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Sample records for direct recombination system

  1. Extended recombinant bacterial ghost system.

    Science.gov (United States)

    Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

    1999-08-20

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri

  2. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

  3. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells.

    Science.gov (United States)

    Choulika, A; Perrin, A; Dujon, B; Nicolas, J F

    1994-11-01

    Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.

  4. Direct facile screening of recombinant DNA vector constructs.

    Science.gov (United States)

    Winnard, Paul T; Challa, Rushi; Bhujwalla, Zaver M; Raman, Venu

    2014-04-01

    Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.

  5. Recombinant expression systems for allergen vaccines.

    Science.gov (United States)

    Singh, Mohan B; Bhalla, Prem L

    2006-01-01

    Allergen immunotherapy of future is likely to be based on allergy vaccines that contain engineered allergens modified to abolish or substantially reduce their IgE-binding activity in order to remove the risk of unwanted anaphylactic responses. The development of efficient systems for the production of recombinant allergens in sufficient quantities is requirement for establishing use of engineered allergens as components of allergy vaccines. This review outlines relative advantages and disadvantages of various heterologous systems for production of recombinant allergens. Microbial systems are most convenient and cost effective platforms for the production of recombinant allergens. However, lack of post-translational processing implies that some allergens have to be expressed in eukaryotic systems for proper folding and post-translational modifications such as glycosylation. Yeast systems can yield high levels of recombinant allergens but often are associated with hyper- glycosylation problems. Mammalian cell culture systems offer suitable post -translational modifications but are nearly hundred fold more expensive than microbial systems. The use of plants as bio-factories for production of recombinant allergens is emerging as a very attractive option as plants-based production system offer several advantages over other expression systems such as post translational processing of proteins, low production costs, scale up ability and enhanced safety due to absence of animal or human pathogens.

  6. The homologous recombination system of Ustilago maydis.

    Science.gov (United States)

    Holloman, William K; Schirawski, Jan; Holliday, Robin

    2008-08-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

  7. Recombinant human-like collagen directed growth of hydroxyapatite nanocrystals

    Science.gov (United States)

    Zhai, Y.; Cui, F. Z.

    2006-05-01

    Bones are biocomposites with hierarchical structure that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Type I collagen proteins, acidic extracellular matrix proteins, play a critical role in mineral formation and many researches on artificial bones have been made inspired by nature using type I collagen derived from animal tissues. Here we report that recombinant human-like type I collagen, an acidic protein, can direct growth of hydroxyapatite (HA) nanocrystals in vitro in the form of self-assembly of nano-fibrils of mineralized collagen resembling extracellular matrix. The mineralized collagen fibrils aligned parallel to each other to form mineralized collagen fibers. HA nanocrystals grew on the surface of these collagen fibrils with the c-axis of nanocrystals of HA orienting along the longitudinal axis of the fibrils. These artificial analogs of bone have a potential clinical application in bone repair.

  8. Minimum length of direct repeat sequences required for efficient homologous recombination induced by zinc finger nuclease in yeast.

    Science.gov (United States)

    Ren, ChongHua; Yan, Qiang; Zhang, ZhiYing

    2014-10-01

    Zinc finger nuclease (ZFN) technology is a powerful molecular tool for targeted genome modifications and genetic engineering. However, screening for specific ZFs and validation of ZFN activity are labor intensive and time consuming. We previously designed a yeast-based ZFN screening and validation system by inserting a ZFN binding site flanked by a 164 bp direct repeat sequence into the middle of a Gal4 transcription factor, disrupting the open reading frame of the yeast Gal4 gene. Expression of the ZFN causes a double stranded break at its binding site, which promotes the cellular DNA repair system to restore expression of a functional Gal transcriptional factor via homologous recombination. Expression of Gal4 transcription factor leads to activation of three reporter genes in an AH109 yeast two-hybrid strain. However, the 164 bp direct repeat appears to generate spontaneous homologous recombination frequently, resulting in many false positive ZFNs. To overcome this, a series of DNA fragments of various lengths from 10 to 150 bp with 10 bp increase each and 164 bp direct repeats flanking the ZFN binding site were designed and constructed. The results demonstrated that the minimum length required for ZFN-induced homologous recombination was 30 bp, which almost eliminated spontaneous recombination. Using the 30 bp direct repeat sequence, ZFN could efficiently induce homologous recombination, while false positive ZFNs resulting from spontaneous homologous recombination were minimized. Thus, this study provided a simple, fast and sensitive ZFN screening and activity validation system in yeast.

  9. CRISPR-directed mitotic recombination enables genetic mapping without crosses.

    Science.gov (United States)

    Sadhu, Meru J; Bloom, Joshua S; Day, Laura; Kruglyak, Leonid

    2016-05-27

    Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR (clustered, regularly interspaced, short palindromic repeats) to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences.

  10. A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

    Science.gov (United States)

    Alejska, Magdalena; Figlerowicz, Magdalena; Malinowska, Nelli; Urbanowicz, Anna; Figlerowicz, Marek

    2005-07-07

    At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

  11. The homologous recombination system of Ustilago maydis

    OpenAIRE

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we...

  12. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

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    C.P. Nunes

    2000-12-01

    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  13. Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes.

    Science.gov (United States)

    Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan; Frock, Richard L; Du, Zhou; Meyers, Robin M; Meng, Fei-long; Schatz, David G; Alt, Frederick W

    2015-11-05

    RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Recombinant expression of placental growth factor in baculovirus expression system

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    Narges Arbabi

    2016-12-01

    Full Text Available Background: Angiogenesis or formation of new blood vessels is the most important factor in physiological and pathological conditions. Human Placental growth factor (hPLGF protein in is one of the most important proteins which stimulate angiogenesis. Baculovirus expression system has been used successfully to over express eukaryotic proteins in insect cells. This system uses a very strong viral promoter, AcNPV polyhedrin, for high level of protein expression. Methods: hPLGF gene cloned in pFastBac-HT vector and transformed in DH10Bac.The recombinant bacmid was extracted and used in SF9 insect cells and transfected by cellfectin method. Target protein expression was confirmed with Western blot. Results: Transferring of the recombinant vector into Bacmid was successful and the PLGF gene sequence was confirmed. PLGF and recombinant protein expression by Western blotting was confirmed. Conclusion: Baculovirus protein expression system expresses PLGF strongly and recombinant protein can be used in different tests.

  15. DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

    Science.gov (United States)

    Morrical, Scott W.

    2015-01-01

    The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species. PMID:25646379

  16. Two portable recombination enhancers direct donor choice in fission yeast heterochromatin

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Holm, Lærke Rebekka; Hansen, Janne Verhein

    2013-01-01

    of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were...... transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps...

  17. A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.

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    Seong-Bin Kim

    Full Text Available Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega. To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

  18. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.; Haitjema, Charles H.

    2017-02-21

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  19. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  20. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Science.gov (United States)

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  1. Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins.

    Science.gov (United States)

    Kurtzman, A L; Govindarajan, S; Vahle, K; Jones, J T; Heinrichs, V; Patten, P A

    2001-08-01

    Recent developments in directed evolution technologies combined with innovations in robotics and screening methods have revolutionized protein engineering. These methods are being applied broadly to many fields of biotechnology, including chemical engineering, agriculture and human therapeutics. More specifically, DNA shuffling and other methods of genetic recombination and mutation have resulted in the improvement of proteins of therapeutic interest. Optimizing genetic diversity and fitness through iterative directed evolution will accelerate improvements in engineered protein therapeutics.

  2. Two portable recombination enhancers direct donor choice in fission yeast heterochromatin

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Holm, Lærke Rebekka; Hansen, Janne Verhein;

    2013-01-01

    Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable...... of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were...

  3. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III.

    Science.gov (United States)

    Richard-Fogal, Cynthia L; San Francisco, Brian; Frawley, Elaine R; Kranz, Robert G

    2012-06-01

    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  4. The Fission Yeast FANCM Ortholog Directs Non-Crossover Recombination During Meiosis

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    Lorenz, Alexander; Osman, Fekret; Sun, Weili; Nandi, Saikat; Steinacher, Roland; Whitby, Matthew C.

    2012-01-01

    The formation of healthy gametes depends on programmed DNA double strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favour of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to Humans suggests that this interplay may be a general feature of meiotic recombination. PMID:22723423

  5. Making recombinant proteins in animals--different systems, different applications.

    Science.gov (United States)

    Dyck, Michael K; Lacroix, Dan; Pothier, François; Sirard, Marc-André

    2003-09-01

    Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.

  6. Xer Site Specific Recombination: Double and Single Recombinase Systems

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    Castillo, Fabio; Benmohamed, Amal; Szatmari, George

    2017-01-01

    The separation and segregation of newly replicated bacterial chromosomes can be constrained by the formation of circular chromosome dimers caused by crossing over during homologous recombination events. In Escherichia coli and most bacteria, dimers are resolved to monomers by site-specific recombination, a process performed by two Chromosomally Encoded tyrosine Recombinases (XerC and XerD). XerCD recombinases act at a 28 bp recombination site dif, which is located at the replication terminus region of the chromosome. The septal protein FtsK controls the initiation of the dimer resolution reaction, so that recombination occurs at the right time (immediately prior to cell division) and at the right place (cell division septum). XerCD and FtsK have been detected in nearly all sequenced eubacterial genomes including Proteobacteria, Archaea, and Firmicutes. However, in Streptococci and Lactococci, an alternative system has been found, composed of a single recombinase (XerS) genetically linked to an atypical 31 bp recombination site (difSL). A similar recombination system has also been found in 𝜀-proteobacteria such as Campylobacter and Helicobacter, where a single recombinase (XerH) acts at a resolution site called difH. Most Archaea contain a recombinase called XerA that acts on a highly conserved 28 bp sequence dif, which appears to act independently of FtsK. Additionally, several mobile elements have been found to exploit the dif/Xer system to integrate their genomes into the host chromosome in Vibrio cholerae, Neisseria gonorrhoeae, and Enterobacter cloacae. This review highlights the versatility of dif/Xer recombinase systems in prokaryotes and summarizes our current understanding of homologs of dif/Xer machineries. PMID:28373867

  7. Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

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    Jung Kirsten

    2008-04-01

    Full Text Available Abstract Background Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Results Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Conclusion Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

  8. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

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    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  9. Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease.

    Science.gov (United States)

    Bresson, D; Chardès, T; Chapal, N; Bès, C; Cerutti, M; Devauchelle, G; Bouanani, M; Mani, J C; Péraldi-Roux, S

    2001-01-01

    Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.

  10. Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster

    Science.gov (United States)

    Nagy, Ervin D.; Bennetzen, Jeffrey L.

    2008-01-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site–leucine-rich repeat (NBS–LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the ∼3.7-kb NBS–LRR genes. Because any unequal recombination located within the flanking NBS–LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS–LRR loci. PMID:18719093

  11. Metabolomic analysis of riboswitch containing E. coli recombinant expression system.

    Science.gov (United States)

    Muhamadali, Howbeer; Xu, Yun; Morra, Rosa; Trivedi, Drupad K; Rattray, Nicholas J W; Dixon, Neil; Goodacre, Royston

    2016-02-01

    In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional-translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.

  12. Recombinant AAV delivery to the central nervous system.

    Science.gov (United States)

    Bockstael, Olivier; Foust, Kevin D; Kaspar, Brian; Tenenbaum, Liliane

    2011-01-01

    Recombinant AAV-mediated gene delivery to the CNS can be performed either by direct delivery at the target site or from the periphery, using intramuscular injections and retrograde transport along motor neuron projections or intravenous injections and blood-brain barrier crossing.In this chapter, we describe: 1. Methods for recombinant virus administration, including stereotactic surgery, intramuscular, and intravenous administration. 2. Methods to evaluate the number and biodistribution of brain and spinal cord cells expressing the transgene by immunohistochemisty as well as the amount of transgene product by ELISA in the target region. 3. Methods to characterize the cellular specificity of transgene expression by double immunofluorescence. 4. Methods to quantify the amounts of viral DNA as well as of transgene mRNA by quantitative PCR and RT-PCR, respectively.

  13. Directional dark matter by polar angle direct detection and application of columnar recombination

    CERN Document Server

    Li, Jin

    2015-01-01

    We report a systematic study on the directional sensitivity of a direct dark matter detector that detects the polar angle of a recoiling nucleus. A WIMP-mass independent method is used to obtain the sensitivity of a general detector in an isothermal galactic dark matter halo. By using two-dimensional distributions of energy and polar angle, a detector without head-tail information with 6.3 times the statistics is found to achieve the same performance level as a full three-dimensional tracking dark matter detector. Optimum operation orientations are obtained for various experimental configurations, with detectors that are space- or Earth-fixed, have head-tail capability or not, and use energy information or not. Earth-fixed detectors are found to have best sensitivity when the polar axis is oriented at a 45 degree angle from the Earth's pole. The WIMP-mass dependence of the performance of a detector with a 3 keV energy threshold that uses xenon as target material is reported. We apply realistic experimental re...

  14. Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

    Science.gov (United States)

    Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

    2006-04-28

    Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

  15. Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

    Science.gov (United States)

    Albariño, César G; Uebelhoer, Luke S; Vincent, Joel P; Khristova, Marina L; Chakrabarti, Ayan K; McElroy, Anita; Nichol, Stuart T; Towner, Jonathan S

    2013-11-01

    Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5' and 3' RACE, followed by the cloning of viral proteins NP, VP35, VP30 and L into expression plasmids. Novel conditions were then developed to efficiently replicate virus mini-genomes followed by the construction of full-length genomic clones from which recombinant wild type and GFP-containing MARVs were rescued. Surprisingly, when these recombinant MARVs were propagated in primary human macrophages, a dramatic difference was found in their ability to grow and to elicit anti-viral cytokine responses.

  16. Photoelectrochemical based direct conversion systems

    Energy Technology Data Exchange (ETDEWEB)

    Kocha, S.; Arent, D.; Peterson, M. [National Renewable Energy Lab., Golden, CO (United States)] [and others

    1995-09-01

    The goal of this research is to develop a stable, cost effective, photoelectrochemical based system that will split water upon illumination, producing hydrogen and oxygen directly, using sunlight as the only energy input. This type of direct conversion system combines a photovoltaic material and an electrolyzer into a single monolithic device. We report on our studies of two multifunction multiphoton photoelectrochemical devices, one based on the ternary semiconductor gallium indium phosphide, (GaInP{sub 2}), and the other one based on amorphous silicon carbide. We also report on our studies of the solid state surface treatment of GaInP{sub 2} as well as our continuing effort to develop synthetic techniques for the attachment of transition metal complexes to the surface of semiconductor electrodes. All our surface studies are directed at controlling the interface energetics and forming stable catalytic surfaces.

  17. Dimer-atom-atom recombination in the universal four-boson system

    OpenAIRE

    Deltuva, A.

    2012-01-01

    The dimer-atom-atom recombination process in the system of four identical bosons with resonant interactions is studied. The description uses the exact Alt, Grassberger and Sandhas equations for the four-particle transition operators that are solved in the momentum-space framework. The dimer-dimer and atom-trimer channel contributions to the ultracold dimer-atom-atom recombination rate are calculated. The dimer-atom-atom recombination rate greatly exceeds the three-atom recombination rate.

  18. Cloning of Rabbit HPRT Gene Using the Recombineering System

    Institute of Scientific and Technical Information of China (English)

    Jianjun SHI; Donghui CAI; Xuejin CHEN; Huizheng SHENG

    2007-01-01

    Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

  19. Annealing effects on recombinative activity of nickel at direct silicon bonded interface

    Energy Technology Data Exchange (ETDEWEB)

    Kojima, Takuto, E-mail: tkojima@toyota-ti.ac.jp; Ohshita, Yoshio; Yamaguchi, Masafumi [Toyota Technological Institute, 2-12-1 Hisakata, Tempaku-ku, Nagoya, 468-8511 (Japan)

    2015-09-15

    By performing capacitance transient analyses, the recombination activity at a (110)/(100) direct silicon bonded (DSB) interface contaminated with nickel diffused at different temperatures, as a model of grain boundaries in multicrystalline silicon, was studied. The trap level depth from the valence band, trap density of states, and hole capture cross section peaked at an annealing temperature of 300 °C. At temperatures ⩾400 °C, the hole capture cross section increased with temperature, but the density of states remained unchanged. Further, synchrotron-based X-ray analyses, microprobe X-ray fluorescence (μ-XRF), and X-ray absorption near edge structure (XANES) analyses were performed. The analysis results indicated that the chemical phase after the sample was annealed at 200 °C was a mixture of NiO and NiSi{sub 2}.

  20. Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.

  1. Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Kol, Stefan; Andersen, Mikael Rørdam

    2016-01-01

    for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding...... the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER-directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER-directed mRNA, with no cytoplasmic translation of ER-directed recombinant proteins observed....... These observations indicate that the recombinant mRNA, encoding ER-directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also...

  2. Genome modifications and cloning using a conjugally transferable recombineering system

    Directory of Open Access Journals (Sweden)

    Mohammad J Hossain

    2015-12-01

    Full Text Available The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.

  3. Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

    Science.gov (United States)

    Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing

    2015-03-04

    Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

  4. Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae.

    Science.gov (United States)

    Calado, C R; Hamilton, G E; Cabral, J M; Fonseca, L P; Lyddiatt, A

    2001-01-01

    The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.

  5. 16 CFR 1000.13 - Directives System.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Directives System. 1000.13 Section 1000.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION GENERAL COMMISSION ORGANIZATION AND FUNCTIONS § 1000.13 Directives System. The Commission maintains a Directives System which contains delegations...

  6. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

    Directory of Open Access Journals (Sweden)

    Ali Nazari Shirvan

    2016-06-01

    Full Text Available Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

  7. Direct evidence of recombination in the recA gene of Aeromonas bestiarum.

    Science.gov (United States)

    Sanglas, Ariadna; Albarral, Vicenta; Farfán, Maribel; Lorén, J Gaspar; Fusté, M Carmen

    2016-03-01

    Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.

  8. Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

    Science.gov (United States)

    Oliveira, Pedro H; Lemos, Francisco; Monteiro, Gabriel A; Prazeres, Duarte M F

    2008-09-01

    In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

  9. Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

    Science.gov (United States)

    Butler, James R; Santos, Rafael M N; Martens, Gregory R; Ladowski, Joseph M; Wang, Zheng-Yu; Li, Ping; Tector, Matthew; Tector, A Joseph

    2017-05-15

    Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic

  10. Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

    Directory of Open Access Journals (Sweden)

    Jofre Juan

    2006-09-01

    Full Text Available Abstract Background The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.

  11. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J

    2006-12-01

    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  12. Spectral dependence of direct and trap-mediated recombination processes in lead halide perovskites using time resolved microwave conductivity.

    Science.gov (United States)

    Guse, Joanna A; Soufiani, Arman M; Jiang, Liangcong; Kim, Jincheol; Cheng, Yi-Bing; Schmidt, Timothy W; Ho-Baillie, Anita; McCamey, Dane R

    2016-04-28

    Elucidating the decay mechanisms of photoexcited charge carriers is key to improving the efficiency of solar cells based on organo-lead halide perovskites. Here we investigate the spectral dependence (via above-, inter- and sub-bandgap optical excitations) of direct and trap-mediated decay processes in CH3NH3PbI3 using time resolved microwave conductivity (TRMC). We find that the total end-of-pulse mobility is excitation wavelength dependent - the mobility is maximized (172 cm(2) V(-1) s(-1)) when charge carriers are excited by near bandgap light (780 nm) in the low charge carrier density regime (10(9) photons per cm(2)), and is lower for above- and sub-bandgap excitations. Direct recombination is found to occur on the 100-400 ns timescale across excitation wavelengths near and above the bandgap, whereas indirect recombination processes displayed distinct behaviour following above- and sub-bandgap excitations, suggesting the influence of different trap distributions on recombination dynamics.

  13. Recombinant Functional Human Lactoferrin Expressed in Baculovirus System

    Institute of Scientific and Technical Information of China (English)

    Tao LIU; Yao-Zhou ZHANG; Xiang-Fu WU

    2006-01-01

    Human lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. In this study, we amplified hLfcDNA by reverse transcription-polymerase chain reaction from normal human mammary gland.The nucleotide sequence of the hLf was identical to the known hLf. We constructed a recombinant virus,vBm-hLf, harboring the hLfgene and exploited the BmN cells as host to produce recombinant human lactoferrin(rhLf). It was found that a recombinant protein with a molecular mass of approximately 78 kDa was expressed.Approximately 13.5 μg rhLf was purified from 1-2× 105 BmN cells infected by vBm-hLf and the rhLf proved to be biologically active. This method established in our study will pave the way for efficient production of rhLf for further application of this protein in the future.

  14. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.

    Science.gov (United States)

    Fu, Jun; Bian, Xiaoying; Hu, Shengbaio; Wang, Hailong; Huang, Fan; Seibert, Philipp M; Plaza, Alberto; Xia, Liqiu; Müller, Rolf; Stewart, A Francis; Zhang, Youming

    2012-05-01

    Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

  15. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

    Science.gov (United States)

    Onouchi, H; Yokoi, K; Machida, C; Matsuzaki, H; Oshima, Y; Matsuoka, K; Nakamura, K; Machida, Y

    1991-12-11

    Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

  16. Micro-physics simulations of columnar recombination along nuclear recoil tracks in high-pressure Xe gas for directional dark matter searches

    CERN Document Server

    Nakajima, Y; Long, M; Nygren, D; Oliveira, C; Renner, J

    2015-01-01

    Directional sensitivity is one of the most important aspects of WIMP dark matter searches. Yet, making the direction of nuclear recoil visible with large target masses is a challenge. To achieve this, we are exploring a new method of detecting directions of short nuclear recoil tracks in high-pressure Xe gas, down to a few micron long, by utilizing columnar recombination. Columnar recombination changes the scintillation and ionization yields depending on the angle between a track and the electric field direction. In order to realize this, efficient cooling of electrons is essential. Trimethylamine(TMA) is one of the candidate additives to gaseous Xe in order to enhance the effect, not only by efficiently cooling the electrons, but also by increasing the amount of columnar recombination by Penning transfer. We performed a detailed simulation of ionization electrons transport created by nuclear recoils in a Xe + TMA gas mixture, and evaluated the size of the columnar recombination signal. The results show that ...

  17. A selectable and excisable marker system for the rapid creation of recombinant poxviruses.

    Directory of Open Access Journals (Sweden)

    Julia L Rintoul

    Full Text Available BACKGROUND: Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. CONCLUSION/SIGNIFICANCE: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.

  18. Planar antenna system for direction finding

    Science.gov (United States)

    Mardale, Iulia-Cezara; Cocias, Gabriela; Dumitrascu, Ana; Tamas, Razvan; Berescu, Serban

    2015-02-01

    Applications of direction finding techniques include detection and localization of pulsed electromagnetic sources. This paper presents the design and analysis of a planar antenna system for direction finding. Our proposed system includes 4 hybrid couplers that generate 900 shifted signals, 2 crossover couplers also known as 0dB couplers, two 450 phase shifters, two 00 phase shifters and 4 patch antennas.

  19. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  20. Excitation of the CO fourth positive system by the dissociative recombination of CO2/+/ ions.

    Science.gov (United States)

    Gutcheck, R. A.; Zipf, E. C.

    1973-01-01

    The fourth positive system of CO has been excited in a static afterglow experiment by the dissociative recombination of CO2(+) ions. From combined absolute optical and microwave measurements the specific recombination coefficient for exciting the CO system was found to be (2 plus 1 or minus 0.5) x 10 to the minus 8th cu cm/sec. This value represents approximately 5% of the total recombination coefficient (4.0 plus or minus 0.5) x 10 to the minus 7th cu cm/sec measured in this experiment, implying that CO2(+) dissociative recombination will contribute significantly to the excitation of the CO fourth positive system in the Martian airglow. Corroborative electron heating experiments showed that the magnitude of the specific recombination coefficient decreased as the electron temperature was increased. Evidence was also found for the presence of vibrationally excited ions in the CO2(+) plasma, a result which indicates that analogous laboratory studies on the dissociative recombination of O2(+), N2(+), and NO(+) ions may have also involved vibrationally hot plasmas.

  1. ASSESMENT OF CRYOPRESERVATION SYSTEMS INFLUENCE ON THE SURVAVIAL OF E. COLI RECOMBINANT STRAINS

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available The cryopreservation systems of recombinant bacterial cells based on glycerol were studied in these experiments according to the hypothesis that glycerol is one of the widely used cryoprotective additives in microbiology and a multitude of factors affecting the effectiveness of cryopreservation in microorganisms; the best cryoprotective additive and the optimum concentration for a particular microorganism has to be determined empirically. The results obtained in this experiment are showing that the freezing procedure at -80°C in LB 40% glycerol is the optimum system for the cryopreservation of E. coli DH5α recombinant cells. The use of SOC medium supplemented with 10g/l NaCl provided more proper conditions of culture for the defrosted E. coli DH5α recombinant cells, reducing the osmotic stress during the recovery after thawing. The utilization of this optimum cryopreservation system offer the possibility of preserving the large volume of work and time involved by the recombinant DNA technology procedures applied for obtaining a recombinant strain, avoiding the storage of recombinant strains by costly and time consuming microbiology culturing techniques.

  2. Directional spectral emissivity measurement system

    Science.gov (United States)

    Halyo, Nesim (Inventor); Pandey, Dhirendra K. (Inventor)

    1992-01-01

    Apparatus and process for determining the emissivity of a test specimen including an integrated sphere having two concentric walls with a coolant circulating therebetween, and disposed within a chamber which may be under ambient, vacuum or inert gas conditions. A reference sample is disposed within the sphere with a monochromatic light source in optical alignment therewith. A pyrometer is in optical alignment with the test sample for obtaining continuous test sample temperature measurements during a test. An arcuate slit port is provided through the spaced concentric walls of the integrating sphere with a movable monochromatic light source extending through and movable along the arcuate slit port. A detector system extends through the integrating sphere for continuously detecting an integrated signal indicative of all radiation within its field of view, as a function of the emissivity of the test specimen at various temperatures and various angle position of the monochromatic light source. A furnace for heating the test sample to approximately 3000 K. and control mechanism for transferring the heated sample from the furnace to the test sample port in the integrating sphere is also contained within the chamber.

  3. Expression and export: recombinant protein production systems for Aspergillus.

    Science.gov (United States)

    Fleissner, André; Dersch, Petra

    2010-07-01

    Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

  4. Optical sensing systems based on biomolecular recognition of recombinant proteins

    Science.gov (United States)

    Salins, Lyndon L.; Schauer-Vukasinovic, Vesna; Daunert, Sylvia

    1998-05-01

    SIte-directed mutagenesis and the associated site-specific fluorescent labeling of proteins can be used to rationally design reagentless fluorescent molecular senors. The phosphate binding protein (PBP) and calmodulin (CaM) bind to phosphate and calcium in a highly specific manner. These ions induce a hinge motion in the proteins, and the resultant conformational change constitutes the basis of the sensor development. By labeling each protein at a specific site with environment-sensitive fluorescent probes, these conformational changes can be monitored and related to the amount of analyte ion present. In this study, the polymerase chain reaction was used to construct mutants of PBP and CaM that have a single cysteine at positions 197 and 109, respectively. Each protein was site-specifically labeled through the sulfhydryl group of the introduced cysteine residue at a single location with an environment-sensitive fluorescent probe. Characterization of the steady-state fluorescence indicated an enhancement of signal intensity upon binding of the analyte ion. Highly sensitive and selective and selective sensing systems for phosphate and calcium were obtained by using this approach.

