cast samples. A minor fraction of crystalline phases (oxides and carbides) was detected on the as-cast surface. Differential scanning calorimetry measurements showed a glass transition temperature at 856 K for alloy A and 841 K for alloy B1, and an onset crystallization temperature of 887 K for alloy A and 885 K for alloy B1. In the case of alloy B2 a slightly different crystallization sequence was observed. Values of hardness ({approx}13 GPa) and the Young modulus ({approx}180 GPa) were measured by nanoindentation for both the alloys. The effects of adverse casting conditions (such as air atmosphere, non-conventional injection copper mold casting and partial replacement of pure elements with commercial grade raw materials) on the glass formation and properties of the alloy are discussed.

  2. Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids

    DEFF Research Database (Denmark)

    Jutkina, Jekaterina; Hansen, Lars Hestbjerg; Li, Lili

    2013-01-01

    In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+ C content of 53.75%. A total of 135 open reading frames (ORFs) were ...

  3. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  4. Genomic comparison of archaeal conjugative plasmids from Sulfolobus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2004-01-01

    All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence...

  5. Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Brown, Susan E; Knudson, Dennis L; Ishimaru, Carol A

    2002-05-01

    Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

  6. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  7. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)

    SUHARTONO

    2010-04-01

    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  8. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  9. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  10. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  11. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  12. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

    Science.gov (United States)

    Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

    2016-12-01

    Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

  13. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  14. Deciphering conjugative plasmid permissiveness in wastewater microbiomes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel; Milani, Stefan Morberg

    2017-01-01

    Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via...... still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from...... diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent...

  15. Parametric Grid Information in the DOE Knowledge Base: Data Preparation, Storage and Access.

    Energy Technology Data Exchange (ETDEWEB)

    Hipp, J. R.; Young, C. J.; Moore, S. G.; Shepherd, E. R.; Schultz, C. A.; Myers, S. C.

    1999-10-01

    The parametric grid capability of the Knowledge Base provides an efficient, robust way to store and access interpolatable information which is needed to monitor the Comprehensive Nuclear Test Ban Treaty. To meet both the accuracy and performance requirements of operational monitoring systems, we use a new approach which combines the error estimation of kriging with the speed and robustness of Natural Neighbor Interpolation (NNI). The method involves three basic steps: data preparation (DP), data storage (DS), and data access (DA). The goal of data preparation is to process a set of raw data points to produce a sufficient basis for accurate NNI of value and error estimates in the Data Access step. This basis includes a set of nodes and their connectedness, collectively known as a tessellation, and the corresponding values and errors that map to each node, which we call surfaces. In many cases, the raw data point distribution is not sufficiently dense to guarantee accurate error estimates from the NNI, so the original data set must be densified using a newly developed interpolation technique known as Modified Bayesian Kriging. Once appropriate kriging parameters have been determined by variogram analysis, the optimum basis for NNI is determined in a process we call mesh refinement, which involves iterative kriging, new node insertion, and Delauny triangle smoothing. The process terminates when an NNI basis has been calculated which will fit the kriged values within a specified tolerance. In the data storage step, the tessellations and surfaces are stored in the Knowledge Base, currently in a binary flatfile format but perhaps in the future in a spatially-indexed database. Finally, in the data access step, a client application makes a request for an interpolated value, which triggers a data fetch from the Knowledge Base through the libKBI interface, a walking triangle search for the containing triangle, and finally the NNI interpolation.

  16. Parametric Grid Information in the DOE Knowledge Base: Data Preparation, Storage, and Access

    Energy Technology Data Exchange (ETDEWEB)

    HIPP,JAMES R.; MOORE,SUSAN G.; MYERS,STEPHEN C.; SCHULTZ,CRAIG A.; SHEPHERD,ELLEN; YOUNG,CHRISTOPHER J.

    1999-10-01

    The parametric grid capability of the Knowledge Base provides an efficient, robust way to store and access interpolatable information which is needed to monitor the Comprehensive Nuclear Test Ban Treaty. To meet both the accuracy and performance requirements of operational monitoring systems, we use a new approach which combines the error estimation of kriging with the speed and robustness of Natural Neighbor Interpolation (NNI). The method involves three basic steps: data preparation (DP), data storage (DS), and data access (DA). The goal of data preparation is to process a set of raw data points to produce a sufficient basis for accurate NNI of value and error estimates in the Data Access step. This basis includes a set of nodes and their connectedness, collectively known as a tessellation, and the corresponding values and errors that map to each node, which we call surfaces. In many cases, the raw data point distribution is not sufficiently dense to guarantee accurate error estimates from the NNI, so the original data set must be densified using a newly developed interpolation technique known as Modified Bayesian Kriging. Once appropriate kriging parameters have been determined by variogram analysis, the optimum basis for NNI is determined in a process they call mesh refinement, which involves iterative kriging, new node insertion, and Delauny triangle smoothing. The process terminates when an NNI basis has been calculated which will fir the kriged values within a specified tolerance. In the data storage step, the tessellations and surfaces are stored in the Knowledge Base, currently in a binary flatfile format but perhaps in the future in a spatially-indexed database. Finally, in the data access step, a client application makes a request for an interpolated value, which triggers a data fetch from the Knowledge Base through the libKBI interface, a walking triangle search for the containing triangle, and finally the NNI interpolation.

  17. [Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid].

    Science.gov (United States)

    Madajczak, Grzegorz; Binek, Marian

    2005-01-01

    Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.

  18. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Membrane-based sample preparation for ion chromatography-Techniques, instrumental configurations and applications.

    Science.gov (United States)

    Frenzel, Wolfgang; Markeviciute, Inga

    2017-01-06

    Sample preparation is the bottleneck of many analytical methods, including ion chromatography (IC). Procedures based on the application of membranes are important, yet not well appreciated means for clean-up and analyte preconcentration of liquid samples. Filtration, ultrafiltration, the variety of dialysis techniques, i.e. passive dialysis, Donnan dialysis and electrodialysis, as well as gas-diffusion are being reviewed here with respect to their application in combination with IC. Instrumental aspects including hardware requirements, configuration of membrane separation units and membrane characteristics are presented. Operation in batch and flow-through mode is described with emphasis on the latter to in-line coupling with IC, permitting fully automated operation. Attention is also drawn to dialysis probes and microdialysis both providing options for in-situ measurements with inherent selective sampling of analytes and sample preparation. The respective features of the various techniques are outlined with respect to the possibilities of matrix removal and selectivity enhancement. In this article, we provide examples of application of the diverse membrane separation techniques and discuss the benefits and limitations thereof.