  5. Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

    Science.gov (United States)

    Baum, J A; Kakefuda, M; Gawron-Burke, C

    1996-12-01

    The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.

  6. Coaxial direct-detection lidar-system

    DEFF Research Database (Denmark)

    2014-01-01

    The invention relates to a coaxial direct-detection LIDAR system for measuring velocity, temperature and/or particulate density. The system comprises a laser source for emitting a laser light beam having a lasing center frequency along an emission path. The system further comprises an optical...... frequency and/or the center filter frequency may be scanned. The invention further relates to an aircraft airspeed measurement device, and a wind turbine airspeed measurement device comprising the LIDAR system....... delivery system arranged in the emission path of the laser source, the optical delivery system being arranged for delivering the laser light beam in a measuring direction, the optical delivery system further being configured for collecting a return signal backscattered along the measuring direction...

  7. SDR (Systems Directed Reading): An Overview.

    Science.gov (United States)

    Baugo Community Schools, Elkhart, IN.

    The objective of this project for kindergarten through fifth grade is to interest public and private educational institutions in the systematization of elementary school reading programs. Facets of Systems Directed Reading (SDR) include the use of a differentiated staffing pattern; experienced language arts unit leaders guiding and directing all…

  8. Universal four-boson system: dimer-atom-atom Efimov effect and recombination reactions

    CERN Document Server

    Deltuva, A

    2013-01-01

    Recent theoretical developments in the four-boson system with resonant interactions are described. Momentum-space scattering equations for the four-particle transition operators are used. The properties of unstable tetramers with approximate dimer-atom-atom structure are determined. In addition, the three- and four-cluster recombination processes in the four-boson system are studied.

  9. Direct Numerical Simulation of Reionization II: Recombinations, Clumping Factors, and the Photon Budget for Reionization

    CERN Document Server

    So, Geoffrey C; Reynolds, Daniel R; Harkness, Robert P

    2013-01-01

    In this first of several application papers, we investigate the mechanics of reionization from stellar sources in high-z galaxies, the utility of various clumping factors on estimating the recombination time in the IGM, and the photon budget required to achieve reionization. We test the accuracy of the static and time-dependent models of Madau et al. as predictors of reionization completion/maintenance. We simulate a WMAP7 LCDM cosmological model in a 20 Mpc comoving cube with 800^3 uniform fluid cells and dark matter particles. By tuning our star formation to approximately match the observed star formation rate density and luminosity function, we created a fully coupled radiation-hydro realization of H reionization which begins to ionize at z~10 and completes at z~5.8. We find that roughly 2 ionizing photons per H atom are required to convert the neutral IGM to a highly ionized state, which supports the "photon starved" scenario discussed by Bolton & Haehnelt. The events during reionization that lead to ...

  10. Inhomogeneous recombinations during cosmic reionization

    OpenAIRE

    Sobacchi, Emanuele; Mesinger, Andrei

    2014-01-01

    By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedb...

  11. Excision dynamics of Vibrio pathogenicity island-2 from Vibrio cholerae: role of a recombination directionality factor VefA

    Directory of Open Access Journals (Sweden)

    Napolitano Michael G

    2010-11-01

    Full Text Available Abstract Background Vibrio Pathogenicity Island-2 (VPI-2 is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758 that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate. Results We determined the conditions and the factors that affect excision of VPI-2 in V. cholerae N16961. We demonstrate that excision from chromosome 1 is induced at low temperature and after sublethal UV-light irradiation treatment. In addition, after UV-light irradiation compared to untreated cells, cells showed increased expression of three genes, intV2 (VC1758, and two putative recombination directionality factors (RDFs, vefA (VC1785 and vefB (VC1809 encoded within VPI-2. We demonstrate that along with IntV2, the RDF VefA is essential for excision. We constructed a knockout mutant of vefA in V. cholerae N16961, and found that no excision of VPI-2 occurred, indicating that a functional vefA gene is required for excision. Deletion of the second RDF encoded by vefB did not result in a loss of excision. Among Vibrio species in the genome database, we identified 27 putative RDFs within regions that also encoded IntV2 homologues. Within each species the RDFs and their cognate IntV2 proteins were associated with different island regions suggesting that this pairing is widespread. Conclusions We demonstrate that excision of VPI-2 is induced under some environmental stress conditions and we show for the first time that an RDF encoded within a pathogenicity island in V. cholerae is required for excision of the

  12. Establishment and application of both FLP and Cre site-specific recombination systems at the same position in the genome

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Both FRT-FRT and LoxP-LoxP sites that are the target sequences of site-specific recombinases have been constructed in a vector,called C4LFY,using the recombinant DNA technique.C4LFY also contains P elements,2 exons and 1 intron of Drosophila yellow gene,yellow promoter and enhancers,and flanking DNA.Since C4LFY made use of two pairs of FRT and LoxP sites,this vector included two site-specific recombination systems.C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation.In the presence of the FLP or Cre recombinase,either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome.Using this system,the molecular basis of yellow gene expression and regulation during development have been investigated.Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers.In addition,the 5' and 3' genomic sequences flanking the yellow gene have been preliminarily studied and their potential role is discussed.

  13. Directed animals, quadratic and rewriting systems

    CERN Document Server

    Marckert, Jean-François

    2011-01-01

    A directed animal is a percolation cluster in the directed site percolation model. The aim of this paper is to exhibit a strong relation between in one hand, the problem of computing the generating function $\\G$ of directed animals on the square lattice, counted according to the area and the perimeter, and on the other hand, the problem to find a solution to a system of quadratic equations involving unknown matrices. The matrices solution of this problem can be finite or infinite. We were unable to find finite solutions. We present some solid clues that some infinite explicit matrices, fix points of a rewriting like system are the natural solutions of this system of equations: some strong evidences are given that the problem of finding $\\G$ reduces then to the problem of finding an eigenvector to an explicit infinite matrix. Similar properties are shown for other combinatorial questions concerning directed animals, and for different lattices.

  14. Recombinative reading derived from pseudoword instruction in a miniature linguistic system.

    Science.gov (United States)

    Hanna, Elenice S; Kohlsdorf, Marina; Quinteiro, Regiane S; de Melo, Raquel Maria; de Souza, Deisy das GraCas; de Rose, Julio C; McIlvane, William J

    2011-01-01

    A miniature linguistic system was used to study acquisition of recombinative symbolic behavior. Three studies evaluated the teaching conditions of conditional discriminations with printed and spoken pseudowords that could potentially generate recombinative reading. Fifty-four college students across all studies learned to match 12 printed pseudowords to 12 spoken pseudowords. Some also matched pictures to the same spoken words. Each two-syllable pseudoword was formed by symbols from an arbitrarily created alphabet composed of four vowels and four consonants. Letters had univocal correspondence with phonemes. Recombinative receptive reading, comprehensive reading, and textual responding to pseudowords were periodically assessed. Experiment 1 (n  =  20) showed that recombinative reading increased as the number of trained words composed of the same symbols increased. Experiment 2 (n  =  14) showed that overtraining the same two words did not produce recombinative reading for most participants. Experiment 3 (n  =  20), in which training with pictures was omitted, showed that elemental control by within-syllable units can develop even when the trained pseudowords are meaningless (not related to pictures). The present results support the utility of the miniature linguistic system methodology for identifying and controlling environmental determinants of rudimentary reading skills.

  15. Hydrogen from Water in a Novel Recombinant Cyanobacterial System

    Energy Technology Data Exchange (ETDEWEB)

    Weyman, Philip D [J. Craig Venter Institute; Smith, Hamillton O.

    2014-12-03

    Photobiological processes are attractive routes to renewable H2 production. With the input of solar energy, photosynthetic microbes such as cyanobacteria and green algae carry out oxygenic photosynthesis, using sunlight energy to extract protons and high energy electrons from water. These protons and high energy electrons can be fed to a hydrogenase system yielding H2. However, most hydrogen-evolving hydrogenases are inhibited by O2, which is an inherent byproduct of oxygenic photosynthesis. The rate of H2 production is thus limited. Certain photosynthetic bacteria are reported to have an O2-tolerant evolving hydrogenase, yet these microbes do not split water, and require other more expensive feedstocks. To overcome these difficulties, the goal of this work has been to construct novel microbial hybrids by genetically transferring O2-tolerant hydrogenases from other bacteria into a class of photosynthetic bacteria called cyanobacteria. These hybrid organisms will use the photosynthetic machinery of the cyanobacterial hosts to perform the water-oxidation reaction with the input of solar energy, and couple the resulting protons and high energy electrons to the O2-tolerant bacterial hydrogenase, all within the same microbe (Fig. 1). The ultimate goal of this work has been to overcome the sensitivity of the hydrogenase enzyme to O2 and address one of the key technological hurdles to cost-effective photobiological H2 production which currently limits the production of hydrogen in algal systems. In pursuit of this goal, work on this project has successfully completed many subtasks leading to a greatly increased understanding of the complicated [NiFe]-hydrogenase enzymes. At the beginning of this project, [NiFe] hydrogenases had never been successfully moved across wide species barriers and had never been heterologously expressed in cyanobacteria. Furthermore, the idea that whole, functional genes could be extracted from complicated, mixed-sequence meta-genomes was not

  16. Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

    NARCIS (Netherlands)

    Mlynarova, L.; Conner, A.J.; Nap, J.P.H.

    2006-01-01

    A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either t

  17. Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

    NARCIS (Netherlands)

    Mlynarova, L.; Conner, A.J.; Nap, J.P.H.

    2006-01-01

    A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either

  18. A comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems.

    Science.gov (United States)

    Gecchele, Elisa; Merlin, Matilde; Brozzetti, Annalisa; Falorni, Alberto; Pezzotti, Mario; Avesani, Linda

    2015-03-23

    Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products. In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable. For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein. Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

  19. Trends toward rotary steerable directional systems

    Energy Technology Data Exchange (ETDEWEB)

    Warren, T.M. [Amoco Exploration and Production Technology Group, Tulsa, OK (United States)

    1997-05-01

    Directional drilling will continue to be important in the petroleum industry for the foreseeable future as reserves in offshore locations, environmentally sensitive areas and locations with restricted surface access are developed. Emphasis on re-entries to extend the life of onshore and offshore production facilities and on horizontal completions to improve production rates and ultimate recovery will continue to place demands on directional drilling technology. Efficiency improvements that may be achieved through introduction of a new technology are often not easy to quantify, even though they may be quite significant. As long as the job gets done with the currently used system, and no better system is immediately available, it is natural to concentrate efforts on improving the existing system rather than introducing a new system. Here, the discussion is aimed at showing that drilling with steerable motor directional systems is inefficient, and that a significant improvement could be gained by introduction of rotary steerable systems. The objective of this article is not to put down motors, which have provided the backbone of directional drilling for three decades, but rather to show that considerable incentive exists for pursuing an alternative system.

  20. An epitope delivery system for use with recombinant mycobacteria

    NARCIS (Netherlands)

    Hetzel, C.; Janssen, R.; Ely, S.J.; Kristensen, N.M.; Bunting, K.; Cooper, J.B.; Lamb, J.R.; Young, D.B.; Thole, J.E.R.

    1998-01-01

    We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- G

  1. An epitope delivery system for use with recombinant mycobacteria

    NARCIS (Netherlands)

    Hetzel, C.; Janssen, R.; Ely, S.J.; Kristensen, N.M.; Bunting, K.; Cooper, J.B.; Lamb, J.R.; Young, D.B.; Thole, J.E.R.

    1998-01-01

    We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- G

  2. Direct bacterial killing in vitro by recombinant Nod2 is compromised by Crohn's disease-associated mutations.

    Directory of Open Access Journals (Sweden)

    Laurent-Herve Perez

    Full Text Available BACKGROUND: A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn's disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn's-associated mutations. Wild-type, but not Crohn's-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. CONCLUSIONS/SIGNIFICANCE: The lack of anti-bacterial activity demonstrated with Crohn's-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.

  3. Hydrogen from Water in a Novel Recombinant Oxygen-Tolerant Cyanobacterial System (Presentation)

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Q.; Smith, H. O.; Maness, P.-C.

    2007-05-01

    The objective of this report is to develop an O{sub 2}-tolerant cyanobacterial system for continuous light-driven H{sub 2} production from water. The overall goal is to produce a cyanobacterial recombinant to produce H{sub 2} continuously.

  4. Nonsequential double-recombination high-order-harmonic generation in molecularlike systems

    DEFF Research Database (Denmark)

    Hansen, Kenneth Christian Klochmann; Madsen, Lars Bojer

    2017-01-01

    We present a study of nonsequential double-recombination (NSDR) high-harmonic generation (HHG) in a molecularlike system. We have calculated the HHG spectrum for a wide range of internuclear distances, and using a Coulomb-corrected three-step model we are able to analyze and predict the observed...

  5. Non-Sequential Double Recombination High Harmonic Generation in Molecular-like Systems

    DEFF Research Database (Denmark)

    Hansen, Kenneth Christian Klochmann; Madsen, Lars Bojer

    Non-sequential double recombination (NSDR) high harmonic generation (HHG) is a strongly correlated two-electron HHG process where two electrons combine their potential and kinetic energy into emitting a single photon. We have studied this process in a molecular-like system and found that the two...

  6. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

    Science.gov (United States)

    Thomason, L C; Calendar, R; Ow, D W

    2001-08-01

    The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

  7. New Directions in Modeling the Lighting Systems

    Directory of Open Access Journals (Sweden)

    P. Fiala

    2004-12-01

    Full Text Available This paper presents information about new directions in the modelingof lighting systems, and an overview of methods for the modeling oflighting systems. The new R-FEM method is described, which is acombination of the Radiosity method and the Finite Elements Method. Thepaper contains modeling results and their verification by experimentalmeasurements and by the Matlab simulation for this R-FEM method.

  8. Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1

    DEFF Research Database (Denmark)

    Ellis, April L; Pan, Wensheng; Yang, Guang

    2010-01-01

    BACKGROUND: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human...... perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. RESULTS: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess...... glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary...

  9. A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

    Directory of Open Access Journals (Sweden)

    Marino Gennaro

    2006-12-01

    Full Text Available Abstract Background The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. Results A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. Conclusion Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger.

  10. Hydrogen from Water in a Novel Recombinant Cyanobacterial System

    Energy Technology Data Exchange (ETDEWEB)

    Weyman, Philip D [J. Craig Venter Institute; Smith, Hamillton O.

    2014-12-03

    Photobiological processes are attractive routes to renewable H2 production. With the input of solar energy, photosynthetic microbes such as cyanobacteria and green algae carry out oxygenic photosynthesis, using sunlight energy to extract protons and high energy electrons from water. These protons and high energy electrons can be fed to a hydrogenase system yielding H2. However, most hydrogen-evolving hydrogenases are inhibited by O2, which is an inherent byproduct of oxygenic photosynthesis. The rate of H2 production is thus limited. Certain photosynthetic bacteria are reported to have an O2-tolerant evolving hydrogenase, yet these microbes do not split water, and require other more expensive feedstocks. To overcome these difficulties, the goal of this work has been to construct novel microbial hybrids by genetically transferring O2-tolerant hydrogenases from other bacteria into a class of photosynthetic bacteria called cyanobacteria. These hybrid organisms will use the photosynthetic machinery of the cyanobacterial hosts to perform the water-oxidation reaction with the input of solar energy, and couple the resulting protons and high energy electrons to the O2-tolerant bacterial hydrogenase, all within the same microbe (Fig. 1). The ultimate goal of this work has been to overcome the sensitivity of the hydrogenase enzyme to O2 and address one of the key technological hurdles to cost-effective photobiological H2 production which currently limits the production of hydrogen in algal systems. In pursuit of this goal, work on this project has successfully completed many subtasks leading to a greatly increased understanding of the complicated [NiFe]-hydrogenase enzymes. At the beginning of this project, [NiFe] hydrogenases had never been successfully moved across wide species barriers and had never been heterologously expressed in cyanobacteria. Furthermore, the idea that whole, functional genes could be extracted from complicated, mixed-sequence meta-genomes was not

  11. Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

    Institute of Scientific and Technical Information of China (English)

    ZONG Xiaolin; HA Zhuo; ZHAO Lili; LIU Diqiu; QIAO Xinyuan; JIANG Yanping; GE Junwei; LI Yijing; TANG Lijie

    2011-01-01

    Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin (PLFN). A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

  12. Recombinant production of plant lectins in microbial systems for biomedical application – the frutalin case study

    Directory of Open Access Journals (Sweden)

    Carla eOliveira

    2014-08-01

    Full Text Available Frutalin is a homotetrameric partly-glycosylated alpha-D-galactose-binding lectin of biomedical interest from Artocarpus incisa (breadfruit seeds, belonging to the jacalin-related lectins family. As other plant lectins, frutalin is a heterogeneous mixture of several isoforms possibly with distinct biological activities. The main problem of using such lectins as biomedical tools is that batch-to-batch variation in isoforms content may lead to inconstant results. The production of lectins by recombinant means has the advantage of obtaining high amounts of proteins with defined amino-acid sequences and more precise properties. In this mini review, we provide the strategies followed to produce two different forms of frutalin in two different microbial systems: Escherichia coli and Pichia pastoris. The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit. Emphasis is given particularly to recombinant frutalin produced in P. pastoris, which showed a remarkable capacity as biomarker of human prostate cancer and as apoptosis-inducer of cancer cells. Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

  13. Excision of the Shigella Resistance Locus Pathogenicity Island in Shigella flexneri Is Stimulated by a Member of a New Subgroup of Recombination Directionality Factors

    Science.gov (United States)

    Luck, Shelley N.; Turner, Sally A.; Rajakumar, Kumar; Adler, Ben; Sakellaris, Harry

    2004-01-01

    Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis. PMID:15292162

  14. Direct current power delivery system and method

    Science.gov (United States)

    Zhang, Di; Garces, Luis Jose; Dai, Jian; Lai, Rixin

    2016-09-06

    A power transmission system includes a first unit for carrying out the steps of receiving high voltage direct current (HVDC) power from an HVDC power line, generating an alternating current (AC) component indicative of a status of the first unit, and adding the AC component to the HVDC power line. Further, the power transmission system includes a second unit for carrying out the steps of generating a direct current (DC) voltage to transfer the HVDC power on the HVDC power line, wherein the HVDC power line is coupled between the first unit and the second unit, detecting a presence or an absence of the added AC component in the HVDC power line, and determining the status of the first unit based on the added AC component.

  15. Implementation of passive autocatalytic recombiner system as a hydrogen mitigation system in Korean nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Hyun; Sung, Je Joong; Ha, Sang Jun [Korea Hydro and Nuclear Power Co. Ltd., Central Research Institute, Daejeon (Korea, Republic of); Yeo, In Seon [KEPCO Engineering and Construction Co. Ltd., Gyeonggi-do (Korea, Republic of)

    2015-08-15

    Ensuring the containment integrity during a severe accident in nuclear power reactor by maintaining the hydrogen concentration below an acceptable level has been recognized to be of critical importance since Three Mile Island and Fukushima Daiichi nuclear power plant accidents. Although there exist various mitigation measures for hydrogen risk, a passive autocatalytic recombiner (PAR) has been emphasized as a viable option for the mitigation of hydrogen risk under the extended station blackout conditions due to its passive operation characteristics for the hydrogen removal. To enhance the capability of hydrogen control, the hydrogen mitigation system with various types of PARs has been implemented for all nuclear power plants in Korea. This paper presents an implementation procedure of PAR system and the analysis results to determine the location and capacity of PAR in OPR1000. Various accident scenarios have been adopted considering important event sequences from a combination of probabilistic methods, deterministic methods and sound engineering judgment. A MAAP 4.0.6+ with a multi-compartment model has been used as an analysis tool with conservative hydrogen generation and removal models. The detailed analyses have been performed for selected severe accident scenarios including sensitivity analysis with/without operations of various safety systems. The possibility of global flame acceleration (FA) and deflagration-to-detonation transient (DDT) has been assessed with sigma (flame acceleration potential) and 7-lambda (DDT potential) criterion. It is concluded that the newly designed hydrogen mitigation system with twenty-four (24) PARs can effectively remove hydrogen in the containment atmosphere and prevent global FA and DDT.

  16. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

    OpenAIRE

    Claudia Rabert; Daniel Weinacker; Adalberto Pessoa Jr; Farías,Jorge G.

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expressio...

  17. Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

    Directory of Open Access Journals (Sweden)

    Sina Mirzaahmadi

    2011-01-01

    Full Text Available The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

  18. IRDL cloning: a one-tube, zero-background, easy-to-use, directional cloning method improves throughput in recombinant DNA preparation.

    Science.gov (United States)

    Wang, Jiancai; Xu, Ronghua; Liu, Aizhong

    2014-01-01

    Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.

  19. High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    WANG Xiaoyan; GAO Linlin; DENG Fei; ZHANG Yanfang; LI Yan; LIN Jusheng

    2007-01-01

    HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

  20. Directive Antenna for Ultrawideband Medical Imaging Systems

    Directory of Open Access Journals (Sweden)

    Amin M. Abbosh

    2008-01-01

    Full Text Available A compact and directive ultrawideband antenna is presented in this paper. The antenna is in the form of an antipodal tapered slot with resistive layers to improve its directivity and to reduce its backward radiation. The antenna operates over the frequency band from 3.1 GHz to more than 10.6 GHz. It features a directive radiation with a peak gain which is between 4 dBi and 11 dBi in the specified band. The time domain performance of the antenna shows negligible distortion. This makes it suitable for the imaging systems which require a very short pulse for transmission/reception. The effect of the multilayer human body on the performance of the antenna is also studied. The breast model is used for this purpose. It is shown that the antenna has more than 90% fidelity factor when it works in free space, whereas the fidelity factor decreases as the signal propagates inside the human body. However, even inside the human body, the fidelity factor is still larger than 70% revealing the possibility of using the proposed antenna in biomedical imaging systems.

  1. Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    M Boustanshenas

    2013-01-01

    Full Text Available Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs in Escherichia coli BL21 (DE3. Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F′ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4. The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3 cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3 is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.

  2. New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

    Science.gov (United States)

    Eko, F O; Witte, A; Huter, V; Kuen, B; Fürst-Ladani, S; Haslberger, A; Katinger, A; Hensel, A; Szostak, M P; Resch, S; Mader, H; Raza, P; Brand, E; Marchart, J; Jechlinger, W; Haidinger, W; Lubitz, W

    1999-03-26

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign

  3. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    Science.gov (United States)

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Human olfactory receptors: recombinant expression in the baculovirus/Sf9 insect cell system, functional characterization, and odorant identification.

    Science.gov (United States)

    Matarazzo, Valéry; Ronin, Catherine

    2013-01-01

    Cell surface expression of recombinant olfactory receptors (ORs) is a major limitation in characterizing their functional nature. We have shown that the recombinant expression of a human OR, OR 17-210, in the baculovirus/Sf9 insect cell system allows this protein to be expressed at the cell surface. We used Ca(2+) imaging to demonstrate that recombinant OR 17-210 produces cellular activities upon odorant stimulation with ketones. Furthermore, this expression and functional system has been used to show that the preincubation of Human Odorant Binding Protein 2A decrease the calcium response of OR 17-210 following stimulation by acetophenone and beta ionone.

  5. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    Science.gov (United States)

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

  6. Recombinant Technology in the Development of Materials and Systems for Soft-Tissue Repair.

    Science.gov (United States)

    Girotti, Alessandra; Orbanic, Doriana; Ibáñez-Fonseca, Arturo; Gonzalez-Obeso, Constancio; Rodríguez-Cabello, José Carlos

    2015-11-18

    The field of biomedicine is constantly investing significant research efforts in order to gain a more in-depth understanding of the mechanisms that govern the function of body compartments and to develop creative solutions for the repair and regeneration of damaged tissues. The main overall goal is to develop relatively simple systems that are able to mimic naturally occurring constructs and can therefore be used in regenerative medicine. Recombinant technology, which is widely used to obtain new tailored synthetic genes that express polymeric protein-based structures, now offers a broad range of advantages for that purpose by permitting the tuning of biological and mechanical properties depending on the intended application while simultaneously ensuring adequate biocompatibility and biodegradability of the scaffold formed by the polymers. This Progress Report is focused on recombinant protein-based materials that resemble naturally occurring proteins of interest for use in soft tissue repair. An overview of recombinant biomaterials derived from elastin, silk, collagen and resilin is given, along with a description of their characteristics and suggested applications. Current endeavors in this field are continuously providing more-improved materials in comparison with conventional ones. As such, a great effort is being made to put these materials through clinical trials in order to favor their future use.

  7. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang

    2009-10-01

    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  8. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  9. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  10. The new pLAI (lux regulon based auto-inducible) expression system for recombinant protein production in Escherichia coli.

    Science.gov (United States)

    Nocadello, Salvatore; Swennen, Erwin Frans

    2012-01-05

    After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS) system.The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene independently from external inducers or drastic changes in

  11. Direct measurement of recombination frequency in interspecific hybrids between Hordeum vulgare and H. bulbosum using genomic in situ hybridization.

    Science.gov (United States)

    Zhang, L; Pickering, R; Murray, B

    1999-09-01

    Two different genotypes of diploid Hordeum vulgare x H. bulbosum hybrids, which differ in their pattern of meiotic metaphase pairing behaviour, were investigated at MI and AI by genomic in situ hybridization (GISH). One hybrid, 102C2, showed a high frequency of bivalents at metaphase I whereas the other, 103K5, showed a high frequency of univalents. The GISH analysis of both hybrids established that pairing occurred only between chromosomes of different parental genomes and revealed that pairing frequency greatly exceeded recombination. Hybrid 102C2 had a significantly higher recombination frequency than 103K5, but in both hybrids recombination involved only distal chromosome regions. However, an interesting finding is that the ratio of recombination to pairing frequency in 103K5 (1:8.9) is twice as high compared with 102C2 (1:17). The hybrids also differed in chromosome stability; little chromosome elimination occurred in 102C2 but 103K5 showed extensive chromosome loss. It appears that the high frequency of bound arms at MI favours retention of H. bulbosum chromosomes and maintains stability of chromosome numbers in PMCs. Various ideas are put forward to explain the discrepancy between meiotic pairing frequency and recombination in these hybrids.

  12. Rosa26-GFP direct repeat (RaDR-GFP mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

    Directory of Open Access Journals (Sweden)

    Michelle R Sukup-Jackson

    2014-06-01

    Full Text Available Homologous recombination (HR is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

  13. Analysis Method for Licensing Application of Passive Autocatalytic Recombiner System as a Hydrogen Mitigation System of OPR-1000

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Changhyun; Sung, Jejoong; Ha, Sangjun [Korea Hydro and Nuclear Power Co. Ltd., Daejeon (Korea, Republic of); Yeo, Inseon [KEPCO Engineering and Construction Co. Ltd., Yongin (Korea, Republic of)

    2014-05-15

    As a result, for some plants, dual hydrogen mitigation systems are prepared with a combination of PARs and igniters that each system has a 100% of full capacity for hydrogen control for postulated severe accident conditions. In the original design of OPR-1000, hydrogen mitigation systems consist of a thermal recombiner and twenty glow-type igniters, which are used for design basis accident and severe accident, respectively. This paper presents an analysis method for licensing application in Korea to determine the capacity and locations of PARs for the design of a hydrogen mitigation system with PAR. A licensed analysis method of OPR-1000 has been presented to determine the capacity and locations of PAR for the design of a hydrogen mitigation system with PAR. A lumped parameter code of MAAP 4.0.6+ has been adopted to simulate various severe accident scenarios with a 26 multi-compartment containment model. Hydrogen generations were analysed and required capacity and locations of PAR were determined for six accident scenarios selected from a combination of probabilistic and deterministic considerations. A total of twenty-four PARs in the containment dome, steam generator rooms, annulus and adjacent areas was designed and the adequacy of this system has been confirmed through detailed analyses including sensitivity analyses with/without operations of safety systems such as containment heat removal systems, reactor coolant depressurization system and safety injection by accumulator, etc. Through the assessment on the possibility of global FA and DDT, it has been concluded that new PAR system with twenty-four recombiners can remove hydrogen effectively in the containment atmosphere and prevent from global FA and DDT. Further works are required in the future to develop a well-balanced analysis methodology with a combination of lumped and CFD tools focusing on the optimum locations of recombiners and local hydrogen behaviour in containment compartments.

  14. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems

    Directory of Open Access Journals (Sweden)

    Daryl G.S. Smith

    2015-09-01

    Full Text Available Human serum albumin (HSA is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3 (2012 209–290. Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA (Chen et al., Biochim. Biophys. Acta (BBA—Gen. Subj. 1830(12 (2013 5515–5525; Kobayashi, Biologicals 34(1 (2006 55–59. Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15 (2012 4661–4670, both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9 (2014 e109893. We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.

  15. Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing.

    Science.gov (United States)

    Girardet, A; Lien, S; Leeflang, E P; Beaufrère, L; Tuffery, S; Munier, F; Arnheim, N; Claustres, M; Pellestor, F

    1999-01-01

    In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.

  16. Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli

    DEFF Research Database (Denmark)

    Williamson, M R; Doherty, J P; Woodcock, D M

    1993-01-01

    We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations. We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned......A+ mcr+ hosts compared with a methylation-tolerant host (mcr-). Of the clones recovered in recA+mcr+ hosts, > 20% of clones had an inactivating mutation in tet. The majority of such mutant clones contained deletions that frequently extended into the unmethylated portion of tet and even into the plasmid...

  17. Use of a recombination-deficient phage lambda system to construct wheat genomic libraries.

    Science.gov (United States)

    Murray, M G; Kennard, W C; Drong, R F; Slightom, J L

    1984-10-01

    The poor cloning efficiency of wheat (Triticum aestivum cv. Yamhill) DNA in conventional cloning vectors has previously prevented the preparation of complete genomic libraries. We show here that while wheat DNA does not clone efficiently using the vector Ch4A, it can be cloned efficiently using Ch32. Ch32 clones are red- gam+ and therefore can be propagated on recombination-deficient hosts. These results suggest that instability of wheat sequences in conventional lambda vector systems has frustrated previous attempts to prepare libraries.

  18. Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli

    DEFF Research Database (Denmark)

    Williamson, M R; Doherty, J P; Woodcock, D M

    1993-01-01

    We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations. We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned.......HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were...

  19. An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus

    Institute of Scientific and Technical Information of China (English)

    Ruikun; Du; Manli; Wang; Zhihong; Hu; Hualin; Wang; Fei; Deng

    2015-01-01

    Japanese encephalitis virus(JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus–host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope(E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

  20. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.

    Science.gov (United States)

    Drake, Pascal M W; Barbi, Tommaso; Sexton, Amy; McGowan, Edward; Stadlmann, Johannes; Navarre, Catherine; Paul, Matthew J; Ma, Julian K-C

    2009-10-01

    Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within

  1. ESDIS DOI System, Approach, and Future Direction

    Science.gov (United States)

    Wanchoo, L.; James, N.

    2013-12-01

    that has the data product description. Even though the development and maintenance of DOI landing pages would be the responsibility of the DAACs, ESDIS in consultation with the data providers is currently developing the landing page guidelines that specifies the key data product descriptive elements to be included on each data product's landing page. This poster will describe in detail the unique process and system currently implemented by ESDIS for registering DOIs. In addition, the paper will summarize some of the early implementation shortcomings and how they were addressed, as well as the future directions of the ESDIS DOI system/process.

  2. Tumor immunity within the central nervous system stimulated by recombinant Listeria monocytogenes vaccination.

    Science.gov (United States)

    Liau, Linda M; Jensen, Eric R; Kremen, Thomas J; Odesa, Sylvia K; Sykes, Steven N; Soung, Michael C; Miller, Jeff F; Bronstein, Jeff M

    2002-04-15

    Tumors arising within the central nervous system (CNS) present the immune system with a challenging target, given the heterogeneous nature of these neoplasms and their location within an "immunologically privileged" site. We used the lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a pseudotumor antigen to investigate recombinant Listeria monocytogenes as a tumor vaccine against s.c. and intracerebral challenges with a NP-expressing glioma, 9L-NP. Using Fischer 344 rats, we demonstrate that vaccination with recombinant L. monocytogenes-NP stimulates protection against s.c., but not intracerebral, 9L-NP tumor challenge in an antigen-specific, CD8(+) T-cell-dependent manner. After s.c. tumor rejection, enhanced antitumor immunity is achieved via epitope spreading that permits complete resistance against lethal intracerebral challenge with 9L-NP and with the untransfected parental 9L tumor. Unlike the CD8(+)-dependent immune responses against s.c. 9L-NP tumors, this expanded intracerebral immunity against endogenous tumor-associated antigens is dependent on both CD4(+) and CD8(+) T cells. Taken together, these results demonstrate that the mechanisms of tumor immunity within the brain are different from those elicited against non-CNS tumors. Furthermore, vaccination approaches exploiting the concept of epitope spreading may enhance the efficacy of antitumor immune responses within the immunologically privileged CNS, potentially mediating tumor cell killing through both CD4(+)- and CD8(+)-dependent effector pathways.

  3. Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Julian M Glück

    Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

  4. Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

    DEFF Research Database (Denmark)

    Juel, C; Thomsen, J J; Rentsch, R L;

    2007-01-01

    Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...... on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... received 5,000 IU rHuEpo every second day for 14 days, and subsequently a single dose of 5,000 IU weekly for 12 weeks. Muscle biopsies were obtained before and after 14 weeks of rHuEpo treatment. The treatment increased hematocrit (from 44.7 to 48.8%), maximal oxygen uptake by 8.1%, and submaximal...

  5. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  6. Binary Laser Direct Writing System and Its Applications

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new laser direct writing system is introduced and the potential application of the diffractive optical elements (DOE's) fabricated by applying laser direct writing system are presented. The fabrication techniques by applying the laser direct writing are developed. Experimental results have been obtained by applying laser direct writing machine with line width of 5μm and 10μm.

  7. Construction of recombinant Marek's disease virus (rMDV co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

    Directory of Open Access Journals (Sweden)

    Zhenjie Zhang

    Full Text Available To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA gene from Avian Influenza Virus H9N2 strain and a Fusion (F gene from the Newcastle disease virus (NDV. The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101ΔMeq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the

  8. A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    Full Text Available Avian leukosis virus subgroup J (ALV-J has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP. We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.

  9. A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies.

    Science.gov (United States)

    Wang, Qi; Li, Xiaofei; Ji, Xiaolin; Wang, Jingfei; Shen, Nan; Gao, Yulong; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Zhang, Shide; Wang, Xiaomei

    2014-01-01

    Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.

  10. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

    Science.gov (United States)

    Veitch, N C; Tams, J W; Vind, J; Dalbøge, H; Welinder, K G

    1994-06-15

    Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

  11. Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm.

    Science.gov (United States)

    Usami, Akihiro; Ishiyama, Seiji; Enomoto, Chiaki; Okazaki, Hironobu; Higuchi, Keiko; Ikeda, Mashahiro; Yamamoto, Takeshi; Sugai, Mutsumi; Ishikawa, Yukiko; Hosaka, Yumiko; Koyama, Teruyuki; Tobita, Yoneko; Ebihara, Syoko; Mochizuki, Toshiko; Asano, Yoshimi; Nagaya, Hidekazu

    2011-02-01

    Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.

  12. The charge exchange recombination diagnostic system on the DIII-D tokamak

    Energy Technology Data Exchange (ETDEWEB)

    Gohil, P.; Burrell, K.H.; Groebner, R.J.; Kim, J.; Martin, W.C.; McKee, E.L.; Seraydarian, R.P.

    1991-11-01

    The charge exchange recombination (CER) diagnostic system on the DIII-D tokamak is used to make spatially and temporally resolved measurements of the ion temperature and toroidal and poloidal rotation velocities. This is performed through visible spectroscopic measurements of the Doppler broadened and Doppler shifted HE II 468.6 nm, the CVI 529.1 nm, and the BV 494.5 nm spectral lines which have been excited by charge exchange recombination interactions between the fully stripped ions and the neutral atoms from the heating beams. The plasma viewing optics comprises 32 viewing chords spanning a typical plasma minor radius of 63 cm across the midplane, of which 15 spatial chords span 4.2 cm at the plasma edge just within the separatrix and provide a chord-to-chord spatial resolution of 0.3 cm. Fast camera readout electronics can provide a temporal resolution of 260 {mu}s per time slice, but the effective minimum integration time, at present, is 1 ms which is limited by the detected photon flux from the plasma and the decay times of the phosphors used on the multichannel plate image intensifiers. Significant changes in the edge plasma radial electric field at the L-H transition have been observed, as determined from the CER measurements, and these results are being extensively compared to theories which consider the effects of sheared electric fields on plasma turbulence. 13 refs., 10 figs.

  13. Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.

    Science.gov (United States)

    George, Susan; Bhasker, Salini; Madhav, Harish; Nair, Archana; Chinnamma, Mohankumar

    2014-02-01

    Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.

  14. Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus

    Directory of Open Access Journals (Sweden)

    Santiago Haase

    2015-03-01

    Full Text Available Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh was replaced by a bacterial β-galactosidase (lacZ gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+ AgMNPV expressing the green fluorescent protein gene (gfp. This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

  15. Intratumoral modulation of the inducible co-stimulator ICOS by recombinant oncolytic virus promotes systemic anti-tumour immunity

    Science.gov (United States)

    Zamarin, Dmitriy; Holmgaard, Rikke B.; Ricca, Jacob; Plitt, Tamar; Palese, Peter; Sharma, Padmanee; Merghoub, Taha; Wolchok, Jedd D.; Allison, James P.

    2017-01-01

    Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade. PMID:28194010

  16. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3

    Directory of Open Access Journals (Sweden)

    Su Lingqia

    2012-01-01

    Full Text Available Abstract Background Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3, and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. Results T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3. In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. Conclusions In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.

  17. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

    Directory of Open Access Journals (Sweden)

    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  18. Near Threshold Effects on Recombination and Vibrational Relaxation in Efimov Systems

    CERN Document Server

    Shu, D; Côté, R

    2016-01-01

    We investigate the energy dependence of inelastic processes in systems which possess Efimov states. We consider the three-body recombination rate $K_3$ where three free atoms interact to produce an atom--dimer pair, and the relaxation rate $K_{\\rm rel}$ where an atom quenches a weakly bound state of a dimer near an Efimov resonance to more deeply bound levels. Using a model capturing the key features of the Efimov problem, we identify new energy regimes for $K_3$, namely the NTR (Near Threshold Resonance) regime behavior $E^{-2}$ for negative scattering lengths and the NTS (Near Threshold Suppression) regime behavior $E^2$ for positive scattering lengths. We also confirm a previously found oscillatory behavior of $K_3$ at higher energy $E$. Finally, we find that $K_{\\rm rel}$ behaves as $E^{-1}$ in the NTR regime.

  19. Development of recombinant nucleoprotein-based diagnostic systems for Lassa fever.

    Science.gov (United States)

    Saijo, Masayuki; Georges-Courbot, Marie-Claude; Marianneau, Philippe; Romanowski, Victor; Fukushi, Shuetsu; Mizutani, Tetsuya; Georges, Alain-Jean; Kurata, Takeshi; Kurane, Ichiro; Morikawa, Shigeru

    2007-09-01

    Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses.

  20. Systemic Venous Thrombosis after Recombinant Factor VIIa in the Control of Bleeding after Cardiac Surgery

    OpenAIRE

    Mahmoud, Amr; Al-Ruzzeh, Sharif; McKeague, Helena; Cross, Mike

    2007-01-01

    Recombinant factor VIIa has been licensed in the United Kingdom since 1996 for the control of bleeding in hemophilic patients who are actively bleeding or are about to undergo surgery. Medical practitioners are also applying recombinant factor VIIa toward the control of bleeding in patients without hemophilia. Although the efficacy of recombinant factor VIIa has been shown in many patients, concerns have arisen about the risk of thrombotic adverse events.

  1. Career Directions--Renewable Energy Systems Integrator

    Science.gov (United States)

    Fleeman, Stephen R.

    2012-01-01

    Renewable energy systems are beginning to appear everywhere. Solar modules are creating "blue roofs" that convert the energy from the sun into household electricity. Solar thermal systems on roofs can generate hot water. Wind turbines catch breezes to provide even more electricity. Recommendations for saving energy, specifying systems for…

  2. Career Directions--Renewable Energy Systems Integrator

    Science.gov (United States)

    Fleeman, Stephen R.

    2012-01-01

    Renewable energy systems are beginning to appear everywhere. Solar modules are creating "blue roofs" that convert the energy from the sun into household electricity. Solar thermal systems on roofs can generate hot water. Wind turbines catch breezes to provide even more electricity. Recommendations for saving energy, specifying systems for…

  3. Direct optimization of heterogeneous systems in electrical engineering; Optimisation directe de systemes heterogenes en Genie electrique

    Energy Technology Data Exchange (ETDEWEB)

    Fefermann, Y.

    2002-12-15

    Today, a global viewpoint in the design of devices called 'system approach', is imposed by couplings between physical fields and multiple interactions between sub-systems. Indeed, concerning static and electromechanical converters, or control systems, numerous issues of design are more and more mastered when each element is separately conceived. However, as soon as designing an heterogeneous system seen as a 'complex of elements in interaction' is at stake, arises the problem of the global optimization with respect to multiple criteria which characterize the quality of its behavior. The works realized in this thesis concern the multi-criteria optimization of systems by direct methods not related to the analytical calculation of the gradient. These techniques are applied to a typical electrical vehicle. We propose for each of the sub-systems a modeling adapted to the problem. In particular, an original method based on variations by similarity was elaborated for the design procedure of the PMSM machine. The parameters (DC supply voltage, geometrical parameters,...) can be fitted to all the system levels. They optimize performance criteria: minimization of the losses, of the embedded mass,... The methods of optimization used to resolve this non-linear problem are the Nelder and Mead's Simplex and Hooke and Jeeves algorithm. The constraints of the problem are treated by penalties (exact and quadratic) and a simple heuristic is proposed allowing a constraints normalization and a better algorithm convergence. The optimization results enable to underline the couplings between the various sub-systems, a significant point of the analysis. They have also shown the interest of a global approach in the design and the study of electric systems. (author)

  4. Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems.

    Science.gov (United States)

    Grogan, Dennis W; Stengel, Kristy R

    2008-09-01

    In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20-22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2-5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius. The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius, as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.

  5. 47 CFR 73.150 - Directional antenna systems.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Directional antenna systems. 73.150 Section 73... BROADCAST SERVICES AM Broadcast Stations § 73.150 Directional antenna systems. (a) For each station employing a directional antenna, all determinations of service provided and interference caused shall...

  6. Power electronic converter systems for direct drive renewable energy applications

    DEFF Research Database (Denmark)

    Chen, Zhe

    2013-01-01

    This chapter presents power electronic conversion systems for wind and marine energy generation applications, in particular, direct drive generator energy conversion systems. Various topologies are presented and system design optimization and reliability are briefly discussed....

  7. Nanog reporter system in mouse embryonic stem cells based on highly efficient BAC homologous recombination

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indicating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related factors in mES cells.

  8. Medical Robots: Current Systems and Research Directions

    OpenAIRE

    Beasley, Ryan A.

    2012-01-01

    First used medically in 1985, robots now make an impact in laparoscopy, neurosurgery, orthopedic surgery, emergency response, and various other medical disciplines. This paper provides a review of medical robot history and surveys the capabilities of current medical robot systems, primarily focusing on commercially available systems while covering a few prominent research projects. By examining robotic systems across time and disciplines, trends are discernible that imply future capabilities ...

  9. Double promoter expression systems for recombinant protein production by industrial microorganisms.

    Science.gov (United States)

    Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

    2017-09-12

    Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more

  10. The unconventional Xer recombination machinery of Streptococci/Lactococci.

    Directory of Open Access Journals (Sweden)

    Pascal Le Bourgeois

    2007-07-01

    Full Text Available Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (dif(SL associated with a dedicated tyrosine recombinase (XerS. In contrast to the E. coli Xer system, only a single recombinase is required to recombine dif(SL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when dif(SL sites are located on chromosome dimers. Moreover, the XerS/dif(SL recombination requires the streptococcal protein FtsK(SL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.

  11. A novel and highly efficient production system for recombinant adeno-associated virus vector

    Institute of Scientific and Technical Information of China (English)

    WU; Zhijian(伍志坚); WU; Xiaobing(吴小兵); CAO; Hui(曹晖); DONG; Xiaoyan(董小岩); WANG; Hong(王宏); HOU; Yunde(侯云德)

    2002-01-01

    Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  12. Initiation of V(D)J recombination in a cell-free system

    NARCIS (Netherlands)

    D.C. van Gent (Dik); J.F. McBlane; D.A. Ramsden; M.J. Sadokfsky; J.E. Hesse (Joanne); M. Gellert

    1995-01-01

    textabstractCells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-leng

  13. Medical Robots: Current Systems and Research Directions

    Directory of Open Access Journals (Sweden)

    Ryan A. Beasley

    2012-01-01

    Full Text Available First used medically in 1985, robots now make an impact in laparoscopy, neurosurgery, orthopedic surgery, emergency response, and various other medical disciplines. This paper provides a review of medical robot history and surveys the capabilities of current medical robot systems, primarily focusing on commercially available systems while covering a few prominent research projects. By examining robotic systems across time and disciplines, trends are discernible that imply future capabilities of medical robots, for example, increased usage of intraoperative images, improved robot arm design, and haptic feedback to guide the surgeon.

  14. Twin arginine translocation system in secretory expression of recombinant human growth hormone

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Bagherinejad

    2016-01-01

    Full Text Available Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3 to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

  15. A marine direction finding system based on global positioning system

    Science.gov (United States)

    Dǎnişor, Alin; Izet-Ünsalan, Kunsel-Özel; Ünsalan, Deniz; Tamaş, Razvan; Dǎnişor, Cosmin

    2015-02-01

    Direction finding and attitude determination is of utmost importance for marine, aerial, spatial and land-based navigation [1], as well as control of vehicles, in surveying and in target acquisition of tracking radars. These problems can be solved using dedicated sensors commonly named as compasses and rate gyros. Unfortunately, the classical means of attitude determination both by magnetic and gyrocompasses become unusable at extreme latitudes. Furthermore, gyrocompasses inherently yield erroneous results on high speed craft. Three-axis attitude of a vehicle can be determined using a GPS receiver with multiple antennas, by measuring carrier phases [2], signal strength [3], or integrated INS/GPS systems [4]. This paper proposes a new method of attitude determination using two low-cost GPS receivers.

  16. Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems.

    Science.gov (United States)

    Kenny, Diarmuid T; Gaunitz, Stefan; Hayes, Catherine A; Gustafsson, Anki; Sjöblom, Magnus; Holgersson, Jan; Karlsson, Niclas G

    2013-01-01

    Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.

  17. Direction Identification System of Garlic Clove Based on Machine Vision

    Directory of Open Access Journals (Sweden)

    Gao Chi

    2013-05-01

    Full Text Available In order to fulfill the requirements of seeding direction of garlic cloves, the paper proposed a research method of garlic clove direction identification based on machine vision, it expounded the theory of garlic clove direction identification, stated the arithmetic of it, designed the direction identification device of it, then developed the control system of garlic clove direction identification based on machine vision, at last tested the garlic clove direction identification, and the result of the experiment certificated that the rate of garlic clove direction identification could reach to more than 97%, and it demonstrated that the research is of high feasibility and technological values.

  18. Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

    Science.gov (United States)

    Hansen, Debra T; Thiyagarajan, Thirumagal; Larson, Amy C; Hansen, Jeffrey L

    2016-07-01

    Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.

  19. CRISPR-Cas and contact dependent secretion systems present on excisable pathogenicity islands with conserved recombination modules.

    Science.gov (United States)

    Carpenter, Megan R; Kalburge, Sai S; Borowski, Joseph D; Peters, Molly C; Colwell, Rita R; Boyd, E Fidelma

    2017-03-06

    Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB, via integrase mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic V. cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio Pathogenicity Island-1 (VPI-1) insertion site in nineteen V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in twelve V. cholerae strains on a 68 kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site-specifically from the bacterial chromosome as complete units and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs.Importance This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and

  20. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami

    2017-05-08

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  1. A novel synthetic medium and expression system for subzero growth and recombinant protein production in Pseudoalteromonas haloplanktis TAC125.

    Science.gov (United States)

    Sannino, F; Giuliani, M; Salvatore, U; Apuzzo, G A; de Pascale, D; Fani, R; Fondi, M; Marino, G; Tutino, M L; Parrilli, E

    2017-01-01

    The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a model organism of cold-adapted bacteria. The interest in the study of this psychrophilic bacterium stems from its capability either as a non-conventional system for production of recombinant protein and as a rich source of bioactive compounds. To further explore the biotechnological ability of P. haloplanktis TAC125, we have developed a synthetic medium, containing D-gluconate and L-glutamate (GG), which allows the bacterium to grow even at subzero temperatures. P. haloplanktis TAC125 growing in GG medium at low temperature displays growth kinetic parameters which confirm its spectacular adaptation to cold environment and subzero lifestyle, paving the way to the definition of the underlying molecular strategies. Moreover, in this paper, we report the setup of a finely regulated gene expression system inducible by D-galactose to produce recombinant protein in GG synthetic medium at temperatures as low as -2.5 °C. Thanks to the combination of the novel medium and the new expression system, we obtained for the first time the production of a recombinant protein at subzero temperature, thus providing an innovative strategy for the recombinant production of "difficult" proteins.

  2. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    Science.gov (United States)

    Roth, D B; Wilson, J H

    1985-05-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination.

  3. Direct methanol feed fuel cell and system

    Science.gov (United States)

    Surampudi, Subbarao (Inventor); Frank, Harvey A. (Inventor); Narayanan, Sekharipuram R. (Inventor); Chun, William (Inventor); Jeffries-Nakamura, Barbara (Inventor); Kindler, Andrew (Inventor); Halpert, Gerald (Inventor)

    2009-01-01

    Improvements to non acid methanol fuel cells include new formulations for materials. The platinum and ruthenium are more exactly mixed together. Different materials are substituted for these materials. The backing material for the fuel cell electrode is specially treated to improve its characteristics. A special sputtered electrode is formed which is extremely porous. The fuel cell system also comprises a fuel supplying part including a meter which meters an amount of fuel which is used by the fuel cell, and controls the supply of fuel based on said metering.

  4. Development of a direct contact ice storage system

    Energy Technology Data Exchange (ETDEWEB)

    Poirier, R. [Chicago Bridge & Iron Company, Plainfield, IL (United States)

    1989-03-01

    The program described involves the design, construction, and performance testing of a Direct Freeze Thermal Energy Storage System. Task 1 (Design) has been completed; and Task 2 (construction) is in progress, with equipment procurements presently underway. Once constructed, the system will undergo extensive laboratory performance testing and analysis, followed by an assessment of the system`s cost effectiveness. This study will advance the understanding and development of the direct freeze concept, which offers inherent benefits for thermal energy storage.

  5. Direction finding antenna system for spark detection and localization

    Science.gov (United States)

    Topor, Raluca E.; Bucuci, Stefania C.; Tamas, Razvan D.; Danisor, Alin; Dumitrascu, Ana; Berescu, Serban

    2015-02-01

    This paper proposes a novel UWB antenna system for spark detection and localization by using the amplitude comparison direction finding (DF) method. The proposed design consists of two identical axially crossed "padlock" shaped UWB antennas, with unbalanced feeding. Simulation results show that such radiating systems can be used for assessing the direction of arrival for short pulses.

  6. Directed Transport of Interacting Particle Systems: Recent Progress

    Institute of Scientific and Technical Information of China (English)

    ZHENG Zhi-Gang

    2005-01-01

    Recent developments in studies of directed transport processes in interacting particle systems are retrospected. Due to the interactions among elements, the directed transport process exhibits complicated and novel cooperative dynamics. We considered various possibilities in achieving ratchet motion by breaking different symmetries of many-body systems. It is shown that the directional transport can even be induced by breaking the coupling symmetry and the spatiotemporal symmetries.

  7. Unambiguous identification of recombination lines in single zinc-blende ZnSe nanowires in direct relation to their microstructure.

    Science.gov (United States)

    Saxena, Ankur; Pan, Qi; Ruda, Harry E

    2013-03-15

    We present results on the low-temperature photoluminescence characterization of individual ZnSe nanowires, whose crystal structure was determined to be zinc-blende by transmission electron microscopy on the same individual nanowires as were studied optically. The photoluminescence response from single ZnSe nanowires was found to be dominated by excitonic emission due to native point defects, while no emission peaks related to the unintentional impurities were detected. Two strong photoluminescence lines were observed at 2.785 eV and 2.780 eV, assigned to the excitons bound to deep neutral acceptors related to the vacancies of Zn (V(Zn)) and complexes of V(Zn), respectively. Another recombination peak at 2.800 eV related to the free exciton emission in ZnSe was also observed. Longitudinal optical-phonon replicas of up to three orders were seen for both lines, and the average number of emitted phonons was also determined. The excitonic emission linewidths of 1.5 meV were observed from individual nanowires, which are the narrowest excitonic linewidths reported so far for ZnSe nanowires. The optical response from a single nanowire was also compared to that from a bundle of nanowires, and it was found that the linewidths of excitonic emission from the bundle of nanowires were slightly larger than those from single nanowires, due to the effects of ensemble broadening. It is also suggested that in the case of a bundle of nanowires, the broadening is limited by the nanowire which exhibits the largest excitonic linewidth.