  20. Preparation and Evaluation of Contact Lenses Embedded with Polycaprolactone-Based Nanoparticles for Ocular Drug Delivery.

    Science.gov (United States)

    Nasr, Farzaneh Hashemi; Khoee, Sepideh; Dehghan, Mohammad Mehdi; Chaleshtori, Sirous Sadeghian; Shafiee, Abbas

    2016-02-08

    To improve the efficiency of topical ocular drug administration, we focused on development of a nanoparticles loaded contact lens to deliver the hydrophobic drug over a prolonged period of time. The cross-linked nanoparticles based on PCL (poly ε-caprolactone), 2-hydroxy ethyl methacrylate (HEMA), and poly ethylene glycol diacrylate (PEG-DA) were prepared by surfactant-free miniemulsion polymerization. The lens material was prepared through photopolymerization of HEMA and N-vinylpyrrolidone (NVP) using PEG-DA as the cross-linker. Effects of nanoparticles loading on critical contact lens properties such as transparency, water content, modulus and ion and oxygen permeabilities were studied. Nanoparticles and hydrogel showed high viability, indicating the absence of cytotoxicity and stimulatory effect. Drug release studies revealed that the hydrogel embedded with nanoparticles released the drug for a period of 12 days. The results of this study provide evidence that nanoparticles loaded hydrogels could be used for extended delivery of loteprednol etabonate and perhaps other drugs.

  1. Preparation and Recipe Optimization of Water-based Architectural Heat Insulation Coatings

    Institute of Scientific and Technical Information of China (English)

    CHEN Lijun; SHI Hongxin; XIANG Juping; WU Hongke

    2008-01-01

    Water-based architectural heat insulation coatings were studied to overcome the drawbacks of conventional inorganic silicate heat insulation coatings.The heat insulation coatings were prepared with the method of mechanical agitation when the mixed organic polymer emulsions were used as binder of the coatings and the mixed heat insulating aggregates were applied as powder,and some assistants were also added.Water temperature difference in the plastic container,which was coated with heat insulation coatings,represented the heat-insulating property of the coatings.The influences of components of mixed polymer emulsion,mass ratio of polymer emulsion to powder,particle size of heat insulating aggregates,added amount of air entraining admixture and the match of thickeners on the properties of the coatings were studied.The experimental results show that the heat insulation coatings with good finishing,heat-insulation property and artificial weathering can be prepared when the binder is composed of 66.92% styrene-acrylic emulsion,16.59% elastic emulsion and 16.49% silicone-acrylic emulsion,the mass ratio of polymer emulsion to powder is 0.45,the particle size of heat insulating aggregates is in the rang of 200 and 250 mesh size,the added amount of sericite is 15%,and the added amount of air entraining admixture is in the range of 1.0% and 1.5% and the thickeners are the mixtures of ASE-60 and RM-5000.

  2. Preparation and characterization of ultrafine Fe-Cu-based catalysts for CO hydrogenation

    Institute of Scientific and Technical Information of China (English)

    Yunlai Su; Yingli Wang; Zhongmin Liu

    2008-01-01

    The ultrafine particles of a new style Fe-Cu-based catalysts for CO hydrogenation were prepared by impregnating the organic sol of Fe(OH)3 and Cu(OH)2 onto the activated Al2O3, in which the organic sol of Fe(OH)3 and Cu(OH)2 were pre-pared in the microemulsion of dodecylbenzenesulfonic acid sodium(S)/n-butanol(A)/toluene(O)/water with V(A)/V(O) = 0.25 and W(A)/W(S)=1.50. This catalyst was characterized by particle size analysis, XRD and TG. The results of particle size analysis showed that Fe(OH)3 particles with a mean size of 17.1 nm and Cu(OH)2 particles with an average size of 6.65 μm were obtained. TG analysis and XRD patterns suggested that 673 K is the optimal calcination temperature. CO hydrogenation produced C+2 OH with a high selectivity above 58 wt% by using the ultrafine particles as catalyst, and the total alcohol yield of 0.250 g·ml-1·h-1 was obtained when the contents of Al2O3 and K were 88.61 wt% and 1.60 wt%, respectively.

  3. Influence of surface preparation on fracture load of resin composite-based repairs

    Science.gov (United States)

    Mateos-Palacios, Rocío; Román-Rodríguez, Juan-Luis; Solá-Ruíz, María-Fernanda; Fons-Font, Antonio

    2015-01-01

    The purpose of the present study is to evaluate the fracture load of composite-based repairs to fractured zirconium oxide (Z) crowns and to ceramic-fused-to-metal (CM) crowns, comparing different mechanical surface preparation methods. A total of 75 crowns were repaired; samples then underwent dynamic loading and thermocycling. Final fracture load values for failure of the repaired crowns were measured and the type of fracture registered. Group I: CM: Surface preparation with a diamond bur + 9.5% Hydrofluoric Acid (HF) etching; Group II): CM: air-particle (Al2O3) + 9.5% HF; Group III: CM: Silica coating (SiO2); Group IV): Z: air-particle (Al2O3) + HF 9.5%; Group V) Z: Silica coating (SiO2). Of the three CM groups, Group I (CM-diamond bur) showed the highest mean failure value, with significant difference in comparison with Group III (CM-silica coating). For the zirconia groups, the highest value was obtained by Group V (silica coating). Key words:Crown, ceramic-fused-to-metal, zirconia, resin-composite, ceramic covering. PMID:25810848

  4. Chemical characterization of Klason lignin preparations from plant-based foods.

    Science.gov (United States)

    Bunzel, Mirko; Schüssler, Anne; Tchetseubu Saha, Gérard

    2011-12-14

    To analyze the accuracy of the Klason lignin method as applied for the determination of lignin contents in plant based-food products, Klason lignin preparations from curly kale, pears, whole wheat grains, and corn bran were chemically characterized. Characterization included routine ash and protein determinations and the extraction of fat/waxes as well as cutin/suberin depolymerization and extraction of the liberated monomers. Fat/wax and cutin/suberin amounts in the Klason lignin preparations were determined gravimetrically, and their compositions were analyzed by using GC-MS. Typical fat, wax, and cutin (and suberin) constituents such as saturated and unsaturated fatty acids, hydroxy and/or epoxy fatty acids, and phenolic acids were identified in all samples, whereas the detection of long-chain hydrocarbons, alcohols, and ketones, sterols, stanols, and dioic acids was dependent on the sample analyzed. Estimation of the contribution of non-lignin compounds to the Klason lignin contents reduced the noncorrected Klason lignin contents of the insoluble fibers from 28.7% (kale), 22.8% (pear), 14.8% (wheat), and 9.9% (corn) to maximum lignin contents of 6.5% (kale), 16.4% (pear), 4.9% (wheat), and 2.3% (corn). These data demonstrate that certain commonly used statements such as "cereal brans are highly lignified" need to be revised.

  5. Organic Photovoltaic Cells Based on PbPc Nanocolumns Prepared by Glancing Angle Deposition

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2013-01-01

    Full Text Available Organic small material lead phthalocyanine (PbPc nanocolumns were prepared via glancing angle deposition (GLAD on indium tin oxide (ITO coated glass substrates. Organic electron acceptor materials fullerene (C60 was evaporated onto the nanocolumn PbPc thin films to prepare heterojunction structure ITO/PbPc/C60/Bphen/Al organic photovoltaic cells (OPVs. It is worthwhile to mention that C60 molecules firstly fill the voids between PbPc nanocolumns and then form impact C60 layer. The interpenetrating electron donor/acceptor structure effectively enhances interface between electron donor and electron acceptor, which is beneficial to exciton dissociation. The short circuit current density (Jsc of organic photovoltaic devices (OPVs based on PbPc nanocolumn was increased from 1.19 mA/cm2 to 1.74 mA/cm2, which should be attributed to the increase of interface between donor and acceptor. The effect of illumination intensity on the performance of OPVs was investigated by controlling the distance between light source and sample, and the Jsc of two kind of OPVs was increased along with the increase of illumination intensity.

  6. Modification of inkjet printer for polymer sensitive layer preparation on silicon-based gas sensors

    Directory of Open Access Journals (Sweden)

    Tianjian Li

    2015-04-01

    Full Text Available Inkjet printing is a versatile, low cost deposition technology with the capabilities for the localized deposition of high precision, patterned deposition in a programmable way, and the parallel deposition of a variety of materials. This paper demonstrates a new method of modifying the consumer inkjet printer to prepare polymer-sensitive layers on silicon wafer for gas sensor applications. A special printing tray for the modified inkjet printer to support a 4-inch silicon wafer is designed. The positioning accuracy of the deposition system is tested, based on the newly modified printer. The experimental data show that the positioning errors in the horizontal direction are negligibly small, while the positioning errors in the vertical direction rise with the increase of the printing distance of the wafer. The method for making suitable ink to be deposited to form the polymer-sensitive layer is also discussed. In the testing, a solution of 0.1 wt% polyvinyl alcohol (PVA was used as ink to prepare a sensitive layer with certain dimensions at a specific location on the surface of the silicon wafer, and the results prove the feasibility of the methods presented in this article.

  7. Preparation, Characterization, and Cationic Functionalization of Cellulose-Based Aerogels for Wastewater Clarification

    Directory of Open Access Journals (Sweden)

    Yang Hu

    2016-01-01

    Full Text Available Aerogels are a series of materials with porous structure and light weight which can be applied to many industrial divisions as insulators, sensors, absorbents, and cushions. In this study, cellulose-based aerogels (aerocelluloses were prepared from cellulosic material (microcrystalline cellulose in sodium hydroxide/water solvent system followed by supercritical drying operation. The average specific surface area of aerocelluloses was 124 m2/g. The nitrogen gas (N2 adsorption/desorption isotherms revealed type H1 hysteresis loops for aerocelluloses, suggesting that aerocelluloses may possess a porous structure with cylindrically shaped pores open on both ends. FTIR and XRD analyses showed that the crystallinity of aerocelluloses was significantly decreased as compared to microcrystalline cellulose and that aerocelluloses exhibited a crystalline structure of cellulose II as compared to microcrystalline cellulose (cellulose I. To perform cationic functionalization, a cationic agent, (3-chloro-2-hydroxypropyl trimethylammonium chloride, was used to introduce positively charged sites on aerocelluloses. The cationized aerocelluloses exhibited a strong ability to remove anionic dyes from wastewater. Highly porous and low cost aerocelluloses prepared in this study would be also promising as a fast absorbent for environmental pollutants.

  8. Neoglycopolymers based on 4-vinyl-1,2,3-triazole monomers prepared by click chemistry.

    Science.gov (United States)

    Hetzer, Martin; Chen, Gaojian; Barner-Kowollik, Christopher; Stenzel, Martina H

    2010-02-11

    The synthesis of a new glycomonomer based on mannose, prepared via CuAAC, is reported. The resulting 1,2,3-triazole linkage between mannose and the polymer backbone ensures the formation of highly stable glycopolymers, which will not undergo hydrolysis. The monomer 2'-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-alpha-D-mannopyranoside was polymerized in the presence of a RAFT agent - 3-benzylsulfanylthiocarbonylsulfanyl propionic acid - to yield well-defined polymers with molecular weights up to 51,500 g mol(-1) and a PDI of 1.16. The resulting polymer was employed as a macroRAFT agent in the polymerization of NIPAAm in order to generate thermo-responsive block copolymers, which undergo reversible micelle formation at elevated temperatures. The rapid interaction between the polymers prepared and ConA confirms the high affinity of these structures to proteins. While the linear glycopolymers already undergo a fast complexation with ConA, the reported rates have found to be exceeded by the micellar glycopolymer structure presented in the current contribution.

  9. Preparation, thermo-optic property and simulation of optical switch based on azo benzothiazole polymer

    Science.gov (United States)

    Cao, Zhijuan; Qiu, Fengxian; Wang, Qing; Cao, Guorong; Guan, Yijun; Zhuang, Lin; Xu, Xiaolong; Wang, Jie; Chen, Qian; Yang, Dongya

    2013-04-01

    An azo chromophore molecule 4-[(benzothiazole-2-yl)diazenyl]phenyl-1,3-diamine (BTPD) was prepared with 2-amino benzothiazole and m-phenylenediamine by diazo-coupling reaction. Then, the chromophore molecule BTPD was polymerized with NJ-210 and isophorone diisocyanate (IPDI) to obtain novel azo benzothiazole polymer (BTPU). The structures of BTPD and BTPU were characterized using the Fourier transform infrared, UV-visible spectroscopy, DSC and TGA. The physical properties of the obtained BTPU were investigated. The refractive index ( n) of BTPU was demonstrated at different temperature and wavelength (532, 650 and 850 nm) using attenuated total reflection technique. The transmission loss and dispersion characteristic of BTPU film were investigated using the CCD digital imaging devices and Sellmeyer equation. A Y-branch and 2 × 2 Mach-Zehnder interferometer (MZI) polymeric thermo-optic switches based on the thermo-optic effect of prepared BTPU were proposed and the performance of switches was simulated. The results indicated that the power consumption of the Y-branch thermo-optic switch could be only 0.6 mW. The Y-branch and MZI switching rising and falling times obtained were 8.0 and 1.8 ms.