  8. Refolding of recombinant human granulocyte colony stimulating factor: effect of cysteine/cystine redox system.

    Science.gov (United States)

    Tiwari, Krishnanand; Shebannavar, Sunil; Kattavarapu, Krishna; Pokalwar, Santosh; Mishra, Maheshwari K; Chauhan, Ugam Kumari

    2012-08-01

    Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as inclusion bodies (IBs) in E. coli was optimized using cysteine and cystine redox agents. The refolding to correct native form of G-CSF was assessed by reverse phase high performance liquid chromatography (RP-HPLC). The optimized concentrations of cysteine and cystine for correct refolding of G-CSF were found to be 2 mM and 1 mM, respectively. The correctly refolded G-CSF was detected as early as 4 h of incubation in renaturation buffer containing optimized concentrations of cysteine (2 mM) and cystine (1 mM) redox agents. Refolding of G-CSF in optimized redox system increased with increase in shuffling time. Overall, the results suggested the use of cysteine/cystine redox pair could be an alternative to the costlier redox pairs for successful refolding of G-CSF and possibly other human biotherapeutic proteins of importance.

  9. MuLV packaging systems as models for estimating/measuring retrovirus recombination frequency.

    Science.gov (United States)

    Patience, C; Takeuch, Y; Cosset, F L; Weiss, R A

    2001-01-01

    Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in the formation of recombinant viruses. Using sensitive RT-PCR assays, we have investigated human and murine gene therapy packaging cell lines for the incorporation of endogenous retrovirus transcripts into murine leukaemia virus (MLV) vector particles and whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived -galactosidase beta-Gal vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Non-specific packaging of the MLV gag-pol expression vector transcripts was detected in the FLY virions at a low level (one in 17,000 sequences). In other experiments, gag proteins produced by HERV-K particles present in human teratocarcinoma cells did not appear to package MLV-based vectors that expressed Gal transcripts. These findings indicate that retrovirus vectors interact with human packaging cells to produce retrovirus particles that are far less contaminated by endogenous viral sequences or other types of extraneous particles than murine packaging cells.

  10. Charge recombination versus charge separation in donor-bridge-acceptor systems.

    Science.gov (United States)

    Wiberg, Joanna; Guo, Lijun; Pettersson, Karin; Nilsson, Daniel; Ljungdahl, Thomas; Mårtensson, Jerker; Albinsson, Bo

    2007-01-10

    Optimizing the ratio of the rates for charge separation (CS) over charge recombination (CR) is crucial to create long-lived charge-separated states. Mastering the factors that govern the electron transfer (ET) rates is essential when trying to achieve molecular-scale electronics, artificial photosynthesis, and also for the further development of solar cells. Much work has been put into the question of how the donor-acceptor distances and donor-bridge energy gaps affect the electronic coupling, V(DA), and thus the rates of ET. We present here a unique comparison on how these factors differently influence the rates for CS and CR in a porphyrin-based donor-bridge-acceptor model system. Our system contains three series, each of which focuses on a separate charge-transfer rate-determining factor, the donor-acceptor distance, the donor-bridge energy gap, and last, the influence of the electron acceptor on the rate for charge transfer. In these three series both CS and CR are governed by superexchange interactions which make a CR/CS comparative study ideal. We show here that the exponential distance dependence increases slightly for CR compared to that for CS as a result of the increased tunneling barrier height for this reaction, in accordance with the McConnell superexchange model. We also show that the dependence on the tunneling barrier height is different for CS and CR. This difference is highly dependent on the electron acceptor and thus cannot solely be explained by the differences in the frontier orbitals of the electron donor in these porphyrin systems.

  11. AUTOMATED SYSTEM FOR CELL PHONES PRECISE DIRECTION FINDING

    Directory of Open Access Journals (Sweden)

    H. M. Gadzhiev

    2016-01-01

    Full Text Available It is proved that the navigation system use for direction finding of cell phones will greatly expand the functionality and services of telephone networks mobile operators. The direction-finding system advantages and creation of automated system of direction finding cell phones proposed, which significantly expand the possibilities of services of operators of cellular communication in the cell using standard computer equipment operators themselves have been revelead. The developed system has a modular design, which allows modifying software packages, expanding the functionality. The procedure and algorithm of direction finding, allowing piecewise linear approximation of the graph of the displacement of each subscriber is outlined. The developed direction-finding system application for statistical information and optimization of urban public services processing, choice of speed, route and other activities improving the quality of human life is proposed.

  12. Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: Different requirements for the RAD1, RAD 10, and RAD52 genes

    Energy Technology Data Exchange (ETDEWEB)

    Prado, F.; Aguilera, A. [Universidad de Sevilla (Spain)

    1995-01-01

    We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB)-induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence. 74 refs., 5 figs., 6 tabs.

  13. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  14. RECOMBINANT HORSERADISH PEROXIDASE FOR ANALYTICAL APPLICATIONS

    OpenAIRE

    2013-01-01

    The article deals with prospects of using recombinant horseradish peroxidase in analytical biochemistry and biotechnology. Problems of recombinant horseradish peroxidase cloning in different expression systems, possible approaches to their solution, advantages of recombinant recombinant horseradish peroxidase and recombinant horseradish peroxidase-fusion proteins for immunoassays are considered. Possibility for development of mediatorless bienzyme biosensor for peroxide and metabolites, yield...

  15. The development of direct write technology in BAE Systems

    Science.gov (United States)

    Sidhu, J.; McDonald, J.; Morgan, S.; Wescott, Andrew; O'Driscoll, E. J.

    2006-12-01

    Direct Write (DW) is an emerging group of technologies that allow printing of electronic and other functional components out of vacuum, directly onto structural parts and assemblies. With its ability to deposit a wide range of dissimilar materials, and transfer details directly from CAD/CAM, the process is very flexible, enabling rapid progress from design to fabrication. This paper provides an introduction to direct write, and describes the BAE Systems activities in this field. The paper also describes the use of lasers in direct write, and some provisional results on laser curing are presented.

  16. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhong-Min GUO; Kang XU; Ying YUE; Bing HUANG; Xin-Yan DENG; Nü-Qi ZHONG; Xun HONG; Xi-Gu CHEN; Dong XIAO

    2005-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTAtetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur.Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

  17. A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform.

    Directory of Open Access Journals (Sweden)

    Li Tian

    Full Text Available BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV. The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.

  18. Opto-electro-modulated transient photovoltage and photocurrent system for investigation of charge transport and recombination in solar cells

    Science.gov (United States)

    Shi, Jiangjian; Li, Dongmei; Luo, Yanhong; Wu, Huijue; Meng, Qingbo

    2016-12-01

    An opto-electro-modulated transient photovoltage/photocurrent system has been developed to probe microscopic charge processes of a solar cell in its adjustable operating conditions. The reliability of this system is carefully determined by electric circuit simulations and experimental measurements. Using this system, the charge transport, recombination and storage properties of a conventional multicrystalline silicon solar cell under different steady-state bias voltages, and light illumination intensities are investigated. This system has also been applied to study the influence of the hole transport material layer on charge extraction and the microscopic charge processes behind the widely considered photoelectric hysteresis in perovskite solar cells.

  19. Opto-electro-modulated transient photovoltage and photocurrent system for investigation of charge transport and recombination in solar cells.

    Science.gov (United States)

    Shi, Jiangjian; Li, Dongmei; Luo, Yanhong; Wu, Huijue; Meng, Qingbo

    2016-12-01

    An opto-electro-modulated transient photovoltage/photocurrent system has been developed to probe microscopic charge processes of a solar cell in its adjustable operating conditions. The reliability of this system is carefully determined by electric circuit simulations and experimental measurements. Using this system, the charge transport, recombination and storage properties of a conventional multicrystalline silicon solar cell under different steady-state bias voltages, and light illumination intensities are investigated. This system has also been applied to study the influence of the hole transport material layer on charge extraction and the microscopic charge processes behind the widely considered photoelectric hysteresis in perovskite solar cells.

  20. Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

    Directory of Open Access Journals (Sweden)

    Dennis J. Díaz-Rincón

    2017-01-01

    Full Text Available Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1 were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

  1. High Temperature 300°C Directional Drilling System

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, Kamalesh [Baker Hughes Oilfield Operations, Houston, TX (United States); Aaron, Dick [Baker Hughes Oilfield Operations, Houston, TX (United States); Macpherson, John [Baker Hughes Oilfield Operations, Houston, TX (United States)

    2015-07-31

    Many countries around the world, including the USA, have untapped geothermal energy potential. Enhanced Geothermal Systems (EGS) technology is needed to economically utilize this resource. Temperatures in some EGS reservoirs can exceed 300°C. To effectively utilize EGS resources, an array of injector and production wells must be accurately placed in the formation fracture network. This requires a high temperature directional drilling system. Most commercial services for directional drilling systems are rated for 175°C while geothermal wells require operation at much higher temperatures. Two U.S. Department of Energy (DOE) Geothermal Technologies Program (GTP) projects have been initiated to develop a 300°C capable directional drilling system, the first developing a drill bit, directional motor, and drilling fluid, and the second adding navigation and telemetry systems. This report is for the first project, “High Temperature 300°C Directional Drilling System, including drill bit, directional motor and drilling fluid, for enhanced geothermal systems,” award number DE-EE0002782. The drilling system consists of a drill bit, a directional motor, and drilling fluid. The DOE deliverables are three prototype drilling systems. We have developed three drilling motors; we have developed four roller-cone and five Kymera® bits; and finally, we have developed a 300°C stable drilling fluid, along with a lubricant additive for the metal-to-metal motor. Metal-to-metal directional motors require coatings to the rotor and stator for wear and corrosion resistance, and this coating research has been a significant part of the project. The drill bits performed well in the drill bit simulator test, and the complete drilling system has been tested drilling granite at Baker Hughes’ Experimental Test Facility in Oklahoma. The metal-to-metal motor was additionally subjected to a flow loop test in Baker Hughes’ Celle Technology Center in Germany, where it ran for more than 100

  2. Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system.

    Science.gov (United States)

    Yu, YunZhou; Shi, DanYang; Liu, Si; Gong, Zheng-Wei; Wang, Shuang; Sun, ZhiWei

    2015-01-01

    Although Escherichia coli and yeast were commonly used to express recombinant Hc of botulinum neurotoxins, as an alternative, in current study, a 293E expression system was used to express the Hc of botulinum neurotoxin serotype B (BHc) as soluble recombinant protein for experimental vaccine evaluation. Our results demonstrated that the 293E expression system could produce high level of recombinant secreted BHc protein, which was immunorecognized specifically by anti-botulinum neurotoxin serotype B (BoNT/B) sera and showed ganglioside binding activities. The serological response and efficacy of recombinant BHc formulated with aluminum hydroxide adjuvant were evaluated in mice. Immunization with Alhydrogel-formulated BHc subunit vaccine afforded the effective protection against BoNT/B challenge. A frequency- and dose-dependent effect to immunization with BHc subunit vaccine was observed and the ELISA antibody titers correlated well with neutralizing antibody titers and protection. And a solid-phase assay showed that the neutralizing antibodies from the BHc-immunized mice inhibited the binding of BHc to the ganglioside GT1b. Our results also show that the plasmid pABE293SBHc derived of the 293E expression system as DNA vaccine is capable of inducing stronger humoral response and protective efficacy against BoNT/B than the pVAX1SBHc. In summary, immunization with the 293E-expressed BHc protein generates effective immune protection against BoNT/B as E. coli or yeast-expressed BHc, so the efficient expression of botulinum Hc protein for experimental vaccine can be prepared using the 293E expression system.

  3. Using an OPO laser system for recombination studies in a storage ring: LIR to n = 3 states of deuterium

    Energy Technology Data Exchange (ETDEWEB)

    Justiniano, E.; Asp, S.; DeWitt, D.R.; Schuch, R. [Stockholm Univ. (Sweden). Dept. of Atomic Phys.; Andler, G. [Manne Siegbahn Lab., Stockholm Univ. (Sweden)

    1997-04-01

    We have investigated the use of a one-stage optical parametric oscillator (OPO) laser system for studies of laser induced radiative recombination (LIR) of stored ions. For the test case considered, LIR into n = 3 states of deuterium, we find that the energy resolution is limited by the broad linewidth of the OPO. Within this resolution, our LIR results are in good agreement with a theoretical average field induced threshold shift model. (orig.). 17 refs.

  4. Computer Directed Training System (CDTS), User’s Manual

    Science.gov (United States)

    1983-07-01

    94111447030 OPInONAL FORM 2.2 BACK (4-77) DEPARTMENT OF THE AIR FORCE A AUL5-5 DISRIBceO LIMITED TO DOD, REFR TER REQUESTS TO THE ADPS MANAGER...SYSTEM SUMMARY 2.1 System Aplication . The Computer Directed Training System is used to prepare and present lessons that supplement local on-the-job

  5. Three-body recombination in heteronuclear systems at finite temperature with a large positive scattering length

    Science.gov (United States)

    Emmons, Samuel; Acharya, Bijaya; Platter, Lucas

    2017-01-01

    For an ultracold heteronuclear mixture with a large positive interspecies scattering length and negligible intraspecies scattering length, we determine the three-body recombination rate as a function of collision energy using universal functions of a single scaling variable. We use the zero-range approximation and the Skorniakov -Ter-Martirosian equation to calculate these scaling functions for a range of collision energies. Further, we explore the effects that a nonzero temperature has on three-body recombination, as well as the effects of the formation of deep dimers, for experimentally relevant heteronuclear gases such as the 6Li-133Cs mixture. NSF Grant Nos. PHY-1516077 and PHY-1555030.

  6. 78 FR 13316 - National Forest System Land Management Planning Directives

    Science.gov (United States)

    2013-02-27

    ... Forest Service RIN 0596-AD06 National Forest System Land Management Planning Directives AGENCY: Forest...) land management planning regulation. Issuance of these proposed directives will provide consistent... CONTACT: Annie Eberhart Goode, Planning Specialist, Ecosystem Management Coordination Staff, 202-205-1056...

  7. mathematical model for direct evaporative space cooling systems

    African Journals Online (AJOL)

    eobe

    MATHEMATICAL MODEL FOR DIRECT EVAPORATIVE SPACE COOLING. SYSTEMS ... Water is the working fluid in evaporative cooling thus it is ..... co o lin g efficien cy (%. ) Time (hrs) predicted experimental. 0. 10. 20. 30. 40. 50. 60. 70. 80.

  8. Direct Ethanol Production from Lignocellulosic Sugars and Sugarcane Bagasse by a Recombinant Trichoderma reesei Strain HJ48

    Directory of Open Access Journals (Sweden)

    Jun Huang

    2014-01-01

    Full Text Available Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.

  9. Langevin model for a Brownian system with directed motion

    Science.gov (United States)

    Ambía, Francisco; Híjar, Humberto

    2016-08-01

    We propose a model for an active Brownian system that exhibits one-dimensional directed motion. This system consists of two Brownian spherical particles that interact through an elastic potential and have time-dependent radii. We suggest an algorithm by which the sizes of the particles can be varied, such that the center of mass of the system is able to move at an average constant speed in one direction. The dynamics of the system is studied theoretically using a Langevin model, as well as from Brownian Dynamics simulations.

  10. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  11. Modifying Directionality through Auditory System Scaling in a Robotic Lizard

    DEFF Research Database (Denmark)

    Shaikh, Danish; Hallam, John; Christensen-Dalsgaard, Jakob

    2010-01-01

    The peripheral auditory system of a lizard is strongly directional. This directionality is created by acoustical coupling of the two eardrums and is strongly dependent on characteristics of the middle ear, such as interaural distance, resonance frequency of the middle ear cavity and of the tympanum....... Therefore, directionality should be strongly influenced by their scaling. In the present study, we have exploited an FPGA–based mobile robot based on a model of the lizard ear to investigate the influence of scaling on the directional response, in terms of the robot’s performance in a phonotaxis task...

  12. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  13. High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system

    NARCIS (Netherlands)

    Wiel, van de D.F.M.; Rijn, van P.A.; Meloen, R.H.; Moormann, R.J.M.

    1998-01-01

    Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain

  14. Systemic immune responses to oral administration of recombinant attenuated Salmonella typhimurium expressing Helicobacter pyloriurease in mice

    Institute of Scientific and Technical Information of China (English)

    Xiao-Feng Liu; Jia-Lu Hu; Qi-Zheng Quan; Zi-Qin Sun; Yao-Jun Wang; Feng Qi

    2005-01-01

    AIM: To evaluate whether attenuated Salmonellatyphimurium producing Helicobacter pylori ( H pylori) urease subunit B (UreB) could induce systemic immune responses against H pylori infection.METHODS: Attenuated S. typhimurium SL3261 was used as a live carrier of plasmid pTC01-UreB, which encodes recombinant H pylori UreB protein. Balb/c mice were given oral immunization with two doses of SL3261/pTC01-UreBat a 3-wk interval. Twelve weeks after oral immunization of mice, serum IgG antibodies were evaluated by ELISA assay. Gamma interferon (IFN-γ) and interleukin 10 (IL-10)in the supernatant of spleen cell culture were also assessed by ELISA.RESULTS: After oral immunization of mice, serum specific IgG antibodies against UreB in vaccine group were much higher than that in PBS and native Salmonella SL3261control groups (A450, 0.373±0.100 vs 0.053±0.022, 0.142±0.039, respectively, P<0.01). Moreover, IFN-γ in vaccine group was on average 167.53±29.93 pg/mL, which showed a significant increase vs that of PBS control group (35.68±3.55 pg/mL, P<0.01). There was also a tremendous increase of IL-L0 in vaccine group compared to PBS and SL3261 control groups (275.13±27.65 pg/mL vs 56.00±7.15 pg/mL, 68.02±15.03 pg/mL, respectively, P<0.01). In addition, no obvious side effects in mice and no change in gastric inflammation were observed. CONCLUSION: The multiple oral immunizations with the attenuated S. typhimurium expressing H pylori UreB could induce significant systemic immune responses, suggesting it may be used as oral vaccine against H pylori infection.

  15. Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Sylvia Merkert

    2014-01-01

    Full Text Available Genetic engineering of human induced pluripotent stem cells (hiPSCs via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs. Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

  16. Directed transport of coupled systems in symmetric periodic potentials

    Institute of Scientific and Technical Information of China (English)

    郑志刚; 刘凤芝; 高建

    2003-01-01

    In this paper, we discuss the damped unidirectional motions of a coupled lattice in a periodic potential. Each particle in the lattice is subject to a time-periodic ac force. Our studies reveal that a directed transport process can be observed when the ac forces acting on the coupled lattice have a phase shift (mismatch). This directed motion is a collaboration of the coupling, the substrate potential, and the periodic force, which are all.symmetric. The absence of any one of these three factors will not give rise to a directed current. We discuss the complex relations between the directed current and parameters in the system. Results in this paper can be accomplished in experiments. Moreover,our results can be generalized to the studies of directed transport processes in more complicated spatially extended systems.

  17. Production of a recombinant laccase from Pichia pastoris and biodegradation of chlorpyrifos in a laccase/vanillin system.

    Science.gov (United States)

    Xie, Huifang; Li, Qi; Wang, Minmin; Zhao, Linguo

    2013-06-28

    The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60°C, but it was not stable at high temperature. The enzyme could remain stable at 30°C for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028).

  18. A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

    Science.gov (United States)

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

    2015-03-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.

  19. A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer

    NARCIS (Netherlands)

    Riezebos-Brilman, A.; Walczak, M.; Regts, J.; Rots, Mg; Kamps, G.; Dontje, B.; Haisma, Hy; Wilschut, J.; Daemen, T.

    2007-01-01

    Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical

  20. The differential effects of recombinant brain natriuretic peptide, nitroglycerine and dihydralazine on systemic oxygen delivery and gastric mucosal microvascular oxygenation in dogs*

    NARCIS (Netherlands)

    Schwarte, L. A.; Schwartges, I.; Scheeren, T. W. L.; Schober, P.; Picker, O.

    Brain natriuretic peptide has vasodilatory properties and may thus increase splanchnic perfusion and oxygenation. We compared the effects of recombinant brain natriuretic peptide on gastric mucosal microvascular haemoglobin oxygenation (reflectance spectrophotometry) and systemic variables with

  1. Direct laser additive fabrication system with image feedback control

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, Michelle L. (Albuquerque, NM); Hofmeister, William H. (Nashville, TN); Knorovsky, Gerald A. (Albuquerque, NM); MacCallum, Danny O. (Edgewood, NM); Schlienger, M. Eric (Albuquerque, NM); Smugeresky, John E. (Pleasanton, CA)

    2002-01-01

    A closed-loop, feedback-controlled direct laser fabrication system is disclosed. The feedback refers to the actual growth conditions obtained by real-time analysis of thermal radiation images. The resulting system can fabricate components with severalfold improvement in dimensional tolerances and surface finish.

  2. Scaling of the direct ECC bypass during LBLOCA reflood phase with direct vessel injection system

    Energy Technology Data Exchange (ETDEWEB)

    Yun, B.J.; Kwon, T.S.; Song, C.H.; Jeong, J.J. [Korea Atomic Energy Research Inst., Taejon (Korea, Republic of); Cho, H.K.; Park, G.C. [Seoul National Univ., Dept. of Nuclear Engineering (Korea, Republic of)

    2001-07-01

    As one of the advanced design features of the Korea next generation reactor, direct vessel injection (DVI) system is being considered instead of conventional cold leg injection (CLI) system. It is known that the DVI system greatly enhances the reliability of the emergency core cooling (ECC) system. However, there is still a dispute on its performance in terms of water delivery to the reactor core during the reflood period of a large-break loss-of-coolant accident (LOCA). Thus, experimental validation is under progress. In this paper, a new scaling method, using time and velocity reduced linear scaling law, is suggested for the design of a scaled-down experimental facility to investigate the direct ECC bypass phenomena in PWR downcomer. (authors)

  3. Direct electromechanical driving systems: diversity, constraints and solutions; Les entrainements electromecaniques directs: diversite, contraintes et solutions

    Energy Technology Data Exchange (ETDEWEB)

    Multon, B. [Ecole Normale Superieure, 94 - Cachan (France); Bonal, J. [Promethee Groupe Schneider Electric S.A., 92 - Boulogne-Billancourt (France)

    1999-11-01

    Direct electromechanical drives are the simplest possible transmission systems requiring no additional mechanical components such as gearboxes or rotary to linear converters. This simplicity leads a number of advantages including greater reliability, less maintenance, improved dynamic characteristics and reduced weight and volume. However, the performance specification of direct drives is usually high, with a correspondingly high cost, and often this cannot be justified in complete systems where the other components limit the overall performance. This factor alone has been sufficient to restrict the market penetration of direct drives. The majority of applications are in linear actuators, and in motors operating at very high or very low speeds. New developments are constantly being introduced, especially in relation to structures.

  4. Direct hydrogen fuel cell systems for hybrid vehicles

    Science.gov (United States)

    Ahluwalia, Rajesh K.; Wang, X.

    Hybridizing a fuel cell system with an energy storage system offers an opportunity to improve the fuel economy of the vehicle through regenerative braking and possibly to increase the specific power and decrease the cost of the combined energy conversion and storage systems. Even in a hybrid configuration it is advantageous to operate the fuel cell system in a load-following mode and use the power from the energy storage system when the fuel cell alone cannot meet the power demand. This paper discusses an approach for designing load-following fuel cell systems for hybrid vehicles and illustrates it by applying it to pressurized, direct hydrogen, polymer-electrolyte fuel cell (PEFC) systems for a mid-size family sedan. The vehicle level requirements relative to traction power, response time, start-up time and energy conversion efficiency are used to select the important parameters for the PEFC stack, air management system, heat rejection system and the water management system.

  5. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  6. Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

    Science.gov (United States)

    2008-06-06

    for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever . In this regard...prevention of Lassa fever . In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications...the Arenaviridae family, is the etiologic agent of Lassa fever , which is an acute and often fatal ill- ness endemic to West Africa. There are an

  7. Positioning via Direct Localization in C-RAN Systems

    OpenAIRE

    Jeong, Seongah; Simeone, Osvaldo; Haimovich, Alexander; Kang, Joonhyuk

    2015-01-01

    Cloud Radio Access Network (C-RAN) is a prominent architecture for 5G wireless cellular system that is based on the centralization of baseband processing for multiple distributed radio units (RUs) at a control unit (CU). In this work, it is proposed to leverage the C-RAN architecture to enable the implementation of direct localization of the position of mobile devices from the received signals at distributed RUs. With ideal connections between the CU and the RUs, direct localization is known ...

  8. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an af...

  9. Dual readout 3D direct/induced-signals pixel systems

    CERN Document Server

    Parker, Sherwood; Deile, Mario; Hansen, Thor-Erik; Hasi, Jasmine; Kenney, Christopher; Kok, Angela; Watts, Stephen

    2008-01-01

    In this paper, 3D-electrode pixel detectors are described, in which the bias electrode systems have additional elements. Adding resistors between the bias supply line and each bias electrode together with a signal electrode readout that can measure pulse heights of both polarities could simultaneously provide lower capacitance and improved spatial resolution in both directions. A separate paper (“Dual-readout—strip/pixel systems”) covers an alternative—pixels with an added strip readout in one direction which could be used with either planar or 3D-electrodes, and could simultaneously provide a fast trigger and significantly increase the spatial resolution in both directions.

  10. Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

    Science.gov (United States)

    Kuno, T; Shuntoh, H; Sakaue, M; Saijoh, K; Takeda, T; Fukuda, K; Tanaka, C

    1988-06-30

    The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

  11. Photoelectrochemical based direct conversion systems for hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Kocha, S.; Peterson, M.; Arent, D. [National Renewable Energy Lab., Golden, CO (United States)] [and others

    1996-10-01

    Photon driven, direct conversion systems consist of a light absorber and a water splitting catalyst as a monolithic system; water is split directly upon illumination. This one-step process eliminates the need to generate electricity externally and subsequently feed it to an electrolyzer. These configurations require only the piping necessary for transport of hydrogen to an external storage system or gas pipeline. This work is focused on multiphoton photoelectrochemical devices for production of hydrogen directly using sunlight and water. Two types of multijunction cells, one consisting of a-Si triple junctions and the other GaInP{sub 2}/GaAs homojunctions, were studied for the photoelectrochemical decomposition of water into hydrogen and oxygen from an aqueous electrolyte solution. To catalyze the water decomposition process, the illuminated surface of the device was modified either by addition of platinum colloids or by coating with ruthenium dioxide. These colloids have been characterized by gel electrophoresis.