  10. Influence of surface preparation on fracture load of resin composite-based repairs.

    Science.gov (United States)

    Agustín-Panadero, Rubén; Mateos-Palacios, Rocío; Román-Rodríguez, Juan-Luis; Solá-Ruíz, María-Fernanda; Fons-Font, Antonio

    2015-02-01

    The purpose of the present study is to evaluate the fracture load of composite-based repairs to fractured zirconium oxide (Z) crowns and to ceramic-fused-to-metal (CM) crowns, comparing different mechanical surface preparation methods. A total of 75 crowns were repaired; samples then underwent dynamic loading and thermocycling. Final fracture load values for failure of the repaired crowns were measured and the type of fracture registered. Group I: CM: Surface preparation with a diamond bur + 9.5% Hydrofluoric Acid (HF) etching; Group II): CM: air-particle (Al2O3) + 9.5% HF; Group III: CM: Silica coating (SiO2); Group IV): Z: air-particle (Al2O3) + HF 9.5%; Group V) Z: Silica coating (SiO2). Of the three CM groups, Group I (CM-diamond bur) showed the highest mean failure value, with significant difference in comparison with Group III (CM-silica coating). For the zirconia groups, the highest value was obtained by Group V (silica coating). Key words:Crown, ceramic-fused-to-metal, zirconia, resin-composite, ceramic covering.

  11. Characterization of activated carbon prepared from chlorella-based algal residue.

    Science.gov (United States)

    Chang, Yuan-Ming; Tsai, Wen-Tien; Li, Ming-Hsuan

    2015-05-01

    The chlorella-based microalgal residue (AR) was tested as a novel precursor for preparing activated carbons. A combined carbonization-activation process with flowing N2 and CO2 gases was used to prepare the carbon materials at the activation temperatures of 800-1000 °C and the residence times of 0-30 min in this work. The elemental contents, pore properties and scanning electron microscopy (SEM) observations of the resulting activated carbons have been performed. The results showed that activation temperature may be the most important parameter for determining their pore properties. The maximal Brunauer-Emmett-Teller (BET) surface area and total pore volume of the resulting activated carbon, which was produced at the activation temperature of 950 °C with the residence time of 30 min, were 840 m(2)/g and 0.46 cm(3)/g, respectively. More interestingly, the resulting activated carbons have significant nitrogen contents of 3.6-9.6 wt%, which make them lower carbon contents (i.e., 54.6-68.4 wt%) than those of commercial activated carbons.

  12. Model-based Acceleration of Parameter mapping (MAP) for saturation prepared radially acquired data.

    Science.gov (United States)

    Tran-Gia, Johannes; Stäb, Daniel; Wech, Tobias; Hahn, Dietbert; Köstler, Herbert

    2013-12-01

    A reconstruction technique called Model-based Acceleration of Parameter mapping (MAP) is presented allowing for quantification of longitudinal relaxation time and proton density from radial single-shot measurements after saturation recovery magnetization preparation. Using a mono-exponential model in image space, an iterative fitting algorithm is used to reconstruct one well resolved and consistent image for each of the projections acquired during the saturation recovery relaxation process. The functionality of the algorithm is examined in numerical simulations, phantom experiments, and in-vivo studies. MAP reconstructions of single-shot acquisitions feature the same image quality and resolution as fully sampled reference images in phantom and in-vivo studies. The longitudinal relaxation times obtained from the MAP reconstructions are in very good agreement with the reference values in numerical simulations as well as phantom and in-vivo measurements. Compared to available contrast manipulation techniques, no averaging of projections acquired at different time points of the relaxation process is required in MAP imaging. The proposed technique offers new ways of extracting quantitative information from single-shot measurements acquired after magnetization preparation. The reconstruction simultaneously yields images with high spatiotemporal resolution fully consistent with the acquired data as well as maps of the effective longitudinal relaxation parameter and the relative proton density. Copyright © 2013 Wiley Periodicals, Inc., a Wiley company.

  13. Preparation and chromatographic performance of polymer-based anion exchangers for ion chromatography: A review.

    Science.gov (United States)

    Zatirakha, A V; Smolenkov, A D; Shpigun, O A

    2016-01-21

    In the last decade the developments in the field of ion chromatography (IC) were aimed at increasing the efficiency, sensitivity and rapidity of analysis, as well as on improving separation selectivity. Since selectivity and efficiency to the large extent depend on the surface chemistry of the stationary phase, the development of novel anion exchangers remains one of the priority tasks in modern IC. The exact chemistry of commercially available resins is not known and not many literature data devoted to the procedures of preparing anion exchangers for IC have become available in the last 10-15 years. However, the knowledge about the surface chemistry of anion exchangers can provide understanding of the trends in selectivity and efficiency changes, as well as help with the choice of the stationary phase type suitable for solving a particular analytical task. The current review is devoted to the methods of preparing anion exchangers based on polystyrene-divinylbenzene (PS-DVB) and ethylvinylbenzene-divinylbenzene (EVB-DVB) for IC of inorganic and small organic anions and is aimed at demonstrating the improvement of their performance over the years, which was brought by the development of the new types of stationary phase architecture.

  14. Bio-based polyurethane prepared from Kraft lignin and modified castor oil

    Directory of Open Access Journals (Sweden)

    L. B. Tavares

    2016-11-01

    Full Text Available Current challenges highlight the need for polymer research using renewable natural sources as a substitute for petroleum-based polymers. The use of polyols obtained from renewable sources combined with the reuse of industrial residues such as lignin is an important agent in this process. Different compositions of polyurethane-type materials were prepared by combining technical Kraft lignin (TKL with castor oil (CO or modified castor oil (MCO1 and MCO2 to increase their reactivity towards diphenylmethane diisocyanate (MDI. The results indicate that lignin increases the glass transition temperature, the crosslinking density and improves the ultimate stress especially for those prepared from MCO2 and 30% lignin content from 8.2 MPa (lignin free to 23.5 MPa. Scanning electron microscopy (SEM micrographs of rupture surface after uniaxial tensile tests show ductile-to-brittle transition. The results show the possibility to develop polyurethane-type materials, varying technical grade Kraft lignin content, which cover a wide range of mechanical properties (from large elastic/low Young modulus to brittle/high Young modulus polyurethanes.

  15. Redistribution of mineral elements in wheat grain when applying the complex enzyme preparations based on phytase

    Directory of Open Access Journals (Sweden)

    Elena Kuznetsova

    2016-01-01

    Full Text Available Biogenic minerals play an important role in the whole human nutrition, but they are included in the grain of the phytates that reduces their bioavailability. Whole wheat bread is generally considered a healthy food, but the presence of mineral elements in it is insignificant, because of weak phytate degradation. From all sources of exogenous phytase the most productive are microscopic fungi. To accelerate the process of transition hard mineral elements are mobilized to implement integrated cellulolytic enzyme preparation based on the actions of phytase (producer is Penicillium canescens. Phytase activity was assessed indirectly by the rate of release of phosphate from the substrate. It has been established that the release rate of the phosphoric acid substrate is dependent on the composition of the drug and the enzyme complex is determined by the presence of xylanase. The presented experimental data shows that a cellulase treatment of the grain in conjunction with the β-glucanase or xylanase leading to an increase in phytase activity could be 1.4 - 2.3 times as compared with the individual enzymes. As a result of concerted action of enzymes complex preparation varies topography grain, increase the pore sizes in seed and fruit shells that facilitate the penetration of the enzyme phytase in the aleurone layer to the site of phytin hydrolysis and leads to an increase in phytase activity. In terms of rational parameters of enzymatic hydrolysis, the distribution of mineral elements in the anatomical parts of the grain after processing complex enzyme preparation with the help of X-ray detector EMF miniCup system in a scanning electron microscope JEOL JSM 6390 were investigated. When processing enzyme preparation wheat trend in the distribution of mineral elements, characteristic of grain - the proportion of these elements in the aleurone layer decreases, and in the endosperm increases. Because dietary fiber and phytate found together in the

  16. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  17. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology.

    Science.gov (United States)

    Li, Jian-Feng; Li, Li; Sheen, Jen

    2010-01-14

    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  18. T2 mapping with magnetization-prepared 3D TSE based on a modified BIR-4 T2 preparation.

    Science.gov (United States)

    Weidlich, Dominik; Schlaeger, Sarah; Kooijman, Hendrik; Börnert, Peter; Kirschke, Jan S; Rummeny, Ernst J; Haase, Axel; Karampinos, Dimitrios C

    2017-08-04

    The purpose of this work was to investigate the performance of the modified BIR-4 T2 preparation for T2 mapping and propose a method to remove T2 quantification errors in the presence of large B1 and B0 offsets. The theoretical investigation of the magnetization evolution during the T2 preparation in the presence of B1 and B0 offsets showed deviations from a mono-exponential T2 decay (two parameter fit). A three parameter fit was used to improve T2 accuracy. Furthermore, a two parameter fit with an additional saturation preparation scan was proposed to improve T2 accuracy and precision. These three fitting methods were compared based on simulations, phantom measurements and an in vivo healthy volunteer study of the neck musculature using a 3D TSE readout. The results based upon the pure two parameter fit overestimated T2 in regions with high B0 offsets (up to 40% in phantoms). The three parameter fit T2 values were robust to B0 offsets but with higher standard deviation (up to 40% in simulations). The two parameter fit with the saturation preparation yielded high robustness towards B0 offsets with a noise performance comparable to that of the two parameter fit. In the volunteer study the T2 values obtained by the pure two parameter fit showed a dependence on the field inhomogeneities, whereas the T2 values from the proposed fitting approach were shown to be insensitive to B0 offsets. The proposed method enabled accurate and precise T2 mapping in the presence of large B1 and B0 offsets. Copyright © 2017 John Wiley & Sons, Ltd.

  19. [Acute pancreatitis. Evidence-based practice guidelines, prepared by the Hungarian Pancreatic Study Group].

    Science.gov (United States)

    Hritz, István; Czakó, László; Dubravcsik, Zsolt; Farkas, Gyula; Kelemen, Dezső; Lásztity, Natália; Morvay, Zita; Oláh, Attila; Pap, Ákos; Párniczky, Andrea; Sahin-Tóth, Miklós; Szentkereszti, Zsolt; Szmola, Richárd; Szücs, Ákos; Takács, Tamás; Tiszlavicz, László; Hegyi, Péter

    2015-02-15

    Acute pancreatitis is one of the most common diseases of the gastrointestinal tract associated with significant morbidity and mortality that requires up-to-date and evidence based treatment guidelines. The Hungarian Pancreatic Study Group proposed to prepare evidence based guideline for the medical and surgical management of acute pancreatitis based on the available international guidelines and evidence. The preparatory and consultation task force appointed by the Hungarian Pancreatic Study Group translated and, if it was necessary, complemented and/or modified the international guidelines. All together 42 relevant clinical questions were defined in 11 topics (Diagnosis and etiology, Prognosis, Imaging, Fluid therapy, Intensive care management, Prevention of infectious complications, Nutrition, Biliary interventions, Post-endoscopic retrograde cholangio-pancreatography pancreatitis, Indication, timing and strategy for intervention in necrotizing pancreatitis, Timing of cholecystectomy [or endoscopic sphincterotomy]). Evidence was classified according to the UpToDate® grading system. The draft of the guideline was presented and discussed at the consensus meeting on September 12, 2014. 25 clinical questions with almost total (more than 95%) and 17 clinical questions with strong (more than 70%) agreement were accepted. The present guideline is the first evidence based acute pancreatitis guideline in Hungary. The guideline may provide important help for tuition, everyday practice and for establishment of proper finance of acute pancreatitis. Therefore, the authors believe that these guidelines will widely become as basic reference in Hungary.