  12. Application of GIS in foreign direct investment decision support system

    Science.gov (United States)

    Zhou, Jianlan; Sun, Koumei

    2007-06-01

    It is important to make decisions on how to attract foreign direct investment (FDI) to China and know how the inequality of FDI introduction by locational different provinces. Following background descriptions on China's FDI economic environments and FDI-related policies, this paper demonstrates the uses of geographical information system (GIS) and multi-criterion decision-making (MCDM) framework in solving a spatial multi-objective problem of evaluating and ranking China's provinces for FDI introduction. It implements a foreign direct investment decision support system, which reveals the main determinants of FDI in China and gives some results of regional geographical analysis over spatial data.

  13. Directly modulated cable television transport systems using negative dispersion fiber

    Science.gov (United States)

    Lu, Hai-Han; Liaw, Je-Wei; Lee, Yi-Shiuan; Tsai, Wan-Lin; Ji, Yu-Jie

    2005-03-01

    A directly modulated AM-VSB cable-television transport system using negative dispersion fiber (NDF) as the transmission medium is proposed and successfully demonstrated. Good performances of carrier-to-noise radio, composite second order, and composite triple beat were obtained over a 70-km NDF transport without optical amplification. The directly modulated laser has a positive chirp, while NDF has a negative dispersion property in the transmission fiber. This negative dispersion property compensates for the laser chirp and results in a system with better transmission performance.

  14. Experimental investigation of a directionally enhanced DHX concept for high temperature Direct Reactor Auxiliary Cooling Systems

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, Joel T.; Blandford, Edward D., E-mail: edb@unm.edu

    2016-07-15

    Highlights: • A novel directional heat exchanger design has been developed. • Hydrodynamic tests have been performed on the proposed design. • Heat transfer performance is inferred by hydrodynamic results. • Results are discussed and future work is suggested. - Abstract: The use of Direct Reactor Auxiliary Cooling Systems (DRACSs) as a safety-related decay heat removal system for advanced reactors has developed historically through the Sodium Fast Reactor (SFR) community. Beginning with the EBR-II, DRACSs have been utilized in a large number of past and current SFR designs. More recently, the DRACS has been adopted for Fluoride Salt-Cooled High-Temperature Reactors (FHRs) for similar decay heat removal functions. In this paper we introduce a novel directionally enhanced DRACS Heat Exchanger (DHX) concept. We present design options for optimizing such a heat exchanger so that shell-side heat transfer is enhanced in one primary coolant flow direction and degraded in the opposite coolant flow direction. A reduced-scale experiment investigating the hydrodynamics of a directionally enhanced DHX was built and the data collected is presented. The concept of thermal diodicity is expanded to heat exchanger technologies and used as performance criteria for evaluating design options. A heat exchanger that can perform as such would be advantageous for use in advanced reactor concepts where primary coolant flow reversal is expected during Loss-of-Forced-Circulation (LOFC) accidents where the ability to circulate coolant is compromised. The design could also find potential use in certain advanced Sodium Fast Reactor (SFR) designs utilizing fluidic diode concepts.

  15. Langevin equation for systems with a preferred spatial direction

    Science.gov (United States)

    Belousov, Roman; Cohen, E. G. D.; Rondoni, Lamberto

    2016-09-01

    In this paper, we generalize the theory of Brownian motion and the Onsager-Machlup theory of fluctuations for spatially symmetric systems to equilibrium and nonequilibrium steady-state systems with a preferred spatial direction, due to an external force. To do this, we extend the Langevin equation to include a bias, which is introduced by an external force and alters the Gaussian structure of the system's fluctuations. In addition, by solving this extended equation, we provide a physical interpretation for the statistical properties of the fluctuations in these systems. Connections of the extended Langevin equation with the theory of active Brownian motion are discussed as well.

  16. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  17. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2015-12-29

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  18. A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

    A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

  19. Efficiency of Iepsilon promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightly linked to the strength of transcription.

    Science.gov (United States)

    Zhang, Ke; Zhang, Ling; Yamada, Takechiyo; Vu, Michael; Lee, Anna; Saxon, Andrew

    2002-02-01

    One key unresolved issue in immunoglobulin class switch recombination (CSR) is how the accessibility of the switch region for CSR is controlled. To better understand the nature of accessibility control for human Ig CSR, we developed a novel inducible switch recombination assay based on expression of green fluorescence protein (GFP) from switch constructs undergoing substrate switch recombination (SSR). Efficient SSR depends on the cytokine-inducible Iepsilon promoter and co-stimulation with IL-4+anti-CD40. Characterization of SSR reveals that both S-S deletional recombination and S-S inversion occur. We show that the IL-4-inducible Iepsilon promoter (pIepsilon) selectively determines the efficiency of the accessibility for SSR. However, the pIepsilon-induced transcription, by itself,is not sufficient to direct efficient SSR. For efficient SSR, both pIepsilon-driven transcriptional activity and an additional promoter/enhancer-derived activity are required. The efficiency of SSR is not tightly correlated with the strength of the combined transcriptional activity. Our results suggest that the mechanism(s) underlying the transcriptional activity, e.g. DNA modification is important for controlling the accessibility for efficient switch recombination.

  20. T-cell-directed therapies in systemic lupus erythematosus.

    Science.gov (United States)

    Nandkumar, P; Furie, R

    2016-09-01

    Drug development for the treatment of systemic lupus erythematosus (SLE) has largely focused on B-cell therapies. A greater understanding of the immunopathogenesis of SLE coupled with advanced bioengineering has allowed for clinical trials centered on other targets for SLE therapy. The authors discuss the benefits and shortcomings of focusing on T-cell-directed therapies in SLE and lupus nephritis clinical trials.

  1. Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR

    Directory of Open Access Journals (Sweden)

    Kienle Peter

    2005-01-01

    Full Text Available Abstract Background Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression. The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli. In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated. Results The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD, p53 (Adp53 and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF. Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing. Conclusion In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective.

  2. Effects of Intravenous and Catheter Directed Thrombolytic Therapy with Recombinant Tissue Plasminogen Activator (Alteplase in Non-Traumatic Acute Limb Ischemia; A Randomized Double-Blind Clinical Trial

    Directory of Open Access Journals (Sweden)

    Abbas Saroukhani

    2015-07-01

    Full Text Available Objective: To evaluate the efficacy and safety of intravenous and catheter directed thrombolysis by recombinant tissue plasminogen activator (Alteplase in the patients with non-traumatic acute limb ischemia (ALI. Methods: This was a randomized clinical trial being performed between 2009 and 2011 in Mashhad University of Medical Sciences. We included those patients who were<75 years, with symptoms of less than 14 days duration, ALI of grade IIa and IIb (according to Rutherford classification and absence of distal run off. Baseline assessment of peripheral circulation performed in all the patients. Patients were randomly assigned to undergo intravenous (n=18 or catheter directed thrombolysis (n=20 with Alteplase. The primary endpoint of the study was improvement of clinical status measured by Rutherford classification, ankle brachial index (ABI, visual analogue scale (VAS score measured at 1, 3 and 6 months. The secondary endpoint of the study was complete or near complete recanalization of the occluded artery. Results: A total number of 38 patients with mean age of 54.13±13.5 years were included in the study. There were 23 (60.5% men and 15 (39.5% women among the patients. Overall 3 (7.9% patients had upper and 35 (92.1% lower extremity ischemia. There was no significant difference between two study groups. None of the patients experienced major therapeutic side effects. Both ABI and VAS score improved in patients who have received first dose of t-PA within 24-hourof ALI. There was no significant difference between two study groups regarding the 6-month clinical grade ( p=0.088, VAS score ( p=0.316 and ABI ( p=0.360. The angiographic improvement was significantly higher in CDT group ( p<0.001. Conclusion: Intravenous and catheter directed thrombolysis with t-PA is a safe and effective method in treatment of acute arteriolar ischemia of extremities. However there both intravenous thrombolysis and CDT are comparable regarding the clinical outcome

  3. Recombination monitor

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, S. Y. [Brookhaven National Lab. (BNL), Upton, NY (United States); Blaskiewicz, M. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2017-02-03

    This is a brief report on LEReC recombination monitor design considerations. The recombination produced Au78+ ion rate is reviewed. Based on this two designs are discussed. One is to use the large dispersion lattice. It is shown that even with the large separation of the Au78+ beam from the Au79+ beam, the continued monitoring of the recombination is not possible. Accumulation of Au78+ ions is needed, plus collimation of the Au79+ beam. In another design, it is shown that the recombination monitor can be built based on the proposed scheme with the nominal lattice. From machine operation point of view, this design is preferable. Finally, possible studies and the alternative strategies with the basic goal of the monitor are discussed.

  4. Directional spread of alphaherpesviruses in the nervous system.

    Science.gov (United States)

    Kramer, Tal; Enquist, Lynn W

    2013-02-11

    Alphaherpesviruses are pathogens that invade the nervous systems of their mammalian hosts. Directional spread of infection in the nervous system is a key component of the viral lifecycle and is critical for the onset of alphaherpesvirus-related diseases. Many alphaherpesvirus infections originate at peripheral sites, such as epithelial tissues, and then enter neurons of the peripheral nervous system (PNS), where lifelong latency is established. Following reactivation from latency and assembly of new viral particles, the infection typically spreads back out towards the periphery. These spread events result in the characteristic lesions (cold sores) commonly associated with herpes simplex virus (HSV) and herpes zoster (shingles) associated with varicella zoster virus (VZV). Occasionally, the infection spreads transsynaptically from the PNS into higher order neurons of the central nervous system (CNS). Spread of infection into the CNS, while rarer in natural hosts, often results in severe consequences, including death. In this review, we discuss the viral and cellular mechanisms that govern directional spread of infection in the nervous system. We focus on the molecular events that mediate long distance directional transport of viral particles in neurons during entry and egress.

  5. Directional Spread of Alphaherpesviruses in the Nervous System

    Directory of Open Access Journals (Sweden)

    Lynn W. Enquist

    2013-02-01

    Full Text Available Alphaherpesviruses are pathogens that invade the nervous systems of their mammalian hosts. Directional spread of infection in the nervous system is a key component of the viral lifecycle and is critical for the onset of alphaherpesvirus-related diseases. Many alphaherpesvirus infections originate at peripheral sites, such as epithelial tissues, and then enter neurons of the peripheral nervous system (PNS, where lifelong latency is established. Following reactivation from latency and assembly of new viral particles, the infection typically spreads back out towards the periphery. These spread events result in the characteristic lesions (cold sores commonly associated with herpes simplex virus (HSV and herpes zoster (shingles associated with varicella zoster virus (VZV. Occasionally, the infection spreads transsynaptically from the PNS into higher order neurons of the central nervous system (CNS. Spread of infection into the CNS, while rarer in natural hosts, often results in severe consequences, including death. In this review, we discuss the viral and cellular mechanisms that govern directional spread of infection in the nervous system. We focus on the molecular events that mediate long distance directional transport of viral particles in neurons during entry and egress.

  6. Research on Direct Torque Control System Based on Induction Motor

    Institute of Scientific and Technical Information of China (English)

    康劲松; 陶生桂; 毛明平

    2003-01-01

    The mathematic model of direct torque control (DTC) was deduced. Two simulating models based on the MATLAB & SIMULINK were established. The emphasis is focused on study of the performance difference of the DTC system with stator flux hexagon and circle trajectories. The simulation waveforms of flux, torque and current characters with two flux trajectories were given. Experiments were carried out in an AC drive system based on induction motor and two-level inverter. A dual-CPU structure was used and the communication with two CPUs was obtained by a dual-port RAM in this system.

  7. Estimating coupling directions in the cardiorespiratory system using recurrence properties.

    Science.gov (United States)

    Marwan, Norbert; Zou, Yong; Wessel, Niels; Riedl, Maik; Kurths, Jürgen

    2013-08-28

    The asymmetry of coupling between complex systems can be studied by conditional probabilities of recurrence, which can be estimated by joint recurrence plots. This approach is applied for the first time on experimental data: time series of the human cardiorespiratory system in order to investigate the couplings between heart rate, mean arterial blood pressure and respiration. We find that the respiratory system couples towards the heart rate, and the heart rate towards the mean arterial blood pressure. However, our analysis could not detect a clear coupling direction between the mean arterial blood pressure and respiration.

  8. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

    Science.gov (United States)

    Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

    2014-02-01

    Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.

  9. Direct Problems and Inverse Problems in Biometric Systems

    Directory of Open Access Journals (Sweden)

    Mihailescu Marius Iulian

    2013-10-01

    Full Text Available The article purpose is to describe the two sides of biometrics technologies, direct problems and inverse problems. The advance that we face today in field of Information Technology makes Information Security an inseparable part. The authentication has a huge role when we deal about security. The problems that can appear in implementing and developing biometrics systems is raising many problems, and one of the goal of this article is to focus on direct and inverse problems which is a new and challenging branch in biometrics technologies.

  10. High Efficiency Bi-Directional Converter Used For Renewable System

    Directory of Open Access Journals (Sweden)

    M. Desal Raja

    2014-02-01

    Full Text Available This paper presents an Isolated bi-directional converter for renewable system. Voltage stress and current stress was decreses in switches. Power flows in both side. The proposed converter three phase ac voltage to dc voltage, dc voltage to three phase a.c voltage and synchronous the two voltage source. Silicon carbide diodes and bi-directional switches Mosfet’s are used for decreases the switching loss. LC Series resonant converter connected to the high efficiency. This converter power conversion control by the output voltage information.

  11. Proportional directional valve based automatic steering system for tractors

    Institute of Scientific and Technical Information of China (English)

    Jin-yi LIU; Jing-quan TAN; En-rong MAO; Zheng-he SONG; Zhong-xiang ZHU‡

    2016-01-01

    Most automatic steering systems for large tractors are designed with hydraulic systems that run on either constant flow or constant pressure. Such designs are limited in adaptability and applicability. Moreover, their control valves can unload in the neutral position and eventually lead to serious hydraulic leakage over long operation periods. In response to the problems noted above, a multifunctional automatic hydraulic steering circuit is presented. The system design is composed of a 5-way-3-position proportional directional valve, two pilot-controlled check valves, a pressure-compensated directional valve, a pressure-compensated flow regulator valve, a load shuttle valve, and a check valve, among other components. It is adaptable to most open-center systems with constant flow supply and closed-center systems with load feedback. The design maintains the lowest pressure under load feedback and stays at the neutral position during unloading, thus meeting the requirements for steering. The steering controller is based on proportional-integral-derivative (PID) running on a 51-microcontroller-unit master control chip. An experimental platform is developed to establish the basic characteristics of the system subject to stepwise inputs and sinusoi-dal tracking. Test results show that the system design demonstrates excellent control accuracy, fast response, and negligible leak during long operation periods.

  12. Performance testing of passive autocatalytic recombiners (PARs)

    Energy Technology Data Exchange (ETDEWEB)

    Blanchat, T. [Sandia National Laboratories, Albuquerque, NM (United States); Malliakos, A. [U.S. Nuclear Regulatory Commission, Washington, DC (United States)

    1997-03-01

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  13. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  14. An experimental genetic system using Berberis vulgaris confirms sexual recombination in Puccinia striiformis

    DEFF Research Database (Denmark)

    Rodriguez Algaba, Julian; Walter, Stephanie; Sørensen, Chris Khadgi

    , the use of B. vulgaris plants originating from nature reserves in Sweden and Denmark proved to be successful for infection and selfing a European Pst isolate in the Danish laboratory in 2013. The progeny isolates in the S1 generation were genotyped with microsatellite markers to confirm parental origin...... and to study genotypic diversity. The markers confirmed the parental origin and markers that were heterozygous in the parent generally segregated in the S1 progenies. A largest number of multilocus genotypes observed among the progeny isolates confirmed successful sexual recombination. Segregation...... for avirulence and virulence was investigated using 15 single R-gene wheat lines. The sexual structures and spore forms were documented by microscopic and macroscopic imaging at crucial time points during the life cycle of Pst on the alternate host....

  15. Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4.

    Science.gov (United States)

    Branagan, Amy M; Klein, Jenny A; Jordan, Christian S; Morrical, Scott W

    2014-01-31

    The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.

  16. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    Science.gov (United States)

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  17. Existence of Almost Resolvable Directed 7-Cycle Systems

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Let ARDkCS(v) denote an almost resolvable directed k-cycle system of order v. It is clear that a necessary condition for the existence of an ARDkCS(v) is v≡1(mod k). For k= 3,4,5 and 6, the existence of an ARDkCS(v) had been completely solved. This paper shows that there exists an ARD7CS(v) if and only if v≡1(mod 7) and v≥8.

  18. Novel recombinant sapovirus in Bangladesh.

    Science.gov (United States)

    Dey, Shuvra Kanti; Mizuguchi, Masashi; Okitsu, Shoko; Ushijima, Hiroshi

    2011-01-01

    Recombination of RNA viruses plays an important part in molecular epidemiological study, virus evolution, vaccine design, and viral control programs. Sapovirus, a member of the family Caliciviridae, is one of the major causative agents of viral gastroenteritis affecting all age groups. Sapovirus capsid and polymerase regions were amplified by PCR using specific primers. PCR products were sequenced directly and sequence analysis was performed using CLUSTAL X, SimPlot, and MEGA 4 software package. Based on the genetic analysis, a novel, naturally occurring recombinant sapovirus strain was identified in Bangladesh. Breakpoint analysis of the recombinant sapovirus showed that the recombination site was at the open reading frame ORF1/ORF2 overlap. We described the genetic characterization of a novel, naturally occurring recombinant sapovirus and provided the first evidence of recombination in sapovirus in Bangladesh.

  19. Systemic delivery of recombinant brain derived neurotrophic factor (BDNF) in the R6/2 mouse model of Huntington's disease.

    Science.gov (United States)

    Giampà, Carmela; Montagna, Elena; Dato, Clemente; Melone, Mariarosa A B; Bernardi, Giorgio; Fusco, Francesca Romana

    2013-01-01

    Loss of huntingtin-mediated BDNF gene transcription has been shown to occur in HD and thus contribute to the degeneration of the striatum. Several studies have indicated that an increase in BDNF levels is associated with neuroprotection and amelioration of neurological signs in animal models of HD. In a recent study, an increase in BDNF mRNA and protein levels was recorded in mice administered recombinant BDNF peripherally. Chronic, indwelling osmotic mini-pumps containing either recombinant BDNF or saline were surgically placed in R6/2 or wild-type mice from 4 weeks of age until euthanasia. Neurological evaluation (paw clasping, rotarod performance, locomotor activity in an open field) was performed. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that BDNF- treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as brain volume, striatal atrophy, size and morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. BDNF was effective in increasing significantly the levels of activated CREB and of BDNF the striatal spiny neurons. Moreover, systemically administered BDNF increased the synthesis of BDNF as demonstrated by RT-PCR, and this might account for the beneficial effects observed in this model.

  20. A novel color image encryption algorithm based on genetic recombination and the four-dimensional memristive hyperchaotic system

    Science.gov (United States)

    Chai, Xiu-Li; Gan, Zhi-Hua; Lu, Yang; Zhang, Miao-Hui; Chen, Yi-Ran

    2016-10-01

    Recently, many image encryption algorithms based on chaos have been proposed. Most of the previous algorithms encrypt components R, G, and B of color images independently and neglect the high correlation between them. In the paper, a novel color image encryption algorithm is introduced. The 24 bit planes of components R, G, and B of the color plain image are obtained and recombined into 4 compound bit planes, and this can make the three components affect each other. A four-dimensional (4D) memristive hyperchaotic system generates the pseudorandom key streams and its initial values come from the SHA 256 hash value of the color plain image. The compound bit planes and key streams are confused according to the principles of genetic recombination, then confusion and diffusion as a union are applied to the bit planes, and the color cipher image is obtained. Experimental results and security analyses demonstrate that the proposed algorithm is secure and effective so that it may be adopted for secure communication. Project supported by the National Natural Science Foundation of China (Grant Nos. 61203094 and 61305042), the Natural Science Foundation of the United States (Grant Nos. CNS-1253424 and ECCS-1202225), the Science and Technology Foundation of Henan Province, China (Grant No. 152102210048), the Foundation and Frontier Project of Henan Province, China (Grant No. 162300410196), the Natural Science Foundation of Educational Committee of Henan Province, China (Grant No. 14A413015), and the Research Foundation of Henan University, China (Grant No. xxjc20140006).

  1. Data-Driven Predictive Direct Load Control of Refrigeration Systems

    DEFF Research Database (Denmark)

    Shafiei, Seyed Ehsan; Knudsen, Torben; Wisniewski, Rafal

    2015-01-01

    A predictive control using subspace identification is applied for the smart grid integration of refrigeration systems under a direct load control scheme. A realistic demand response scenario based on regulation of the electrical power consumption is considered. A receding horizon optimal control...... is proposed to fulfil two important objectives: to secure high coefficient of performance and to participate in power consumption management. Moreover, a new method for design of input signals for system identification is put forward. The control method is fully data driven without an explicit use of model...

  2. Sum-Rate Maximization of Coordinated Direct and Relay Systems

    DEFF Research Database (Denmark)

    Sun, Fan; Popovski, Petar; Thai, Chan

    2012-01-01

    Joint processing of multiple communication flows in wireless systems has given rise to a number of novel transmission techniques, notably the two-way relaying based on wireless network coding. Recently, a related set of techniques has emerged, termed coordinated direct and relay (CDR) transmissions......, where the constellation of traffic flows is more general than the two-way. Regardless of the actual traffic flows, in a CDR scheme the relay has a central role in managing the interference and boosting the overall system performance. In this paper we investigate the novel transmission modes, based...

  3. Motion Planning for a Direct Metal Deposition Rapid Prototyping System

    Energy Technology Data Exchange (ETDEWEB)

    AMES,ARLO L.; HENSINGER,DAVID M.; KUHLMANN,JOEL L.

    1999-10-18

    A motion planning strategy was developed and implemented to generate motion control instructions from solid model data for controlling a robotically driven solid free-form fabrication process. The planning strategy was tested using a PUMA type robot arm integrated into a LENS{trademark} (Laser Engineered Net Shape) system. Previous systems relied on a series of x, y, and z stages, to provide a minimal coordinated motion control capability. This limited the complexity of geometries that could be constructed. With the coordinated motion provided by a robotic arm, the system can produce three dimensional parts by ''writing'' material onto any face of existing material. The motion planning strategy relied on solid model geometry evaluation and exploited robotic positioning flexibility to allow the construction of geometrically complex parts. The integration of the robotic manipulator into the LENS{trademark} system was tested by producing metal parts directly from CAD models.

  4. Intranasal immunization of mice with recombinant Streptococcus gordonii expressing NadA of Neisseria meningitidis induces systemic bactericidal antibodies and local IgA.

    Science.gov (United States)

    Ciabattini, Annalisa; Giomarelli, Barbara; Parigi, Riccardo; Chiavolini, Damiana; Pettini, Elena; Aricò, Beatrice; Giuliani, Marzia M; Santini, Laura; Medaglini, Donata; Pozzi, Gianni

    2008-08-05

    NadA and NhhA, two surface proteins of serogroup B Neisseria meningitidis identified as candidate vaccine antigens, were expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant strains were used to immunize BALB/c mice by the intranasal route and the local and systemic immune response was assessed. Mice were inoculated with recombinant bacteria administered alone or with LTR72, a partially inactivated mutant of Escherichia coli heat-labile enterotoxin, as a mucosal adjuvant. Intranasal immunization with live bacteria expressing NadA induced a significant serum antibody response, with a prevalence of the IgG2a subclass, bactericidal activity in the sera of 71% of animals, and a NadA-specific IgA response in nasal and bronchoalveolar lavages. A formalin-inactivated recombinant strain of S. gordonii expressing NadA was also administered intranasally, inducing a systemic and mucosal humoral response comparable to that of live bacteria. The administration of recombinant bacteria with the mucosal adjuvant LTR72 stimulated a stronger systemic antibody response, protective in 85% of sera, while did not increase the local IgA response. Recombinant S. gordonii expressing NhhA induced a systemic but not mucosal antibody response. These data support the role of NadA as vaccine candidate against serogroup B meningococci, and the use of S. gordonii as vector for intranasal vaccination.

  5. Recombination at the DNA level. Abstracts

    Energy Technology Data Exchange (ETDEWEB)

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  6. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    LEI; Wanli

    2001-01-01

    [1]Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. et al., Cucumber mosaic virus, Adv. Virus Res., 1992, 41: 281-348.[2]Hayes, R. J., Buck, K. W., Complete replication of an eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase, Cell, 1990, 63: 363-369.[3]Nitta, N., Takanami, Y., Kuwata, S. et al., Inoculation with RNAs 1 and 2 of cucumber mosaic virus induces viral RNA replicase activity in tobacco mesophyll protoplasts, J. Gen. Virol., 1988, 69: 2695-2700.[4]Suzuki, M., Kuwata, S., Kataoda, J. et al., Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system, Virology, 1991, 183: 106-113.[5]Canto, T., Prior, D. A. M., Hellwald, K. H. et al., Characterization of cucumber mosaic virus (IV)--Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus, Virology, 1997, 237:237-248.[6]Takanami, Y., A striking change in symptoms on cucumber mosaic virus-infected tobacco plants induced by a satellite RNA, Virology, 1981, 109: 120-126.[7]DeBorde, D. C., Naeve, C. W., Herlocher, M. L. et al., Resolution of a common RNA sequencing ambiguity by terminal deoxynucleotidyl transferase, Anal. Biochem., 1986, 157: 275-282.[8]Haseloff, J., Siemering, K. R., Prasher, D. C. et al., Removal of a cryptic intron and subcellular localization of green fluo-rescent protein are required to mark transgenic Arabidopsis plants brightly, Proc. Natl. Acad. Sci. USA, 1997, 94: 2122-2127.[9]Shi, B. J., Ding, S. W., Symons, R. H., Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infec-tivity of cDNA clones of tomato aspermy cucumovirus, J. Gen. Virol., 1997, 78: 1181-1185.[10]Rizzo, T. M., Palukaitis, P., Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: Genera-tion of infectious RNA transcripts, Mol. Gen. Genet., 1990, 222: 249-256.[11]Hall, R. D., The initiation and maintenance of

  7. Characterization of the AlkS/P-alkB-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations

    NARCIS (Netherlands)

    Makart, Stefan; Heinemann, Matthias; Panke, Sven

    2007-01-01

    The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P-alkB expression system in strictly a

  8. A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

    Science.gov (United States)

    Serrano, Mónica; Kint, Nicolas; Pereira, Fátima C.; Saujet, Laure; Boudry, Pierre; Dupuy, Bruno; Martin-Verstraete, Isabelle

    2016-01-01

    The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development. PMID:27631621

  9. Direct Self-Repairing Control for Quadrotor Helicopter Attitude Systems

    Directory of Open Access Journals (Sweden)

    Huiliao Yang

    2014-01-01

    Full Text Available A quadrotor helicopter with uncertain actuator faults, such as loss of effectiveness and lock-in-place, is studied in this paper. An adaptive fuzzy sliding mode controller based on direct self-repairing control is designed for such nonlinear system to track the desired output signal, when any actuator of this quadrotor helicopter is loss of effectiveness or stuck at some place. Moreover, using the Lyapunov stability theory, the stability of the whole system and the convergence of the tracking error can be guaranteed. Finally, the availability of the proposed method is verified by simulation on 3-DOF hover to ensure that the system performance under faulty conditions can be quickly recovered to its normal level. And this proposed method is also proved to be better than that of LQR through simulation.