  20. Chitosan-based hydrogel for dye removal from aqueous solutions: Optimization of the preparation procedure

    Science.gov (United States)

    Gioiella, Lucia; Altobelli, Rosaria; de Luna, Martina Salzano; Filippone, Giovanni

    2016-05-01

    The efficacy of chitosan-based hydrogels in the removal of dyes from aqueous solutions has been investigated as a function of different parameters. Hydrogels were obtained by gelation of chitosan with a non-toxic gelling agent based on an aqueous basic solution. The preparation procedure has been optimized in terms of chitosan concentration in the starting solution, gelling agent concentration and chitosan-to-gelling agent ratio. The goal is to properly select the material- and process-related parameters in order to optimize the performances of the chitosan-based dye adsorbent. First, the influence of such factors on the gelling process has been studied from a kinetic point of view. Then, the effects on the adsorption capacity and kinetics of the chitosan hydrogels obtained in different conditions have been investigated. A common food dye (Indigo Carmine) has been used for this purpose. Noticeably, although the disk-shaped hydrogels are in the bulk form, their adsorption capacity is comparable to that reported in the literature for films and beads. In addition, the bulk samples can be easily separated from the liquid phase after the adsorption process, which is highly attractive from a practical point of view. Compression tests reveal that the samples do not breakup even after relatively large compressive strains. The obtained results suggest that the fine tuning of the process parameters allows the production of mechanical resistant and highly adsorbing chitosan-based hydrogels.

  1. [Handbook for the preparation of evidence-based documents. Tools derived from scientific knowledge].

    Science.gov (United States)

    Carrión-Camacho, M R; Martínez-Brocca, M A; Paneque-Sánchez-Toscano, I; Valencia-Martín, R; Palomino-García, A; Muñoz-Durán, C; Tamayo-López, M J; González-Eiris-Delgado, C; Otero-Candelera, R; Ortega-Ruiz, F; Sobrino-Márquez, J M; Jiménez-García-Bóveda, R; Fernández-Quero, M; Campos-Pareja, A M

    2013-01-01

    This handbook is intended to be an accessible, easy-to-consult guide to help professionals produce or adapt Evidence-Based Documents. Such documents will help standardize both clinical practice and decision-making, the quality always being monitored in such a way that established references are complied with. Evidence-Based Health Care Committee, a member of "Virgen del Rocío" University Hospital quality structure, proposed the preparation of a handbook to produce Evidence-Based Documents including: a description of products, characteristics, qualities, uses, methodology of production, and application scope of every one of them. The handbook consists of seven Evidence-Based tools, one chapter on critical analysis methodology of scientific literature, one chapter with internet resources, and some appendices with different assessment tools. This Handbook provides general practitioners with a great opportunity to improve quality and as a guideline to standardize clinical healthcare, and managers with a strategy to promote and encourage the development of documents in an effort to reduce clinical practice variability, as well as giving patients the opportunity of taking part in planning their own care. Copyright © 2011 SECA. Published by Elsevier Espana. All rights reserved.

  2. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    Science.gov (United States)

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  3. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available Global mass spectrometry (MS-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC or metal oxide affinity chromatography (MOAC. We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

  4. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  5. Sealing of silorane-based composite in laser-prepared primary teeth: effect of acid etching.

    Science.gov (United States)

    Shafiei, Fereshteh; Memarpour, Mahtab; Fekrazad, Reza

    2014-01-01

    The purpose of this study was to evaluate the influence of prior etching on the sealing of silorane composite restorations in Class V cavity preparations prepared with an Er,Cr:YSGG laser compared to bur preparation in primary teeth. Standard Class V cavity preparations were prepared on the buccal surfaces of 56 extracted primary canines and randomly divided into four groups of 14 teeth each, according to laser or bur preparation and the use of prior acid etching in the preparation. All cavity preparations were restored with silorane adhesive and silorane composite. After water storage and thermocycling, the specimens were placed in 0.5 percent basic fuchsin dye solution. Dye penetration was evaluated with a stereomicroscope. The data were analyzed with nonparametric tests (P=.05). Laser preparation and prior etching in bur-prepared cavity preparation had no significant effect on enamel sealing (P>.05), but significantly increased microleakage at the dentin margin (Pacid etching may be necessary to ensure good marginal sealing when laser preparation is used. However, this step is not required when bur cutting is used in primary teeth.

  6. FragIt: A Tool to Prepare Input Files for Fragment Based Quantum Chemical Calculations

    CERN Document Server

    Steinmann, Casper; Hansen, Anne S; Jensen, Jan H

    2012-01-01

    Near linear scaling fragment based quantum chemical calculations are becoming increasingly popular for treating large systems with high accuracy and is an active field of research. However, it remains difficult to set up these calculations without expert knowledge. To facilitate the use of such methods, software tools need to be available for support, setup and lower the barrier of entry for usage by non-experts. We present a fragmentation methodology and accompanying tools called FragIt to help setup these calculations. It uses the SMARTS language to find chemically appropriate substructures in structures and is used to prepare input files for the fragment molecular orbital method in the GAMESS program package. We present patterns of fragmentation for proteins and polysaccharides, specifically D-galactopyranose for use in cyclodextrins.

  7. Preparation and characterization of polystyrene based Nickel molybdate composite membrane electrical–electrochemical properties

    Directory of Open Access Journals (Sweden)

    Urfi Ishrat

    2016-09-01

    Full Text Available The functional properties of the polystyrene based Nickel Molybdate composite membrane prepared by applying 70 MPa pressure are described. The fabricated membrane was characterized by using Fourier Transform Infrared, X-ray diffraction, particle size analyzer and Scanning electron microscopy technique and has been investigated for its functional, diffusive, electrochemical and electrical properties. The impedance data of membrane having capacitive and resistive components are plotted, which show the sequence of semicircles representing an electrical phenomenon due to grain material, grain boundary and interfacial phenomenon. The diffusion of electrolytes was determined by the TMS method revealing dependence of membrane potential on the charge on the membrane matrix, charge and size of permeating ions. The membrane determined the activity of cations with good accuracy in the higher concentration range and shows a great selectivity for K+. Other electrochemical properties like transport number have been discussed its selectivity.

  8. PREPARATION AND CHARACTERIZATION OF ION EXCHANGE MEMBRANES BASED ON POLYVINYLIDENE FLUORIDE

    Institute of Scientific and Technical Information of China (English)

    Bo Tian; Chuan-wei Yan; Fu-hui Wang

    2004-01-01

    A new ion exchange membrane based on polyvinylidene fluoride (PVDF) and sulfonated poly(styrenedivinylbenzene) was prepared by in-situ polymerization. The incorporation of sulfonic groups into the polyvinylidene fluoride composite membrane was confirmed by infrared spectroscopy (IR), ion exchange capacity (IEC) and energy dispersive X-ray analysis (EDAX). Area resistance, IEC and water uptake of the treated membrane were evaluated. When area resistance in NaCl aqueous solution at 25℃, IEC is as high as 2.43 millimoles per gram of the wet membrane. The hydrophilicity of PVDF membrane is also significantly improved after treatment. When 60% of crosslinked membrane was sulfonated at 80℃ for 6 h, water uptake of the treated membrane can attain 64.7%.

  9. Chalcogenide-based thin film sensors prepared by pulsed laser deposition technique

    Science.gov (United States)

    Schubert, J.; Schöning, M. J.; Schmidt, C.; Siegert, M.; Mesters, St.; Zander, W.; Kordos, P.; Lüth, H.; Legin, A.; Mourzina, Yu. G.; Seleznev, B.; Vlasov, Yu. G.

    One advantage of the pulsed laser deposition (PLD) method is the stoichiometric transfer of multi-component target material to a given substrate. This advantage of the PLD determined the choice to prepare chalco-genide-based thin films with an off-axis geometry PLD. Ag-As-S and Cu-Ag-As-Se-Tetargets were used to deposit thin films on Si substrates for an application as a heavy metal sensing device. The films were characterized by means of Rutherford backscattering spectrometry (RBS), transmission electron microscopy (TEM), and electrochemical measurements. The same stoichiometry of the films and the targets was confirmed by RBS measurements. We observed a good long-term stability of more than 60 days and a nearly Nernstian sensitivity towards Pb and Cu, which is comparable to bulk sensors.

  10. Preparation and magnetic properties of phthalocyanine-based carbon materials containing transition metals

    Science.gov (United States)

    Honda, Z.; Sato, S.; Hagiwara, M.; Kida, T.; Sakai, M.; Fukuda, T.; Kamata, N.

    2016-07-01

    A simple method for the preparation of bulk quantities of magnetic carbon materials, which contain uniformly dispersed transition metals (M = Fe, Co, Ni, and Cu) as the magnetic components, is presented. By using highly chlorinated metal phthalocyanine as the building block and potassium as the coupling reagent, phthalocyanine-based carbon materials (PBCMs) containing transition metals were obtained. Our experiments demonstrate the structure of these PBCMs consists of transition metals embedded in graphitic carbon that includes a square planar MN4 magnetic core and the Fe and Co-PBCM possess spontaneous magnetization at room temperature. In addition, carbon-coated transition metal particles were obtained by the Wurtz-type reaction with excess amount of potassium coupling agent. The large transition metal surface area and magnetization of these M-PBCMs are useful for spintronic and catalytic applications.

  11. Carbon-based Composite Electrodes: Preparation, Characterization and Application in Electroanalysis

    Directory of Open Access Journals (Sweden)

    Joop Schoonman

    2007-11-01

    Full Text Available Electrodes based on carbon, i.e., expanded graphite (20%, wt.-epoxy composite(20EG-Epoxy and expanded graphite (20%, wt.-polystyrene composite (20EG-PS havebeen prepared, characterized using scanning electron microscopy (SEM and cyclicvoltammetry (CV, and tested as anodic sensors. The electrodes exhibited good mechanicalresistance and low electrical resistances. Scan rate dependent cyclic voltammetry responsesat 20EG-Epoxy and 20EG-PS composite electrodes, which were exemplified for thiourea(TU, a toxic sulphur organic compound selected as testing target analyte in 0.1 M Na2SO4 supporting electrolyte, were investigated. The obtained voltammetric data were inaccordance with those for a random array of microelectrodes. The voltammetric andchronoamperometric detection results of TU in tap water samples, without a supplementaryaddition of supporting electrolyte, at 20EG-Epoxy electrode proved its use for directanalysis of environmental samples.

  12. Optical Characterization of SERS Substrates Based on Porous Au Films Prepared by Pulsed Laser Deposition

    Directory of Open Access Journals (Sweden)

    V. V. Strelchuk

    2015-01-01

    Full Text Available The SERS (surface enhanced Raman spectroscopy substrates based on nanocomposite porous films with gold nanoparticles (Au NPs arrays were formed using the method of the pulsed laser deposition from the back low-energy flux of erosion torch particles on the glass substrate fixed at the target plain. The dependencies of porosity, and morphology of the surface of the film regions located near and far from the torch axis on the laser ablation regime, laser pulses energy density, their number, and argon pressure in the vacuum chamber, were ascertained. The Au NPs arrays with the controllable extinction spectra caused by the local surface plasmon resonance were prepared. The possibility of the formation of SERS substrates for the detection of the Rhodamine 6G molecules with the concentration 10−10 Mol/L with the enhancement factor 4·107 was shown.

  13. Laser-based surface preparation of composite laminates leads to improved electrodes for electrical measurements

    KAUST Repository

    Almuhammadi, Khaled

    2015-10-19

    Electrical impedance tomography (EIT) is a low-cost, fast and effective structural health monitoring technique that can be used on carbon fiber reinforced polymers (CFRP). Electrodes are a key component of any EIT system and as such they should feature low resistivity as well as high robustness and reproducibility. Surface preparation is required prior to bonding of electrodes. Currently this task is mostly carried out by traditional sanding. However this is a time consuming procedure which can also induce damage to surface fibers and lead to spurious electrode properties. Here we propose an alternative processing technique based on the use of pulsed laser irradiation. The processing parameters that result in selective removal of the electrically insulating resin with minimum surface fiber damage are identified. A quantitative analysis of the electrical contact resistance is presented and the results are compared with those obtained using sanding.

  14. A novel colloid probe preparation method based on chemical etching technique

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Several fundamental problems in hydrophobic force measurements using atomic force microscope (AFM) are discussed in this paper. A novel method for colloid probe preparation based on chemical etching technology is proposed, which is specially fit for the unique demands of hydrophobic force measurements by AFM. The features of three different approaches for determining spring constants of rectangular cantilevers, including geometric dimension, Cleveland and Sader methods are compared. The influences of the sizes of the colloids on the measurements of the hydrophobic force curves are investigated. Our experimental results showed that by selecting colloid probe with proper spring constant and tip size, the hydrophobic force and the complete hydrophobic interaction force curve can be measured by using AFM.