  10. Detection of directional energy damping in vibrating systems

    Indian Academy of Sciences (India)

    B R Amruth; Souri Banerjee

    2009-04-01

    The transmission efficiency, frequency and amplitude alteration have been measured by a simple technique of coupled oscillators with a frequency gradient and in a system of non-Newtonian fluid in the form of corn-flour slime. The system of coupled oscillators was found to exhibit preferential energy transfer towards the low frequency end with the reverse propagation severely damped. Energy transfer in all directions was damped in the non-Newtonian fluid in comparison with water. Also the damping in non-Newtonian fluids works only after a lower limit for input amplitude. While most of the previous studies focussed on dissipation of energy within shock-absorbing systems, we demonstrate the contribution of re-distribution of energy reaching the output end to achieve shock absorbing.

  11. An Active Endoscopy Robotic System for Direct Tracheal Inspection

    Institute of Scientific and Technical Information of China (English)

    YU Lian-zhi; YAN Guo-zheng; MA Guan-ying; ZAN Peng

    2007-01-01

    The development of active endoscopy techniques is one important area of medical robot. This paper designed a new flexible and active endoscopy robotic system for direct tracheal inspection. The mobile mechanism of the robot is based on the inchworm movement actuated by pneumatic rubber actuator. There are five air chambers controlled independently, by adjusting pressures in air chambers, the robot can move in a straight mode or in a bending mode. The inspection sensors and some therapy surgery tools can be equipped in the front of the robot.The prototype was made and its mechanical characteristics were analyzed. The robot could move smoothly in a small plastic tube, and the robot is respectable to be used for inspection in human trachea directly.

  12. RECOMBINANT HORSERADISH PEROXIDASE FOR ANALYTICAL APPLICATIONS

    Directory of Open Access Journals (Sweden)

    А.M. Egorov

    2012-08-01

    Full Text Available The article deals with prospects of using recombinant horseradish peroxidase in analytical biochemistry and biotechnology. Problems of recombinant horseradish peroxidase cloning in different expression systems, possible approaches to their solution, advantages of recombinant recombinant horseradish peroxidase and recombinant horseradish peroxidase-fusion proteins for immunoassays are considered. Possibility for development of mediatorless bienzyme biosensor for peroxide and metabolites, yielding hydrogen peroxide during their transformations, based on co-adsorption of recombinant horseradish peroxidase and the appropriate oxidase was demonstrated. The possibility to produce a fully active recombinant conjugate of recombinant horseradish peroxidase with human heart-type fatty acid binding protein, which may be used in competitive immunoassay for clinical diagnosis of acute myocardial infarction, and recombinant conjugates (N- and C-terminus of recombinant horseradish peroxidase with Fab-fragments of the antibody against atrazine, which may be applied for atrazine pesticides detection, are demonstra ted for the first time.

  13. The study on space-flight induced DNA damage in Arabidopsis thaliana using the related homologous recombination reporter system

    Science.gov (United States)

    Sun, Qiao; Nechitailo, Galina S.; Lu, Jinying; Liu, Min; Li, Huasheng

    Usually, phenotype changes of plants were used to analayze the responding genetic damages. However, this method is time-consuming, laborious and needs a long period. Here, we developed an Arabidopsis thaliana homologous recombination reporter system, in which HR frequency and HR-related AtRAD54 gene expression level were used as mutagenic end points. Based on the system, effect of DNA damage by space-flight during the Shenzhou-9 mission was investigated. In this study, an Arabidopsis thaliana-line transgenic for GUS recombination substrates (R3L66, AtRAD54promoter:: GFP + GUS) was used to study the mutagenicity of space-flight, and the results showed that 13 days space-flight exposure of seedlings induced a significant increase in HRF compared with its ground-base three-dimensional clinostat (generally called a random positioning machine or RPM, an effective simulator of microgravity) controls and ground 1g controls. We also observed three-dimensional clinostat induced a significant increase in HRF and HR-related AtRAD54 gene expression level compared with ground 1g controls. Treatment with the ROS scavenger DMSO dramatically reduced the effects of simulated microgravity on the induction of HR and expression of the AtRAD54 gene, suggesting that ROS play a critical role in mediating the simulated microgravity mutagenic effects in plants. In order to understand the combined effects of radiation and microgravity (the main factors in space environment) on DNA damage, we further investigated the effects of modeled microgravity on radiation-induced bystander effects (RIBE) n vivo in A. thaliana plants using the expression level of the HR-related AtRAD54 gene as mutagenic end points. The results showed that the modeled microgravity significantly inhibited the up-regulated expression of the AtRAD54 gene in bystander aerial plants after root irradiation, suggesting a repressive effect of microgravity on RIBE.

  14. Novel Design for Direct Torque Control System of PMSM

    Directory of Open Access Journals (Sweden)

    HUANG Xu-chao

    2013-04-01

    Full Text Available Nowadays, with the rapid development of high-performance servo system, The conventional permanent magnet synchronous motor (PMSM Direct Torque Control (DTC system has large torque ripple in low speed which cannot be well adapted to today`s development. The main reason is because the number of voltage vectors provided by the two-level inverter is only six and the relationship between voltage vector and torque is not clear[1-5.10-12]. In this paper, the basic concept of direct torque control of permanent magnet synchronous motor is investigated in order to emphasize the effects produced by a given voltage vector on stator and torque variations in this paper. Modified the voltage sector switching table, a novel DTC scheme for the permanent magnet synchronous motor is proposed which is using a novel three-level inverter. An improvement of the drive performance can be obtained by using the novel DTC scheme. The simulation results showed that the scheme could reduce the torque ripple in low speed and improved the stability of the motor under the condition of keeping the system dynamic performance.

  15. Beamforming Design for Coordinated Direct and Relay Systems

    DEFF Research Database (Denmark)

    Sun, Fan; De Carvalho, Elisabeth; Thai, Chan

    2012-01-01

    Joint processing of multiple communication flows in wireless systems has given rise to a number of novel transmission techniques, notably the two-way relaying based on wireless network coding. Recently, a related set of techniques has emerged, termed coordinated direct and relay (CDR) transmissions......, where the constellation of traffic flows is more general than the two-way. Regardless of the actual traffic flows, in a CDR scheme the relay has a central role in managing the interference. In this paper we investigate the novel transmission modes, based on amplify-and-forward, that arise when the relay...

  16. Several Modulation in Direct-Sequence Spectrum-Spread Systems

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Performances of BPSK, balanced-quaternary modulation, dual-quaternary modulation and complex-quaternary modulation in direct-sequence spectrum-spread (DS-SS) system are discussed in this paper. Based on the criterion the power of original signal is the same, it is shown that complex-quaternary modulation has the best performance, and dual-quaternary modulation degrades 2dB. Moreover, the frequency efficient of the two modulations is 2bit/Hz. Balanced-quaternary has the same performance as BPSK with frequency efficient 1bit/Hz.

  17. DIRECT INTEGRATION METHODS WITH INTEGRAL MODEL FOR DYNAMIC SYSTEMS

    Institute of Scientific and Technical Information of China (English)

    吕和祥; 于洪洁; 裘春航

    2001-01-01

    A new approach which is a direct integration method with integral model ( DIM IM) to solve dynamic governing equations is presented. The governing equations are integrated into the integral equations. An algorithm with explicit and predict-correct and selfstarting and fourth-order accuracy to integrate the integral equations is given.Theoretical analysis and numerical examples show that DIM-IM discribed in this paper suitable for strong nonlinear and non-conservative system have higher accuracy than central difference, Houbolt , Newmark and Wilson- Theta methods.

  18. Estimation of the Ideal Binary Mask using Directional Systems

    DEFF Research Database (Denmark)

    Boldt, Jesper; Kjems, Ulrik; Pedersen, Michael Syskind

    2008-01-01

    and the requirements to enable calculations of the ideal binary mask using a directional system without the availability of the unmixed signals. The proposed method has a low complexity and is verified using computer simulation in both ideal and non-ideal setups showing promising results.......The ideal binary mask is often seen as a goal for time-frequency masking algorithms trying to increase speech intelligibility, but the required availability of the unmixed signals makes it difficult to calculate the ideal binary mask in any real-life applications. In this paper we derive the theory...

  19. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli.

    Science.gov (United States)

    Albiniak, Anna M; Matos, Cristina F R O; Branston, Steven D; Freedman, Robert B; Keshavarz-Moore, Eli; Robinson, Colin

    2013-08-01

    The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.

  20. Economics of Direct Hydrogen Polymer Electrolyte Membrane Fuel Cell Systems

    Energy Technology Data Exchange (ETDEWEB)

    Mahadevan, Kathyayani

    2011-10-04

    Battelle's Economic Analysis of PEM Fuel Cell Systems project was initiated in 2003 to evaluate the technology and markets that are near-term and potentially could support the transition to fuel cells in automotive markets. The objective of Battelle?s project was to assist the DOE in developing fuel cell systems for pre-automotive applications by analyzing the technical, economic, and market drivers of direct hydrogen PEM fuel cell adoption. The project was executed over a 6-year period (2003 to 2010) and a variety of analyses were completed in that period. The analyses presented in the final report include: Commercialization scenarios for stationary generation through 2015 (2004); Stakeholder feedback on technology status and performance status of fuel cell systems (2004); Development of manufacturing costs of stationary PEM fuel cell systems for backup power markets (2004); Identification of near-term and mid-term markets for PEM fuel cells (2006); Development of the value proposition and market opportunity of PEM fuel cells in near-term markets by assessing the lifecycle cost of PEM fuel cells as compared to conventional alternatives used in the marketplace and modeling market penetration (2006); Development of the value proposition of PEM fuel cells in government markets (2007); Development of the value proposition and opportunity for large fuel cell system application at data centers and wastewater treatment plants (2008); Update of the manufacturing costs of PEM fuel cells for backup power applications (2009).

  1. Impact of plasma noise on a direct thrust measurement system

    Science.gov (United States)

    Pottinger, S. J.; Lamprou, D.; Knoll, A. K.; Lappas, V. J.

    2012-03-01

    In order to evaluate the accuracy and sensitivity of a pendulum-type thrust measurement system, a linear variable differential transformer (LVDT) and a laser optical displacement sensor have been used simultaneously to determine the displacement resulting from an applied thrust. The LVDT sensor uses an analog interface, whereas the laser sensor uses a digital interface to communicate the displacement readings to the data acquisition equipment. The data collected by both sensors show good agreement for static mass calibrations and validation with a cold gas thruster. However, the data obtained using the LVDT deviate significantly from that of the laser sensor when operating two varieties of plasma thrusters: a radio frequency (RF) driven plasma thruster, and a DC powered plasma thruster. Results establish that even with appropriate shielding and signal filtering the LVDT sensor is subject to plasma noise and radio frequency interactions which result in anomalous thrust readings. Experimental data show that the thrust determined using the LVDT system in a direct current plasma environment and a RF discharge is approximately a factor of three higher than the thrust values obtained using a laser sensor system for the operating conditions investigated. These findings are of significance to the electric propulsion community as LVDT sensors are often utilized in thrust measurement systems and accurate thrust measurement and the reproducibility of thrust data is key to analyzing thruster performance. Methods are proposed to evaluate system susceptibility to plasma noise and an effective filtering scheme presented for DC discharges.

  2. Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-alpha correlates with a reduced La protein.

    Science.gov (United States)

    Shimazaki, Takeo; Honda, Masao; Kaneko, Shuichi; Kobayashi, Kenichi

    2002-01-01

    Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5'-nontranslated region (5'NTR). We investigated the effect of interferon alfa (IFN-alpha) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-alpha or poly(I)-poly(C), HCV-IRES-directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-alpha and 45% at 40 microg/mL of poly(I)-poly(C). Thus, IFN-alpha or poly(I)-poly(C) inhibited HCV-IRES-directed translation more efficiently than a cellular cap-dependent translation. 2',5'-oligoadenylate synthetase (2',5'AS) protein level in cells analyzed significantly increased after the administration of IFN-alpha, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES-directed translation in the transformant cells, suggesting that neither the 2',5'AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES-directed translation, was significantly reduced after the administration of IFN-alpha and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES-directed translation by IFN-alpha and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-alpha in that IFN-alpha or poly(I)-poly(C) selectively inhibited HCV-IRES-directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein.

  3. Directional pattern measurement of the BRAMS beacon antenna system

    Science.gov (United States)

    Martínez Picar, A.; Marqué, C.; Anciaux, M.; Lamy, H.

    2015-01-01

    The typical methods for measuring antenna characteristics are mostly based on the use of remote transmitters or receivers. For antennas used in radio communications, calibrations are usually done on an antenna test stand using transmitters with known power output. In order to minimize the ground effects while performing measurements, it is necessary to place the transmitter or receiver high above ground with the aid of aircrafts. It is, however, necessary to determine precisely the coordinates of the airborne devices as well as to maintain high stability. This used to be excessively difficult to carry out, but recent advances in Unmanned Aerial Vehicle (UAV) technologies have brought a feasible option. In this paper, the results of using a low-cost system for measuring the directional pattern of BRAMS beacon antenna system based on an UAV are presented.

  4. Directional energy focusing on monolayer graphene coupling system

    Science.gov (United States)

    Wei, Buzheng; Yang, Yuguang; Yao, Shuzhi; Xiao, Han; Jian, Shuisheng

    2017-03-01

    A directional energy focusing system based on parallel-monolayer graphene sheets is proposed and is analytically and numerically investigated in this paper. By properly designing the chemical potential distributions, we obtain a ˜0.8-nm-size focusing point at desired positions with energy enhancement factor of over 2410. The flexible tunability of the transmission properties enables us to shut one parallel pair propagation down and guide the waves to the other branch. The light signal at the focal point is efficiently slowed down to over 10,000 times the speed in vacuum as well. The proposed structure may find potential applications in integrated circuits, on-chip systems or energy storage.

  5. Robust direct adaptive fuzzy control for nonlinear MIMO systems

    Institute of Scientific and Technical Information of China (English)

    ZHANG Huaguang; ZHANG Mingjun

    2006-01-01

    For a class of nonlinear multi-input multi-output systems with uncertainty, a robust direct adaptive fuzzy control scheme was proposed. The feedback control law and adaptive law for parameters were derived based on Lyapunov design approach. The overall control scheme can guarantee that the tracking error converges in the small neighborhood of origin, and all signals of the closed-loop system are uniformly bounded. The main advantage of the proposed control scheme is that in each subsystem only one parameter vector needs to be adjusted on-line in the adaptive mechanism, and so the on-line computing burden is reduced. In addition, the proposed control scheme is a smooth control with no chattering phenomena. A simulation example was proposed to demonstrate the effectiveness of the proposed control algorithm.

  6. A Direct Diode Laser System Using a Planar Lightwave Circuit

    Science.gov (United States)

    Hasegawa, Kazuo; Matsubara, Hiroyuki; Ichikawa, Tadashi; Maeda, Mitsutoshi; Ito, Hiroshi

    2008-08-01

    In this paper we propose a direct diode laser (DDL) system consisting of laser diode (LD) bars, a planar lightwave circuit (PLC), and an optical fiber. We have developed a PLC as an optical power combiner and an LD mounting technology that is suitable for coupling to the PLC. A DDL system is presented that consists of six LD-PLC optical modules for the laser-welding of highly heat-resistant plastics. The total output power is in the 200 W class, with a spot diameter of 5.52 mm for the major axis and 5.00 mm for the minor axis at a focal length of 50 mm. The total output efficiency is 60.9% from the laser diode to the welding torch.

  7. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  8. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules...... of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect...... as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics...

  9. The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

    Directory of Open Access Journals (Sweden)

    Abbas Mohajeri

    2017-04-01

    Full Text Available Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

  10. Direct and Indirect Pathogenic of Autoantibodies in Systemic Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Takao Fujii

    2014-01-01

    Full Text Available Autoantibody (autoAb production in patients with systemic autoimmune diseases is a hallmark of disease entity, activity and prognosis. Although a large number of autoAbs have been discovered to date, there is a limited number of autoAbs whose pathogenic roles have been clearly determined. However, intriguing evidence has recently been provided of possible pathogenic roles for anti-neutrophil cytoplasmic Abs (ANCAs against myeloperoxidase (MPO in ANCA-associated vasculitides (AAV and for anti-citrullinated protein Abs (ACPAs in rheumatoid arthritis (RA. Of note, these autoAbs are thought to display both direct and indirect effects on organ failure. Additionally, some autoAbs have been reported to play pathogenic roles in brain damage in patients with neuropsychiatric systemic lupus erythematosus (NPSLE, which is one of the most refractory autoimmune disorders. Thus the binding of autoAbs to a certain sequence of the N-methyl-D-aspartate receptor subunit NR2 (anti-NR2 Abs may directly induce hippocampal neuronal injury. On the other hand, anti-U1 ribonucleoprotein (RNP Abs might be pathogenic by inducing neurotoxic inflammatory mediators intrathecally. Such autoAb measurements are also clinically meaningful for treatment selection.

  11. An affinity-directed protein missile system for targeted proteolysis

    Science.gov (United States)

    Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

    2016-01-01

    The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

  12. SIM_EXPLORE: Software for Directed Exploration of Complex Systems

    Science.gov (United States)

    Burl, Michael; Wang, Esther; Enke, Brian; Merline, William J.

    2013-01-01

    Physics-based numerical simulation codes are widely used in science and engineering to model complex systems that would be infeasible to study otherwise. While such codes may provide the highest- fidelity representation of system behavior, they are often so slow to run that insight into the system is limited. Trying to understand the effects of inputs on outputs by conducting an exhaustive grid-based sweep over the input parameter space is simply too time-consuming. An alternative approach called "directed exploration" has been developed to harvest information from numerical simulators more efficiently. The basic idea is to employ active learning and supervised machine learning to choose cleverly at each step which simulation trials to run next based on the results of previous trials. SIM_EXPLORE is a new computer program that uses directed exploration to explore efficiently complex systems represented by numerical simulations. The software sequentially identifies and runs simulation trials that it believes will be most informative given the results of previous trials. The results of new trials are incorporated into the software's model of the system behavior. The updated model is then used to pick the next round of new trials. This process, implemented as a closed-loop system wrapped around existing simulation code, provides a means to improve the speed and efficiency with which a set of simulations can yield scientifically useful results. The software focuses on the case in which the feedback from the simulation trials is binary-valued, i.e., the learner is only informed of the success or failure of the simulation trial to produce a desired output. The software offers a number of choices for the supervised learning algorithm (the method used to model the system behavior given the results so far) and a number of choices for the active learning strategy (the method used to choose which new simulation trials to run given the current behavior model). The software

  13. Initiation of meiotic recombination in Ustilago maydis

    National Research Council Canada - National Science Library

    Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José; Holloman, William K

    2013-01-01

    .... Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U...

  14. Intrarectal vaccination with recombinant vaccinia virus expressing carcinoembronic antigen induces mucosal and systemic immunity and prevents progression of colorectal cancer.

    Science.gov (United States)

    Kim-Schulze, Seunghee; Kim, Hong Sung; Wainstein, Alberto; Kim, Dae Won; Yang, Wein Cui; Moroziewicz, Dorota; Mong, Phyllus Y; Bereta, Michal; Taback, Bret; Wang, Qin; Kaufman, Howard L

    2008-12-01

    The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

  15. Biosensor for Direct Determination of Fenitrothion and EPN Using Recombinant Pseudomonas putida JS444 with Surface Expressed Organophosphorus Hydrolase. 1. Modified Clark Oxygen Electrode

    OpenAIRE

    2006-01-01

    This paper reports a first microbial biosensor for rapid and cost-effective determination of organophosphorus pesticides fenitrothion and EPN. The biosensor consisted of recombinant PNP-degrading/oxidizing bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorus hydrolase (OPH) on its cell surface as biological sensing element and a dissolved oxygen electrode as the transducer. Surface-expressed OPH catalyzed the hydrolysis of fenitrothion and EPN to release 3-methyl-4-nitr...

  16. [Implant fixation strength and osseointegration following systemic administration of recombinant factor XIII and factor XIII concentrate. Animal experiment with implant fixation strength and osseointegration of porous surface implants].

    Science.gov (United States)

    Kienapfel, H; Wilke, A; Dörner, P; Jürgensen, R; Prinz, H; Hettel, A; Swain, R; Griss, P

    1995-01-01

    30 cylindrical commercially pure titanium fiber porous coated Ti6A14V implants were inserted press-fit into the proximal humeral portion of 30 sheep humeri to determine the systemic effect of recombinant factor XIII and placenta-derived factor XIII concentrate on bone ingrowth and on strength of fixation. For both the recombinant factor XIII and the factor XIII concentrate group the volume fraction of bone ingrowth and the strength of fixation was higher when compared with the control specimens. However the difference was only significant for the factor XIII concentrate group.

  17. Recombinant human activated protein C inhibits local and systemic activation of coagulation without influencing inflammation during Pseudomonas aeruginosa pneumonia in rats

    NARCIS (Netherlands)

    Choi, Goda; Hofstra, Jorrit-Jan H; Roelofs, Joris J T H; Florquin, Sandrine; Bresser, Paul; Levi, Marcel; van der Poll, Tom; Schultz, Marcus J

    OBJECTIVE: Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that recombinant human activated protein C exerts lung-protective effects via anticoagulant and anti-inflammatory pathways. We investigated the role of the protein C system in pneumonia caused by Pseudomonas

  18. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Hansen, Line V.; Jørgensen, Thomas J D

    2007-01-01

    The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a conveni...

  19. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

    Energy Technology Data Exchange (ETDEWEB)

    Lu Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo{sup R} marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53{sup +/-} mouse fibroblasts show elevated levels of homologous recombination compared to their p53{sup +/+} counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  20. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

    Science.gov (United States)

    Lu, Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  1. Recombinant protein blends: silk beyond natural design.

    Science.gov (United States)

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production.

  2. Sex in a test tube: testing the benefits of in vitro recombination.

    Science.gov (United States)

    Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

    2016-10-19

    The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'.

  3. A universal system for interactive image-directed neurosurgery.

    Science.gov (United States)

    Maciunas, R J; Galloway, R L; Fitzpatrick, J M; Mandava, V R; Edwards, C A; Allen, G S

    1992-01-01

    Stereotactic methods confer great accuracy to intracranial target localization, but require strict adherence to a complex program of mechanical and computational maneuvers. A computerized, articulated, localizing 'arm' has been developed that frees the neurosurgeon of these constraints and provides a completely intuitive, 'user-friendly' interface. This universal system is independent of whatever localizing fiducial system is selected. The arm may be sterilized for intracranial use. A variety of intraoperative end effectors may be selected. The patient's CT/MR/PET scans are loaded into computer memory and a three-dimensional shaded surface wireframe diagram of the patient's head is displayed simultaneously with up to 3 independent sets of cross-referenced CT/MR/PET scan images on the intraoperative video screen. The arm's endpoint location and the directional vector are shown as cursors on the relevant scan slices, and change continuously as the surgeon moves the arm. Because the information is continuously updated, an unlimited number of targets and trajectories may be displayed throughout the operation. The arm has an ultimate design accuracy for end-point localization to within 0.1 mm throughout a target volume of 40 x 40 x 40 cm. The tested application accuracy of the first prototype model is 0.31 mm. In clinical use during 30 surgeries, its real-world application accuracy is 0.9 mm. This system provides stereotactic accuracy and universally compatible, intuitive, interactive operation.

  4. Dosimetry and image quality assessment in a direct radiography system

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Bruno Beraldo; Paixao, Lucas; Nogueira, Maria do Socorro, E-mail: boliveira.mg@gmail.com [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Oliveira, Marcio Alves de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Fac. de Medicina. Dept. de Anatomia e Imagem; Teixeira, Maria Helena Araujo [Clinica Dra. Maria Helena Araujo Teixeira, Belo Horizonte, MG (Brazil)

    2014-11-15

    Objective: to evaluate the mean glandular dose with a solid state detector and the image quality in a direct radiography system, utilizing phantoms. Materials and methods: Irradiations were performed with automatic exposure control and polymethyl methacrylate slabs with different thicknesses to calculate glandular dose values. The image quality was evaluated by means of the structures visualized on the images of the phantoms. Results: considering the uncertainty of the measurements, the mean glandular dose results are in agreement with the values provided by the equipment and with internationally adopted reference levels. Results obtained from images of the phantoms were in agreement with the reference values. Conclusion: the present study contributes to verify the equipment conformity as regards dose values and image quality. (author)

  5. The spectrum for overlarge sets of directed triple systems

    Institute of Scientific and Technical Information of China (English)

    Zi-hong; TIAN; Li-jun; Ji

    2007-01-01

    A directed triple system of order v, denoted by DTS(v, λ), is a pair (X,B) where X is a v-set and B is a collection of transitive triples on X such that every ordered pair of X belongs to λ triples of B. An overlarge set of disjoint DTS(v, λ), denoted by OLDTS(v, λ), is a collection {(Y\\{y}, Ai)}i,such that Y is a (v + 1)-set, each (Y\\{y}, Ai) is a DTS(v, λ) and all Ai's form a partition of all transitive triples of Y. In this paper, we shall discuss the existence problem of OLDTS(v, λ) and give the following conclusion: there exists an OLDTS(v, λ) if and only if either λ = 1 and v = 0, 1 (mod 3), or λ = 3 and v≠2.

  6. Hierarchical cooperative control for multiagent systems with switching directed topologies.

    Science.gov (United States)

    Hu, Jianqiang; Cao, Jinde

    2015-10-01

    The hierarchical cooperative control problem is concerned for a two-layer networked multiagent system under switching directed topologies. The group cooperative objective is to achieve finite-time formation control for the upper layer of leaders and containment control for the lower layer of followers. Two kinds of cooperative strategies, including centralized-distributed control and distributed-distributed control, are proposed for two types of switching laws: 1) random switching law with the dwell time and 2) Markov switching law with stationary distribution. Utilizing the state transition matrix methods and matrix measure techniques, some sufficient conditions are derived for asymptotical containment control and exponential almost sure containment control, respectively. Finally, some numerical examples are provided to demonstrate the effectiveness of the proposed control schemes.

  7. Effects of cryoprotectants on the viability and activity of freeze dried recombinant yeasts as novel oral drug delivery systems assessed by an artificial digestive system.