  15. Agarose- and alginate-based biopolymers for sample preparation: Excellent green extraction tools for this century.

    Science.gov (United States)

    Sanagi, Mohd Marsin; Loh, Saw Hong; Wan Ibrahim, Wan Nazihah; Pourmand, Neda; Salisu, Ahmed; Wan Ibrahim, Wan Aini; Ali, Imran

    2016-03-01

    Recently, there has been considerable interest in the use of miniaturized sample preparation techniques before the chromatographic monitoring of the analytes in unknown complex compositions. The use of biopolymer-based sorbents in solid-phase microextraction techniques has achieved a good reputation. A great variety of polysaccharides can be extracted from marine plants or microorganisms. Seaweeds are the major sources of polysaccharides such as alginate, agar, agarose, as well as carrageenans. Agarose and alginate (green biopolymers) have been manipulated for different microextraction approaches. The present review is focused on the classification of biopolymer and their applications in multidisciplinary research. Besides, efforts have been made to discuss the state-of-the-art of the new microextraction techniques that utilize commercial biopolymer interfaces such as agarose in liquid-phase microextraction and solid-phase microextraction.

  16. ANTIBACTERIAL THERMOPLASTIC STARCH-CHITOSAN BASED MATERIALS PREPARED BY MELT-MIXING

    Directory of Open Access Journals (Sweden)

    Liliana C. Tomé,

    2012-06-01

    Full Text Available Antibacterial thermoplastic starch-chitosan based materials were successfully prepared by melt-mixing. The effect of chitosan modification (quaternization and grafting of long carbon chains on the properties of the materials was also studied. All the ensuing materials were characterized in terms of thermal stability, crystalline structure, mechanical performance, and antibacterial activity. The incorporation of chitosan and its derivatives into the thermoplastic starch matrices resulted in an increment in tensile strengths (up to 85%; however for higher chitosan contents (5 and 7.5 wt.%, a decrease on the Young’s modulus (around 50% was observed, together with an increment in the elongation at break, which can be attributed to the prevention of the retrogradation process. Finally, the thermoplastic starch materials incorporated with 7.5 wt% of the unmodified chitosan and of the water-soluble chitosan derivative had partial (about 20% reduction of CFU and total bactericidal effect against S. aureus.Keywords

  17. PREPARATION AND CHARACTERIZATION OF ACETIC ACID LIGNIN-BASED EPOXY BLENDS

    Directory of Open Access Journals (Sweden)

    Fangeng Chen

    2012-05-01

    Full Text Available Lignin-based epoxy resin (LER was prepared from phenolated lignin (PL and epichlorohydrin (ECH in the presence of sodium hydroxide. The eucalyptus acetic acid lignin (AAL was first reacted with phenol in the presence of sulfuric acid to obtain PL. Then, PL was reacted with ECH in aqueous sodium hydroxide to obtain LER. LER was mixed with diglycidyl ether of bisphenol A (E-44 and then cured with triethylenetetramine (TETA. The initial thermal degradation temperature (Td of the cured epoxy blends decreased with the increase in LER content. The residue ratio at 500 °C of the cured epoxy blends (R500, however, increased with the LER content. The maximum adhesive shear strength of the cured epoxy blends was obtained at 20 wt% of LER. The water absorption of epoxy blends increased with increasing the content of LER. SEM photos showed that increasing the content of LER increased inhomogeneity and porosity of epoxy blends.

  18. Ground-based activities in preparation of SELENE ISS experiment on self-rewetting fluids

    Science.gov (United States)

    Savino, R.; Abe, Y.; Castagnolo, D.; Celata, G. P.; Kabov, O.; Kawaji, M.; Sato, M.; Tanaka, K.; Thome, J. R.; Van Vaerenbergh, S.

    2011-12-01

    SELENE (SELf rewetting fluids for thermal ENErgy management) is a microgravity experiment proposed to the European Space Agency (ESA) in response to the Announcement of Opportunities for Physical Sciences. Main objectives of the microgravity research onboard ISS include the quantitative investigation of heat transfer performances of "self-rewetting fluids" and "nano self-rewetting fluids" in model heat pipes and validation of adequate theoretical and numerical modelling able to predict their behaviour in microgravity conditions. This article summarizes the results of ground-based research activities in preparation of the microgravity experiments. They include: 1) thermophysical properties measurements; 2) study of thermo-soluto-capillary effects in micro-channels; 3) numerical modelling; 4) thermal and concentration distribution measurements with optical (e.g. interferometric) and intrusive techniques; 5) surface tension-driven effects and thermal performances test on different capillary structures and heat pipes; 6) breadboards development and support to definition of scientific requirements.

  19. Preparation of Bio-Based Polyamide Elastomer by Using Green Plasticizers

    Directory of Open Access Journals (Sweden)

    Miaomiao He

    2016-07-01

    Full Text Available The purpose of this work was to study the effects of three green plasticizers H2O, glycerol, and soybean oil, on the properties of bio-based BDIS polyamides. The BDIS polyamides synthesized from the following biomass monomers: 1,4-butanediamine (BD, 1,10-decanediamine (DD, itaconic acid (IA, and sebacic acid (SA. It is interesting to note that the amorphous BDIS (IA-80% polyamide was changed from the glassy state to the rubbery state after water soaking and induced crystallization at the same time. The H2O-plasticized non-crosslinked BDIS (IA-80% polyamides can be very useful for the preparation of physical water gel. The glycerol- and soybean oil-plasticized BDIS (IA-80% polyamides displayed excellent toughness. The plasticized BDIS (IA-80% polyamides were characterized by Fouriertransform infrared spectroscopy (FTIR, differential scanning calorimetry (DSC, thermogravimetric analysis (TGA, mechanical testing, and X-ray diffraction (XRD.

  20. Sorbents based on crown ethers: preparation and application for the sorption of strontium

    Science.gov (United States)

    Bezhin, N. A.; Dovhyi, I. I.

    2015-12-01

    The key approaches to the synthesis of crown ether-based sorbents, including immobilization both with and without covalent bonding, are reviewed. Examples of sorbent preparation using anodic oxidation, chemical modification of polymers, polycondensation reactions, chemical modification of inorganic supports and radiochemical synthesis for covalent bonding of crown ether moieties are considered. Immobilization methods without covalent bonding including support synthesis in the presence of crown ethers, impregnation of supports with a crown ether solution and the use of powdered crown ether as a sorbent are presented. The applications of sorbents for selective removal of strontium from solutions of radioactive waste and spent nuclear fuel, for radiochemical analysis (determination of strontium in water, soil and biological materials) and for separation of strontium and yttrium isotopes are discussed. The bibliography includes 114 references.