    Science.gov (United States)

    Blanquet, Stéphanie; Garrait, Ghislain; Beyssac, Erick; Perrier, Céline; Denis, Sylvain; Hébrard, Géraldine; Alric, Monique

    2005-09-01

    The aim of this study was to investigate, in a gastric-small intestinal system TIM-1, the effect of cryoprotectants on the survival of freeze-dried Saccharomyces cerevisiae expressing the heterologous P450 73A1 and their ability to convert trans-cinnamic acid into p-coumaric acid. Yeasts were lyophilized in suspensions of trehalose, maltose, lactose, or a milk proteins/trehalose mix. Freeze-dried or native yeasts and trans-cinnamic acid were introduced simultaneously into TIM-1 at the beginning of digestion. Yeast survival rate was evaluated by cell counting in the ileal effluents. P450 73A1 activity was followed by HPLC assay of p-coumaric acid. Freeze-dried yeasts showed high tolerance to digestive conditions. Nevertheless, their survival rate was lower than that of non-dried cells (around 80% whatever the protective agent vs. 96%). The ability of recombinant freeze-dried S. cerevisiae to perform a bioconversion reaction in the digestive tract was shown with all the protectants. The highest trans-cinnamic acid conversion rate (24 vs. 41% for native yeasts) was obtained with the milk proteins/trehalose mix. These results show that freeze-drying might be considered for the pharmaceutical formulation of new drug delivery systems based on orally administered recombinant yeasts and that TIM-1 could be a helpful tool for the pre-screening of oral dosage forms.

  8. Dissociating object directed and non-object directed action in the human mirror system; implications for theories of motor simulation.

    Directory of Open Access Journals (Sweden)

    Zarinah K Agnew

    Full Text Available Mirror neurons are single cells found in macaque premotor and parietal cortices that are active during action execution and observation. In non-human primates, mirror neurons have only been found in relation to object-directed movements or communicative gestures, as non-object directed actions of the upper limb are not well characterized in non-human primates. Mirror neurons provide important evidence for motor simulation theories of cognition, sometimes referred to as the direct matching hypothesis, which propose that observed actions are mapped onto associated motor schemata in a direct and automatic manner. This study, for the first time, directly compares mirror responses, defined as the overlap between action execution and observation, during object directed and meaningless non-object directed actions. We present functional MRI data that demonstrate a clear dissociation between object directed and non-object directed actions within the human mirror system. A premotor and parietal network was preferentially active during object directed actions, whether observed or executed. Moreover, we report spatially correlated activity across multiple voxels for observation and execution of an object directed action. In contrast to predictions made by motor simulation theory, no similar activity was observed for non-object directed actions. These data demonstrate that object directed and meaningless non-object directed actions are subserved by different neuronal networks and that the human mirror response is significantly greater for object directed actions. These data have important implications for understanding the human mirror system and for simulation theories of motor cognition. Subsequent theories of motor simulation must account for these differences, possibly by acknowledging the role of experience in modulating the mirror response.

  9. A modified direct preconditioner for indefinite symmetric Toeplitz systems

    Energy Technology Data Exchange (ETDEWEB)

    Concus, P. [Lawrence Berkeley Lab., CA (United States); Saylor, P. [Univ. of Illinois, Urbana, IL (United States)

    1994-12-31

    A modification is presented of the classical $O(n{sup 2})$ algorithm of Trench for the direct solution of Toeplitz systems of equations. The Trench algorithm can be guaranteed to be stable only for matrices that are (symmetric) positive definite; it is generally unstable otherwise. The modification permits extension of the algorithm to compute an approximate inverse in the indefinite symmetric case, for which the unmodified algorithm breaks down when principal submatrices are singular. As a preconditioner, this approximate inverse has an advantage that only matrix-vector multiplications are required for the solution of a linear system, without forward and backward solves. The approximate inverse so obtained can be sufficiently accurate, moreover that, when it is used as a preconditioner for the applications investigated, subsequent iteration may not even be necessary. Numerical results are given for several test matrices. The perturbation to the original matrix that defines the modification is related to a perturbation in a quantity generated in the Trench algorithm; the associated stability of the Trench algorithm is discussed.

  10. Symbiotic commensal bacteria direct maturation of the host immune system.

    Science.gov (United States)

    Edelman, Sanna M; Kasper, Dennis L

    2008-11-01

    Although commensal bacteria are known to play an important role in the proper maturation of the immune system of their mammalian hosts, the molecular mechanisms underlying this immunomodulation are poorly characterized. The present review summarizes recent findings in the field and describes new knowledge on the interplay of the innate and adaptive arms of the immune response induced by symbiotic bacterial carbohydrate antigens. Commensal bacteria in the intestine not only interact directly with dendritic cells but also engage in cross-talk with epithelial cells. These interactions lead to the induction of tolerogenic antigen-presenting cells in the lamina propria and ultimately to the regulation of functional maturation of effector T cells. Upon recognition of capsular polysaccharide antigens of commensal bacteria by dendritic cells (through toll-like receptor 2), innate immune responses facilitate and act in conjunction with adaptive responses to promote optimal Th1 polarization. In contrast, adaptive immunoglobulin A responses to symbiotic bacteria regulate the magnitude of oxidative innate immune responses in the mucosa as well as bacterial epitope expression in the lumen. Accumulating evidence is elucidating surface carbohydrate structures of symbiotic bacteria that drive the modulation of the intestinal immune system, resulting in mature, balanced immune responses and oral tolerance.

  11. HR 8799: The Benchmark Directly-Imaged Planetary System

    CERN Document Server

    Currie, Thayne

    2016-01-01

    HR 8799 harbors arguably the first and best-studied directly-imaged planets. In this brief article, I describe how the HR 8799 planetary system is a benchmark system for studying the atmospheres, orbital properties, dynamical stability, and formation of young superjovian planets. Multi-wavelength photometry and spectroscopy show that HR 8799 bcde appear to have thicker clouds than do field brown dwarfs of similar effective temperatures and exhibit evidence for non-equilibrium carbon chemistry, features that are likely connected to the planets' low surface gravities. Over 17 years of astrometric data constrain the planets' orbits to not be face on but possibly in multiple orbital resonances. At orbital separations of 15--70 au and with masses of $\\approx$ 5--7 $M_{\\rm J}$, HR 8799 bcde probe the extremes of jovian planet formation by core accretion: medium-resolution spectroscopy may provide clues about these planets' formation conditions. Data from the next generation of 30 m-class telescopes should better co...

  12. A modified direct preconditioner for indefinite symmetric Toeplitz systems

    Energy Technology Data Exchange (ETDEWEB)

    Concus, P. (Lawrence Berkeley Lab., CA (United States) California Univ., Berkeley, CA (United States). Dept. of Mathematics); Saylor, P. (Illinois Univ., Urbana, IL (United States). Dept. of Computer Science)

    1992-11-01

    A modification is presented of the classical O(n[sup 2]) algorithm of Trench for the direct solution of Toeplitz systems of equations. The Trench algorithm can be guaranteed to be stable only for matrices that are (symmetric) positive definite; it is generally unstable otherwise. The modification permits extension of the algorithm to compute an approximate inverse in the indefinite symmetric case, for which the unmodified algorithm breaks down when principal submatrices are singular. As a preconditioner, this approximate inverse has an advantage that only matrix-vector multiplications are required for the solution of a linear system, without forward and backward solves. The approximate inverse so obtained can be sufficiently accurate, moreover, that, when it is used as a preconditioner for the applications investigated, subsequent iteration may not even be necessary. Numerical results are given for several test matrices. The perturbation to the original matrix that defines the modification is related to a perturbation in a quantity generated in the Trench algorithm; the associated stability of the Trench algorithm is discussed.

  13. A modified direct preconditioner for indefinite symmetric Toeplitz systems

    Energy Technology Data Exchange (ETDEWEB)

    Concus, P. [Lawrence Berkeley Lab., CA (United States)]|[California Univ., Berkeley, CA (United States). Dept. of Mathematics; Saylor, P. [Illinois Univ., Urbana, IL (United States). Dept. of Computer Science

    1992-11-01

    A modification is presented of the classical O(n{sup 2}) algorithm of Trench for the direct solution of Toeplitz systems of equations. The Trench algorithm can be guaranteed to be stable only for matrices that are (symmetric) positive definite; it is generally unstable otherwise. The modification permits extension of the algorithm to compute an approximate inverse in the indefinite symmetric case, for which the unmodified algorithm breaks down when principal submatrices are singular. As a preconditioner, this approximate inverse has an advantage that only matrix-vector multiplications are required for the solution of a linear system, without forward and backward solves. The approximate inverse so obtained can be sufficiently accurate, moreover, that, when it is used as a preconditioner for the applications investigated, subsequent iteration may not even be necessary. Numerical results are given for several test matrices. The perturbation to the original matrix that defines the modification is related to a perturbation in a quantity generated in the Trench algorithm; the associated stability of the Trench algorithm is discussed.

  14. Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

    Science.gov (United States)

    Pu, Qiang-Hong; Shi, Liang; Yu, Chao

    2015-03-01

    1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

  15. Encapsulated Cellular Implants for Recombinant Protein Delivery and Therapeutic Modulation of the Immune System

    Directory of Open Access Journals (Sweden)

    Aurélien Lathuilière

    2015-05-01

    Full Text Available Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines.

  16. Internal quantum efficiency and Auger recombination in green, yellow and red InGaN-based light emitters grown along the polar direction

    Science.gov (United States)

    Ngo, Thi Huong; Gil, Bernard; Damilano, Benjamin; Lekhal, Kaddour; De Mierry, Philippe

    2017-03-01

    We comparatively study the onset of photo-induced non-radiative intrinsic Auger recombination processes for red, yellow and green light emitting InGaNsbnd GaN hetero-structures grown along the polar orientation. We find a dramatic reduction of the photo excitation densities triggering the domination of Auger effect with increasing emission wavelength; that is to say in concert with the enhancement of the internal electric field in the structure. In long wavelength emitters, the internal electric field is stronger, and hence reducing the impact of the internal electric field is more critical.

  17. Direct Sensitivity Test of the MB/BacT System

    Directory of Open Access Journals (Sweden)

    Barreto Angela Maria Werneck

    2002-01-01

    Full Text Available In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35°C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 µg/ml, Isoniazid - 0.2 µg/ml, Pyrazinamide - 100 µg/ml, Ethambutol - 2.5 µg/ml, Ethionamide - 1.25 µg/ml, and Streptomycin - 2 µg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100 and 100% specificity at 95% Confidence Interval (84.5-100, followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.

  18. A novel and simple method for construction of recombinant adenoviruses.

    Science.gov (United States)

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin

    2006-07-19

    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

  19. Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems : A model for biodetoxication in the human digestive environment

    NARCIS (Netherlands)

    Blanquet, S.; Meunier, J.P.; Minekus, M.; Marol-Bonnin, S.; Alric, M.

    2003-01-01

    The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with

  20. Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems : A model for biodetoxication in the human digestive environment

    NARCIS (Netherlands)

    Blanquet, S.; Meunier, J.P.; Minekus, M.; Marol-Bonnin, S.; Alric, M.

    2003-01-01

    The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with

  1. A System for Surveillance Directly from the EMR

    Science.gov (United States)

    Davies, Richard F.; Morin, Jason; Bhatia, Ramanjot S.; de Bruijn, Lambertus

    2013-01-01

    Objective Our objective was to conduct surveillance of nosocomial infections directly from multiple EMR data streams in a large multi-location Canadian health care facility. The system developed automatically triggers bed-day-level-location-aware reports and detects and tracks the incidents of nosocomial infections in hospital by ward. Introduction Hospital acquired infections are a major cause of morbidity, mortality and increased resource utilization. CDC estimates that in the US alone, over 2 million patients are affected by nosocomial infections costing approximately $34.7 billion to $45 billion annually (1). The existing process of detection and reporting relies on time consuming manual processing of records and generation of alerts based on disparate definitions that are not comparable across institutions or even physicians. Methods A multi-stakeholder team consisting of experts from medicine, infection control, epidemiology, privacy, computing, artificial intelligence, data fusion and public health conducted a proof of concept from four complete years of admission records of all patients at the University of Ottawa Heart Institute. Figure 1 lists the data elements investigated. Our system uses an open source enterprise bus ‘Mirth Connect’ to receive and store data in HL7 format. The processing of information is handled by individual components and alerts are pushed back to respective locations. The free text components were classified using natural language processing. Negation detection was performed using NegEx (2). Data-fusion algorithms were used to merge information to make it meaningful and allow complex syndrome definitions to be mapped onto the data. Results The system monitors: Ventilator Associated Pneumonia (VAP), Central Line Infections (CLI), Methicillin Resistant Staph Aureus (MRSA), Clostridium difficile (C. Diff) and Vancomycin resistant Enterococcus (VRE). 21452 hospital admissions occurred in 17670 unique patients over four years. There

  2. Alterations of systemic and muscle iron metabolism in human subjects treated with low-dose recombinant erythropoietin.

    Science.gov (United States)

    Robach, Paul; Recalcati, Stefania; Girelli, Domenico; Gelfi, Cecilia; Aachmann-Andersen, Niels J; Thomsen, Jonas J; Norgaard, Anne M; Alberghini, Alessandra; Campostrini, Natascia; Castagna, Annalisa; Viganò, Agnese; Santambrogio, Paolo; Kempf, Tibor; Wollert, Kai C; Moutereau, Stéphane; Lundby, Carsten; Cairo, Gaetano

    2009-06-25

    The high iron demand associated with enhanced erythropoiesis during high-altitude hypoxia leads to skeletal muscle iron mobilization and decrease in myoglobin protein levels. To investigate the effect of enhanced erythropoiesis on systemic and muscle iron metabolism under nonhypoxic conditions, 8 healthy volunteers were treated with recombinant erythropoietin (rhEpo) for 1 month. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt and considerable decrease in urinary hepcidin and a slight transient increase in GDF-15. The increased iron use and reduced hepcidin levels suggested increased iron mobilization, but the treatment was associated with increased muscle iron and L ferritin levels. The muscle expression of transferrin receptor and ferroportin was up-regulated by rhEpo administration, whereas no appreciable change in myoglobin levels was observed, which suggests unaltered muscle oxygen homeostasis. In conclusion, under rhEpo stimulation, the changes in the expression of muscle iron proteins indicate the occurrence of skeletal muscle iron accumulation despite the remarkable hepcidin suppression that may be mediated by several factors, such as rhEpo or decreased transferrin saturation or both.

  3. Unique sex chromosome systems in Ellobius: How do male XX chromosomes recombine and undergo pachytene chromatin inactivation?

    Science.gov (United States)

    Matveevsky, Sergey; Bakloushinskaya, Irina; Kolomiets, Oxana

    2016-07-18

    Most mammalian species have heteromorphic sex chromosomes in males, except for a few enigmatic groups such as the mole voles Ellobius, which do not have the Y chromosome and Sry gene. The Ellobius (XX ♀♂) system of sex chromosomes has no analogues among other animals. The structure and meiotic behaviour of the two X chromosomes were investigated for males of the sibling species Ellobius talpinus and Ellobius tancrei. Their sex chromosomes, despite their identical G-structure, demonstrate short synaptic fragments and crossover-associated MLH1 foci in both telomeric regions only. The chromatin undergoes modifications in the meiotic sex chromosomes. SUMO-1 marks a small nucleolus-like body of the meiotic XX. ATR and ubiH2A are localized in the asynaptic area and the histone γH2AFX covers the entire XX bivalent. The distribution of some markers of chromatin inactivation differentiates sex chromosomes of mole voles from those of other mammals. Sex chromosomes of both studied species have identical recombination and meiotic inactivation patterns. In Ellobius, similar chromosome morphology masks the functional heteromorphism of the male sex chromosomes, which can be seen at meiosis.

  4. Production of biologically active recombinant avidin in baculovirus-infected insect cells.

    Science.gov (United States)

    Airenne, K J; Oker-Blom, C; Marjomäki, V S; Bayer, E A; Wilchek, M; Kulomaa, M S

    1997-02-01

    An efficient lepidopteran insect cell system was established for the expression of a recombinant form of chicken egg-white avidin. The gene product was obtained in both secreted and intracellular forms, and biologically active recombinant avidin was isolated using affinity chromatography on an iminobiotin-agarose column. Similar to the known quaternary structure of the native egg-white protein, the purified recombinant protein was glycosylated and assembled mainly into tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin. The biotin-binding and structural properties of the recombinant avidin are thus similar to those of the natural egg-white protein, and the insect system is appropriate both for future site-directed mutagenesis studies and for the production of avidin fusion proteins.

  5. Bimolecular recombination in organic photovoltaics.

    Science.gov (United States)

    Lakhwani, Girish; Rao, Akshay; Friend, Richard H

    2014-01-01

    The recombination of electrons and holes is a major loss mechanism in photovoltaic devices that controls their performance. We review scientific literature on bimolecular recombination (BR) in bulk heterojunction organic photovoltaic devices to bring forward existing ideas on the origin and nature of BR and highlight both experimental and theoretical work done to quantify its extent. For these systems, Langevin theory fails to explain BR, and recombination dynamics turns out to be dependent on mobility, temperature, electric field, charge carrier concentration, and trapped charges. Relationships among the photocurrent, open-circuit voltage, fill factor, and morphology are discussed. Finally, we highlight the recent emergence of a molecular-level picture of recombination, taking into account the spin and delocalization of charges. Together with the macroscopic picture of recombination, these new insights allow for a comprehensive understanding of BR and provide design principles for future materials and devices.

  6. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  7. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an NsiⅠ site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants ex-pressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination between the CMV vector and the CP transgene was proved by retro-transcriptional polymerase chain reaction (RT-PCR) and verified by DNA sequencing. Our results argue against the feasibility of the CMV-based replace-ment vector trans-complemented by the CP transgene, and at the same time, enlighten ways to improve the CMV-based expression vector and the biosafety of CMV CP-mediated virus resistant transgenic plants.

  8. Numerical GPR Imaging through Directional Antenna Systems in Complex Scenarios

    Science.gov (United States)

    Comite, Davide; Murgia, Federica; Barbara, Martina; Catapano, Ilaria; Soldovieri, Francesco; Galli, Alessandro

    2017-04-01

    The capability of imaging hidden targets and interfaces in non-accessible and complex scenarios is a topic of increasing interest for several practical applications, such as civil engineering, geophysics, and planetary explorations [1]. In this frame, Ground Penetrating Radar (GPR) has been proven as an efficient and reliable technique, also thanks to the development of effective imaging procedures based on linear modeling of the scattering phenomenon, which is usually considered as activated by ideal sources [1],[2]. Actually, such modeling simplifications are rarely verified in typical operative scenarios, when a number of heterogeneous targets can interact each other and with the surrounding environment, producing undesired contributions such as clutter and ghosts targets. From a physical viewpoint, these phenomena are mainly due to multipath contributions at the receiving antenna system, and different solutions have been proposed to mitigate these effects on the final image reconstruction (see, e.g., [2] and references therein). In this work we investigate on the possible improvements achievable when the directional features of the transmitting antenna system are taken into account in the imaging algorithm. Following and extending the recent investigations illustrated in [2] and [3], we consider in particular arrays of antennas, made by arbitrary types of elements, as activating the scattering phenomenon: hence, the effects of neglecting or accounting for the inherent directional radiation of the considered array are investigated as regards the accuracy of the final reconstruction of targets. Taking into account the resolution losses linked to the relevant synthetic aperture, we analyze the possibility of improving the quality of imaging, mitigating the presence of spurious contributions. By implementing a 'synthetic setup' that analyzes the scenarios under test through different electromagnetic CAD tools (mainly CST Microwave Studio and gprMax), it has been

  9. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    Science.gov (United States)

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

  10. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    Science.gov (United States)

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

  11. Fast Decoupled Power Flow for Power System with High Voltage Direct Current Transmission Line System

    Directory of Open Access Journals (Sweden)

    Prechanon Kumkratug

    2010-01-01

    Full Text Available Problem statement: High voltage direct current transmission line system has been widely applied for control power flow in power system. The power flow analysis was the one of powerful tools by which the power system equipped was analyzed both for planning and operation strategies. Approach: This study presented the method to analyze power flow of power system consisted of HVDC system. HVDC was modeled as the complex power injections. The presented complex power injected was incorporated into the existing power flow program based on fast decoupled method. The presented method was tested on the multimachine power system. Results: The transmission line loss of the system with and without HVDC was compared. Conclusion: From the simulation results, the HVDC can reduce transmission line loss of power system.

  12. Biosensor for Direct Determination of Fenitrothion and EPN Using Recombinant Pseudomonas putida JS444 with Surface Expressed Organophosphorus Hydrolase. 1. Modified Clark Oxygen Electrode

    Directory of Open Access Journals (Sweden)

    Ashok Mulchandani

    2006-04-01

    Full Text Available This paper reports a first microbial biosensor for rapid and cost-effectivedetermination of organophosphorus pesticides fenitrothion and EPN. The biosensorconsisted of recombinant PNP-degrading/oxidizing bacteria Pseudomonas putida JS444anchoring and displaying organophosphorus hydrolase (OPH on its cell surface asbiological sensing element and a dissolved oxygen electrode as the transducer. Surface-expressed OPH catalyzed the hydrolysis of fenitrothion and EPN to release 3-methyl-4-nitrophenol and p-nitrophenol, respectively, which were oxidized by the enzymaticmachinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen,which was measured and correlated to the concentration of organophosphates. Under theoptimum operating conditions, the biosensor was able to measure as low as 277 ppb offenitrothion and 1.6 ppm of EPN without interference from phenolic compounds and othercommonly used pesticides such as carbamate pesticides, triazine herbicides andorganophosphate pesticides without nitrophenyl substituent. The applicability of thebiosensor to lake water was also demonstrated.

  13. High-Speed Optical Local Access Network System Using Bi-Directional Polarization Multiplexing

    Institute of Scientific and Technical Information of China (English)

    Mitsuru Miyauchi; Yanjun Sun

    2003-01-01

    A high-speed and economical optical local access network system is proposed where bi-directional polarization multiplexing is applied to a bi-directional transmission. Experimental results using a prototype system confirmlow optical loss and environmental stabilities.

  14. High-Speed Optical Local Access Network System Using Bi-Directional Polarization Multiplexing

    Institute of Scientific and Technical Information of China (English)

    Mitsuru; Miyauchi; Yanjun; Sun

    2003-01-01

    A high-speed and economical optical local access network system is proposed where bi-directional polarization multiplexing is applied to a bi-directional transmission. Experimental results using a prototype system confirm low optical loss and environmental stabilities.

  15. Inspiration of Direct Grain Production Subsidy System in Typical Countries and Regions

    Institute of Scientific and Technical Information of China (English)

    Shunqiang; SUN; Lu; CHEN; Ya; LI

    2014-01-01

    Through comparison of direct grain production subsidy system in typical countries and regions,such as the United States,EU,Japan and South Korea,this paper evaluated the performance of direct grain production subsidy system. On the basis of comparison and analysis,it summed up experience of the direct grain production subsidy system in typical countries and regions. Finally,it came up with countermeasures and recommendations for improving the direct grain production subsidy system in China.

  16. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  17. Ferritin nanocontainers that self-direct in synthetic polymer systems

    Science.gov (United States)

    Sengonul, Merih C.

    Currently, there are many approaches to introduce functionality into synthetic polymers. Among these, for example, are copolymerization, grafting, and blending methods. However, modifications made by such methods also change the thermodynamics and rheological properties of the polymer system of interest, and each new modification often requires a costly reoptimization of polymer processing. Such a reoptimalization would not be necessary if new functionality could be introduced via a container whose external surface is chemically and physically tuned to interact with the parent polymer. The contents of the container could then be changed without changing other important properties of the parent polymer. In this context this thesis project explores an innovative nanocontainer platform which can be introduced into phase-separating homopolymer blends. Ferritin is a naturally existing nanocontainer that can be used synthetically to package and selectively transport functional moieties to a particular phase that is either in the bulk or on the surface of a homopolymer blend system. The principal focus of this work centers on modifying the surface of wild ferritin to: (1) render modified ferritin soluble in a non-aqueous solvent; and (2) impart it with self-directing properties when exposed to a homopolymer blend surface or incorporated into the bulk of a homopolymer blend. Wild ferritin is water soluble, and this research project successfully modified wild ferritin by grafting either amine-functional poly(ethylene glycol) (PEG) or short-chain alkanes to carbodiimide activated carboxylate groups on ferritin's surface. Such modified ferritin is soluble in dichloromethane (DCM). Modification was confirmed by ion-exchange chromatography, zeta-potential measurements, and electrospray mass spectroscopy. FT-IR was used to quantify the extent of PEGylation of the reaction products through area ratios of the -C-O-C asymmetric stretching vibration of the grafted PEG chains to the

  18. Effects of systemic domestic recombinant human erythropoietin on HIF-1α expression in the retina in a rabbit model of acute high intraocular pressure

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To observe the expression of hypoxia inducible factor-1α (HIF-1α) in the retina of rabbits with acute high intraocular pressure and to investigate the mechanism of systemic domestic recombinant human erythropoietin (rhEPO) protecting the retina from ischemia-reperfusion injury. Methods First,control group and model group were established in rabbit eyes. The acute high intraocular pressure model was established by saline perfusion into anterior chamber,and then hypodermic injection of domestic rhEP...

  19. Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system.

    Science.gov (United States)

    Lan, John Chi-Wei; Yeh, Chun-Yi; Wang, Chih-Chi; Yang, Yu-Hsuan; Wu, Ho-Shing

    2013-10-01

    Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.

  20. Restraining Sodium Volatilization in the Ferric Bauxite Direct Reduction System

    Directory of Open Access Journals (Sweden)

    Wentao Hu

    2016-03-01

    Full Text Available Direct reduction is an emerging utilization technology of ferric bauxite. However, it requires much more sodium carbonate than ordinary bauxite does. The volatilization is one of the most significant parts of sodium carbonate consumption, as reported in previous studies. Based on the new direct reduction method for utilization of ferric bauxite, this paper has systematically investigated factors including heating temperature, heating time, and sodium carbonate dosage influencing sodium volatilization. For the purpose of reducing sodium volatilization, the Box–Benhken design was employed, and the possibility of separating iron and sodium after direct reduction was also investigated.

  1. Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production.

    Science.gov (United States)

    Li, Zhilin; Li, Xun; Wang, Ye; Wang, Youdong; Wang, Fei; Jiang, Jianchun

    2011-10-01

    The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.

  2. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    Science.gov (United States)

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  3. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  4. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  5. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

    Science.gov (United States)

    Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

    2015-05-21

    β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

  6. Transcript-RNA-templated DNA recombination and repair.

    Science.gov (United States)

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  7. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    Directory of Open Access Journals (Sweden)

    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  8. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli

    Science.gov (United States)

    Costa, Sofia J; Silva, Pedro; Almeida, André; Conceição, Antónia; Domingues, Lucília; Castro, António

    2013-01-01

    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen. PMID:23941978

  9. Alterations of systemic and muscle iron metabolism in human subjects treated with low-dose recombinant erythropoietin

    DEFF Research Database (Denmark)

    Robach, Paul; Recalcati, Stefania; Girelli, Domenico;

    2009-01-01

    healthy volunteers were treated with recombinant erythropoietin (rhEpo) for 1 month. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt and considerable decrease in urinary hepcidin and a slight transient increase...

  10. Basal buffer systems for a newly glycosylated recombinant human interferon-β with biophysical stability and DoE approaches.

    Science.gov (United States)

    Kim, Nam Ah; Song, Kyoung; Lim, Dae Gon; Hada, Shavron; Shin, Young Kee; Shin, Sangmun; Jeong, Seong Hoon

    2015-10-12

    The purpose of this study was to develop a basal buffer system for a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a), termed R27T, to optimize its biophysical stability. The protein was pre-screened in solution as a function of pH (2-11) using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the result, its experimental pI and optimal pH range were 5.8 and 3.6-4.4, respectively. Design of experiment (DoE) approach was developed as a practical tool to aid formulation studies as a function of pH (2.9-5.7), buffer (phosphate, acetate, citrate, and histidine), and buffer concentration (20 mM and 50 mM). This method employed a weight-based procedure to interpret complex data sets and to investigate critical key factors representing protein stability. The factors used were Tm, enthalpy, and relative helix contents which were obtained by DSC and Fourier Transform Infrared spectroscopy (FT-IR). Although the weights changed by three responses, objective functions from a set of experimental designs based on four buffers were highest in 20 mM acetate buffer at pH 3.6 among all 19 scenarios tested. Size exclusion chromatography (SEC) was adopted to investigate accelerated storage stability in order to optimize the pH value with susceptible stability since the low pH was not patient-compliant. Interestingly, relative helix contents and storage stability (monomer remaining) increased with pH and was the highest at pH 4.0. On the other hand, relative helix contents and thermodynamic stability decreased at pH 4.2 and 4.4, suggesting protein aggregation issues. Therefore, the optimized basal buffer system for the novel biobetter was proposed to be 20 mM acetate buffer at pH 3.8±0.2. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Dissociative recombination of HCl+

    Science.gov (United States)

    Larson, Åsa; Fonseca dos Santos, Samantha; E. Orel, Ann

    2017-08-01

    The dissociative recombination of HCl+, including both the direct and indirect mechanisms, is studied. For the direct process, the relevant electronic states are calculated ab initio by combining electron scattering calculations to obtain resonance positions and autoionization widths with multi-reference configuration interaction calculations of the ion and Rydberg states. The cross section for the direct dissociation along electronic resonant states is computed by solution of the time-dependent Schrödinger equation. For the indirect process, an upper bound value for the cross section is obtained using a vibrational frame transformation of the elements of the scattering matrix at energies just above the ionization threshold. Vibrational excitations of the ionic core from the ground vibrational state, v = 0 , to the first three excited vibrational states, v = 1 , v = 2 , and v = 3 , are considered. Autoionization is neglected and the effect of the spin-orbit splitting of the ionic potential energy upon the indirect dissociative recombination cross section is considered. The calculated cross sections are compared to measurements.

  12. Secretion of Recombinant Proteins in Mammalian Cells Directed by Growth Hormone Signal Peptide%生长激素信号肽可诱导重组蛋白外分泌表达

    Institute of Scientific and Technical Information of China (English)

    张志谦; 李金萍; 胡颖

    2005-01-01

    Signal peptide capable of efficiently directing many protein secretion in mammalian cells is one ot the key elements in recombinant protein production, gene therapy and the development of DNA vaccines. In order to explore the possibility of rat growth hormone signal peptide as such an element, a new vector based on the mammalian expression vector pcDNA3 was constructed by employing rat growth hormone (rGH) signal peptide as leading sequence, followed by multiple cloning sites, the myc epitope-tag and 6 × his purification tag in the expression cassette. The vector was validated by successfully expressing and secretion of chick MMP-2 Cterminal PEX domain, a potential angiogenesis inhibitor, and tandem peptide repeats of myc epitope-tag in COS-7 cells. These results suggest that rat growth hormone signal peptide is effective in the mediation of recombinant protein expression and secretion, and this vector provides a new tool for universal cloning and secretion of exogenous proteins in mammalian cells.

  13. Direct Measurement of the Density Matrix of a Quantum System

    Science.gov (United States)

    Thekkadath, G. S.; Giner, L.; Chalich, Y.; Horton, M. J.; Banker, J.; Lundeen, J. S.

    2016-09-01

    One drawback of conventional quantum state tomography is that it does not readily provide access to single density matrix elements since it requires a global reconstruction. Here, we experimentally demonstrate a scheme that can be used to directly measure individual density matrix elements of general quantum states. The scheme relies on measuring a sequence of three observables, each complementary to the last. The first two measurements are made weak to minimize the disturbance they cause to the state, while the final measurement is strong. We perform this joint measurement on polarized photons in pure and mixed states to directly measure their density matrix. The weak measurements are achieved using two walk-off crystals, each inducing a polarization-dependent spatial shift that couples the spatial and polarization degrees of freedom of the photons. This direct measurement method provides an operational meaning to the density matrix and promises to be especially useful for large dimensional states.

  14. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

    Directory of Open Access Journals (Sweden)

    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  15. Analysis of interchromosomal mitotic recombination.

    Science.gov (United States)

    McGill, C B; Shafer, B K; Higgins, D R; Strathern, J N

    1990-07-01

    A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

  16. NASA's Earth Science Data Systems - Lessons Learned and Future Directions

    Science.gov (United States)

    Ramapriyan, Hampapuram K.

    2010-01-01

    In order to meet the increasing demand for Earth Science data, NASA has significantly improved the Earth Science Data Systems over the last two decades. This improvement is reviewed in this slide presentation. Many Earth Science disciplines have been able to access the data that is held in the Earth Observing System (EOS) Data and Information System (EOSDIS) at the Distributed Active Archive Centers (DAACs) that forms the core of the data system.

  17. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    Science.gov (United States)

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  18. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    Science.gov (United States)

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  19. Recombinant protein expression in Nicotiana.

    Science.gov (United States)

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  20. Pattern Switchable Antenna System Using Inkjet-Printed Directional Bow-Tie for Bi-Direction Sensing Applications

    Directory of Open Access Journals (Sweden)

    Seung-Hyun Eom

    2015-12-01

    Full Text Available In this paper, we propose a paper-based pattern switchable antenna system using inkjet-printing technology for bi-direction sensor applications. The proposed antenna system is composed of two directional bow-tie antennas and a switching network. The switching network consists of a single-pole-double-throw (SPDT switch and a balun element. A double-sided parallel-strip line (DSPSL is employed to convert the unbalanced microstrip mode to the balanced strip mode. Two directional bow-tie antennas have different radiation patterns because of the different orientation of the reflectors and antennas. It is demonstrated from electromagnetic (EM simulation and measurement that the radiation patterns of the proposed antenna are successfully switched by the SPDT switch.

  1. Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic investigation of the purified recombinant enzyme.

    Science.gov (United States)

    Knecht, W; Bergjohann, U; Gonski, S; Kirschbaum, B; Löffler, M

    1996-08-15

    Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme-activity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 microM and 13.7 microM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihytdroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2-hydroxyethyliden-cyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the isoxazole derivative leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC50 value of 1 microM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kiu = 0.94 microM) and non

  2. Demonstration of luteotrophic responses of human recombinant gamma interferon in porcine corpora lutea using an in-vivo microdialysis system.

    Science.gov (United States)

    Prakash, B S; Pedina, J; Steiner, A; Wuttke, W

    1997-01-01

    Conceptuses from several mammalian species prior to implantation secrete proteins belonging to the family of interferons. The main species of interferons known to be secreted by the pig blastocyst is interferon gamma (IFNgamma), the precise role of which is unclear. We decided to explore its effects on corpus luteum (CL) function using the novel microdialysis technique in vivo. Six cycling miniature pigs were monitored for estrus by daily plasma progesterone analysis and visual symptoms. On day nine of the cycle (day zero being the day of ovulation) the animals underwent surgery, and microdialysis tubing (vitafiber, Amicon U.S.A, cut off mol. wt. 1 million) were implanted in 17 corpora lutea. The inlets and outlets of all tubings were exteriorized and the entry and exit points of tubings in the CLs sealed with tissue glue. The afferent extension tubings were connected to a fraction collector and the system was continuously flushed with Ringer at a flow rate of 2.4 ml/h. After an initial flushout phase of 8 h, fractions were collected every half hour over 3 days. On days 10, 11 and 12 post estrus 12 CLs were stimulated for 4 h with 10(-7) M, 2 x 10(7) M and 4 x 10(-7) M human recombinant IFNgamma (Pharma Biotechnologie) respectively. Simultaneously, fractions were also collected from the remaining five unstimulated corpora lutea which served as controls. Progesterone concentrations in the dialysates were estimated by a sensitive enzymeimmunoassay (EIA). A significant increase (P < 0.01) in progesterone release was observed in all 3 days following stimulation. The progesterone increase was more marked on the first day of stimulation (1 x 10[-7] M) with the hormone levels rising further even after the end of stimulation. The overall increase in progesterone concentration was 2-fold on day 10 in comparison to 15-30% on subsequent days even though IFN concentrations for stimulation were 2- and 4-fold higher. In the unstimulated CLs, a gradual decline (P < 0.01) in

  3. Colloidal drug delivery systems: current status and future directions.

    Science.gov (United States)

    Garg, Tarun; Rath, Goutam; Goyal, Amit Kumar

    2015-01-01

    In this paper, we provide an overview an extensive range of colloidal drug delivery systems with special focus on vesicular and particulates systems that are being used in research or might be potentially useful as carriers systems for drug or active biomolecules or as cell carriers with application in the therapeutic field. We present some important examples of commercially available drug delivery systems with applications in research or in clinical fields. This class of systems is widely used due to excellent drug targeting, sustained and controlled release behavior, higher entrapment efficiency of drug molecules, prevention of drug hydrolysis or enzymatic degradation, and improvement of therapeutic efficacy. These characteristics help in the selection of suitable carrier systems for drug, cell, and gene delivery in different fields.

  4. A rapid screening method to monitor expression of various recombinant proteins from prokaryotic and eukaryotic expression systems using MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Jebanathirajah, J.A.; Andersen, S.; Blagoev, B.;

    2002-01-01

    Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive...... expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high...

  5. Turbidite systems: State of the art and future directions

    Science.gov (United States)

    Normark, W.R.; Posamentier, H.; Mutti, E.

    1993-01-01

    The study of turbidite systems covering a wide range of physical scales has led to confus ion regarding the use of certain key terms and hence a breakdown in communication between workers involved in turbidite research. There are three fundamentally different scales and types of observations derived from the study of outcrop data (ancient systems), high-resolution seismic reflection and side scan sonar data (modern systems), and multichannel seismic reflection data (modern and older buried systems). Despite the variability of scale the same terms are used to describe features that may have little in common. Consequently, turbidite system terminology has become imprecise and even misleading in some cases, thus providing impediments to developing useful predictive models for processes, depositional environments, and lateral and vertical distribution of sand bodies within turbidite systems. To address this concern, we review the principal elements critical to deepwater systems: slump scars, submarine canyons, channels, channel fill deposits, overbank deposits, and lobes and discuss some of their recognition criteria with each different type of data base. Local and regional tectonic setting, relative sea level variations, and bottom current activity are probably the main factors that control size, external geometry, internal stratal configuration, and facies characteristics of both modern and ancient turbidite systems. These factors ultimately control the timing and bounding characteristics between stages of growth of deepwater systems. If comparison of elements from different turbidite deposits using various data types is carried out at similar physical and temporal scales, predictive models eventually may be improved.

  6. Direct coal liquefaction baseline design and system analysis

    Energy Technology Data Exchange (ETDEWEB)

    1991-04-01

    The primary objective of the study is to develop a computer model for a base line direct coal liquefaction design based on two stage direct coupled catalytic reactors. This primary objective is to be accomplished by completing the following: a base line design based on previous DOE/PETC results from Wilsonville pilot plant and other engineering evaluations; a cost estimate and economic analysis; a computer model incorporating the above two steps over a wide range of capacities and selected process alternatives; a comprehensive training program for DOE/PETC Staff to understand and use the computer model; a thorough documentation of all underlying assumptions for baseline economics; and a user manual and training material which will facilitate updating of the model in the future.

  7. Direct coal liquefaction baseline design and system analysis

    Energy Technology Data Exchange (ETDEWEB)

    1990-09-01

    The primary objective of the study is to develop a computer model for a base line direct coal liquefaction design based on two stage direct coupled catalytic reactors. This primary objective is to be accomplished by completing the following: a base line design based on previous DOE/PETC results from Wilsonville pilot plant and other engineering evaluations; a cost estimate and economic analysis; a computer model incorporating the above two steps over a wide range of capacities and selected process alternatives; a comprehensive training program for DOE/PETC Staff to understand and use the computer model; a thorough documentation of all underlying assumptions for baseline economics; and a user manual and training material which will facilitate updating of the model in the future.

  8. Development of a Waste Vault Directional Assay System (VDAS)

    Energy Technology Data Exchange (ETDEWEB)

    LaFleur, Adrienne Marie [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-11-08

    We are developing a portable neutron and gamma detector called VDAS with sufficient sensitivity and directional capability such that it could operate in high gamma dose rate environments. This detector will be designed such that it can be remotely deployed and adaptable to a broad range of possible facility infrastructure to record a 3D array of gross neutron and gamma measurements, including axial and radial rotation, for identifying potential SNM material locations.

  9. Dielectronic recombination theory

    Energy Technology Data Exchange (ETDEWEB)

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  10. Direct and indirect coupling mechanisms in a chiral plasmonic system

    Science.gov (United States)

    Wang, Yongkai; Wen, Xiaojing; Qu, Yu; Fu, Tong; Zhang, Zhongyue

    2016-10-01

    Artificial chiral plasmonic nanostructures (ACPNs) are widely studied and used in biological monitoring, analytical chemistry, and negative-refractive-index media. The mechanism of direct coupling between two twist metal nanorods has been obtained in usual ACPNs. In this work, we proposed a nanosystem of twist nanorods separated by a metal film (TNMF). By analyzing the charge distributions, a new indirect coupling mechanism is found. According to the equivalent LC resonant circuits, gold nanorods on the two sides of the gold film can be regarded as a receiver and an emitter. These components enhanced transmittance and provided direct and indirect coupling mechanisms for the circular dichroism (CD). The direct coupling mode cannot be explained by impedance matching and can be tuned monotonously by monotonously varying geometric dimensions. However, the CD signal of indirect coupling can be explained by impedance matching and can be tuned to its maximum by varying geometric dimensions when the impedances of both sides of the gold film match. These results can help design novel chiral optical structures and promote combined applications between photons and electrons when a gold film is powered on.

  11. 270 Volt Direct Current Generating System Design, Development and Test

    Science.gov (United States)

    1991-08-01

    is greatly influenced by the load (provides bias to the undervoltage detection circuit) the specific system generator carries. Therefore, when the...1 in the following positions. SI, S2 - Position I S3, S4 - Position 2 GCSv - "ONO Connect the system ( Generator . GCU, CS #1 V #2, LC and BTC) as

  12. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  13. PURIFICATION AND ANALYSIS OF CARBOHYDRATE CLUSTER FROM ERYTHROPOIETIN RECOMBINANT EXPRESSION RESULT ON THE LEAVEN SYSTEM OF Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Adi Santoso

    2011-04-01

    Full Text Available For clinical purposes, pure protein and identification of carbohydrate structure from erythropoietin recombinant are needed. Purification was done with His-Trap affinity chromatography method and continued with gel filtration chromatography column to get purer protein. The carbohydrate cluster from the resulting pure protein then can be recognized by using N- and O-glycosidase and can be compared to EPO recombinant from mammal cells. The result showed similarity on the declining trend of the protein molecule’s weight, which could be seen using the Western blot method. Pure oligosaccharide was hydrolyzed to produce various monosaccharide through incubation with HCl 4 N in 100 oC temperature for 6 hours and the result was applied on high intensity liquid chromatography incubator to learn the composition of its monosaccharide.

  14. FY 2000 Project of developing international standards for supporting new industries. Standardization of systems for detecting gene recombination products; 2000 nendo shinki sangyo shiengata kokusai hyojunka kaihatsu jigyo seika hokokusho. Idenshi kumikae seihin kenshutsu system no hyojunka

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Described herein are the FY 2000 results of the studies on the basic conditions for gene recombination products, and methods of determining content of the purchased gene recombination products in agricultural products, conducted to promote the standardization of the gene recombination product detection systems. Soybean is taken up as the gene recombination product to be studied, in which the samples containing the gene recombination product at 5, 0.5, 0.1 and 0% are prepared, and qualitatively analyzed by the DNA extraction and PCR method. No band is detected. For the quantitative examination by the real-time PCR method, the SYBRGeen, Taqman and hybridization probe methods are used to study the PCR conditions. The sample DNA extracted by the DNA Extraction kit for GMO Detection Ver.2 from the sample containing the RoundupReady{sup TM} soybean is quantitatively analyzed by the real-time PCR method for GMO content, which has produced the results for promotion of the standardization. (NEDO)

  15. Generic approach for designing and implementing a passive autocatalytic recombiner PAR-system in nuclear power plant containments

    Energy Technology Data Exchange (ETDEWEB)

    Bachellerie, E. E-mail: bachelle@tecatom.fr; Arnould, F.; Auglaire, M.; Boeck, B. de; Braillard, O.; Eckardt, B.; Ferroni, F.; Moffett, R

    2003-04-01

    PARSOAR project is one of the scientific projects on hydrogen risk in nuclear power plants co-sponsored by the European Commission in the Fifth Euratom Framework Program and the Swiss government. It is one of the components of the 'Safety Accident Management' (SAM) cluster that is focusing on review of severe accident risks and on development of countermeasures for defence-in-depth. The first objective is to carry out a state-of-the-art on passive autocatalytic recombiners (PAR) to constitute a database helpful for the PAR-designers, nuclear power plant designers and utilities, and safety authorities. The second objective is to elaborate a handbook guide that defines an approach for implementing catalytic recombiners in nuclear power plants, and that proposes recommended practices to support the detailed implementation by individual nuclear actors. The third objective is to identify the needs in terms of complementary experimental qualification or computer code developments about the hydrogen risk in nuclear power plants. It is also recommended that future experimental tests and numerical modelling should concentrate on the remaining issues of (a) lowering of existing uncertainties about hydrogen sources as core reflood or the reactions between boron carbide and steam or uranium and steam, (b) completing the experimental qualification of catalytic recombiners for existing and future nuclear power plant applications, and (c) performing well-qualified couplings between computational fluid dynamics computer codes and numerical models simulating PAR-behaviour (thanks to some global and well-instrumented experiments reproducing a complete reference accident sequence in presence of catalytic recombiners). Such areas to be considered for future work could be integrated in the scope of a European hydrogen network of excellence, in the framework of the Sixth Euratom Framework Program (2002-2006)

  16. IMPROVEMENT DIRECTIONS OF MAINTENANCE SYSTEM OF MULTIPLE UNIT

    Directory of Open Access Journals (Sweden)

    O. S. Krasheninin

    2014-07-01

    Full Text Available Purpose. Efficiency of function multiple units park (MUP as a complex system considerably depends on the modes and conditions of its operation, which is defined as the state of their eigenstate and state of the system content. The purpose is the decision scientifically - a technical problem of system of the content of maintenance operation and repair of a rolling stock. Methodology. Article is devoted to a question of the maintenance operation and repair of a rolling stock of Ukraine. On the basis of theoretical probe, the situation which has developed on maintenance operation and repair of MVPS is analysed. The comparative analysis of a railway, motor and air transportation is provided. On the basis of the analysis and comparison by authors it is offered transition from scheduled preventive to system of service of railway transport to service on an actual state. Findings. According to given theoretical comparison of the put perspective in different types of transport it is possible to note that experience and features of operation rolling stock in allied industries is the base for revision and improvement of system of the maintenance rolling stock, including at various stages of its operation. Originality. This article provides the comparative theoretical analysis, concerning contents and repair system between railway, automobile and by air. Practical value. Introduction new approaches of the maintenance operation and rolling stocks repair will allow carrying out gradual transition from maintenance operation and repairing existing scheduled preventive system in repair system «on an actual state». Thus it is possible maximum cutting-down of number of sudden refusals in operation, providing high coefficient of operational readiness of a rolling stock (to 0.95. A comparative analysis of the maintenance system and repair is the basis of the transition feasibility.

  17. Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

    Science.gov (United States)

    Liu, Yuxiu; Sato, Hiroki; Hamana, Masahiro; Moonan, Navita Anisia; Yoneda, Misako; Xia, Xianzhu; Kai, Chieko

    2014-09-01

    Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

  18. Recombinant Technology and Probiotics

    OpenAIRE

    Icy D’Silva

    2011-01-01

    Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

  19. Influence of Functionality on Direct Arylation of Model Systems as a Route Toward Fluorinated Copolymers via Direct Arylation Polymerization (DArP)

    DEFF Research Database (Denmark)

    Livi, Francesco; Gobalasingham, Nemal S.; Bundgaard, Eva

    2015-01-01

    A screening of direct arylation conditions on amodel small molecule system is carried out to develop suitableconditions for the direct arylation polymerization (DArP) of fluorinatedcopolymers, which are incompatible with conditionspreviously utilized successfully for nonfluorinated systems...

  20. National Wilderness Preservation System of the United States - Direct Download

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — This map layer consists of National Wilderness Preservation System areas of 640 acres or more, in the United States, Puerto Rico, and the U.S. Virgin Islands. The...

  1. High Efficiency Direct Methane Solid Oxide Fuel Cell System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — NASA has a defined need for energy dense and highly efficient energy storage and power delivery systems for future space missions. Compared to other fuel cell...

  2. Directed structure at infinity for infinite-dimensional systems

    Science.gov (United States)

    Laakkonen, Petteri; Pohjolainen, Seppo

    2011-04-01

    In this article the structure at infinity of infinite-dimensional linear time invariant systems with finite-dimensional input and output spaces is discussed. It is shown that a diagonal form describing behaviour near infinity can be found. This diagonal form is a generalisation of the Smith-McMillan form at infinity for rational matrices. It is then used to simplify certain solvability conditions of a regulation problem. Examples on time-delay and distributed parameter systems are given.

  3. Direct Interaction between Autonomic Nerves and the Immune System.

    Science.gov (United States)

    1986-08-05

    ablation of the coeliac /superior mesenteric plexus results in total depletion of NE from the spleen and the disappearance of fluorescent profiles. We...substantiation to our anatomical data. Anatomical tracing studies have revealed the coeliac /superior mesenteric ganglionic system as the source of both the...system. In: Psychological and Biological Approaches to the Understanding of Human Disease , Martinus-Nijhof, in press. S13

  4. Photoelectrochemical based direct conversion systems for hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Khaselev, O.; Bansal, A.; Kocha, S.; Turner, J.A. [National Renewable Energy Lab., Golden, CO (United States)

    1998-08-01

    With an eye towards developing a photoelectrochemical system for hydrogen production using sunlight as the only energy input, two types of systems were studied, both involving multijunction devices. One set of cells consisted of a-Si triple junctions and the other a GaInP{sub 2}/GaAs tandem cell combination. Additional investigations were carried out on semiconductor surface modifications to move semiconductor band edges to more favorable energetic positions.

  5. Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity

    Institute of Scientific and Technical Information of China (English)

    Ya-Fei Mao; Jie Yan

    2004-01-01

    AIM: To clone ureB gene from a clinical isolate ofHelicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein.METHODS: ureB gene from a clinical H pyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates.RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureBgene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%.The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins. rUreB specifically combined with the commercial antibodies against whole cell of H pyloriand strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein.Serum samples from all H pyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested H pyloriisolates.CONCLUSION: A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different H

  6. Detecting the cosmological recombination signal from space

    CERN Document Server

    Desjacques, Vincent; Silk, Joseph; de Bernardis, Francesco; Doré, Olivier

    2015-01-01

    Spectral distortions of the CMB have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift $z\\simeq 1100$. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm--dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination histo...

  7. SYSTEME DE COMMUNICATION A ETALEMENT DE BANDE A SEQUENCE DIRECTE

    Directory of Open Access Journals (Sweden)

    M.L BOUCHAREB

    2001-06-01

    Full Text Available Le présent article donne une vue d'ensemble sur la notion et le principe de la communication à étalement de bande. L'étude porte essentiellement sur un type de système de communication dit à référence emmagasinée utilisant la modulation à séquence directe comme technique de modulation pour étaler davantage la bande de  transmission. En premier lieu, nous définissons les structures de ces systèmes de communication à étalement de bande et leurs domaines d'application. Ensuite, nous présentons les notions de la communication à étalement de bande à séquence directe ainsi qu'un aperçu sur la génération des séquences pseudo-aléatoires. Finalement, nous calculons la probabilité d'erreur du système traité.

  8. AGRICULTURAL TAX SYSTEM IN POLAND AND DIRECTIONS OF THE CHANGES

    Directory of Open Access Journals (Sweden)

    Marzena Malewska

    2014-03-01

    Full Text Available A research problem taken in the article is a question, whether legal solutions applied in the Polish system of the taxation of agriculture have an appropriate shape and whether this system is optimal? If not, what are possible options of changes and whether the in-come tax is good for agricultural activities. A method of analysis of secondary sources was used in order to solve the research problem. Required data was taken from the base of the Główny Urząd Statystyczny and scientific publications of the publishing universi-ty. In the article were formulated the following hypotheses: 1.\tThe system of the taxation of agriculture is different than other existing Polish law and it is regarded as unfair in relation to other social groups. 2.\tChanging the tax system in agriculture is necessary and would lead to positive chang-es in the structure of agriculture. 3.\tThe reform of the taxation system of agricultural production is in many aspects neces-sary and beneficial, but it should nevertheless take into account the specificities of the agricultural activity. 4.\tThe introduction of the income tax has its bad side and carries the disadvantages for both farmers and municipalities.

  9. Uncertain rule-based fuzzy systems introduction and new directions

    CERN Document Server

    Mendel, Jerry M

    2017-01-01

    The second edition of this textbook provides a fully updated approach to fuzzy sets and systems that can model uncertainty — i.e., “type-2” fuzzy sets and systems. The author demonstrates how to overcome the limitations of classical fuzzy sets and systems, enabling a wide range of applications from time-series forecasting to knowledge mining to control. In this new edition, a bottom-up approach is presented that begins by introducing classical (type-1) fuzzy sets and systems, and then explains how they can be modified to handle uncertainty. The author covers fuzzy rule-based systems – from type-1 to interval type-2 to general type-2 – in one volume. For hands-on experience, the book provides information on accessing MatLab and Java software to complement the content. The book features a full suite of classroom material. Presents fully updated material on new breakthroughs in human-inspired rule-based techniques for handling real-world uncertainties; Allows those already familiar with type-1 fuzzy se...

  10. Global Positioning System: Political Support, Directions of Development, and Expectations

    Directory of Open Access Journals (Sweden)

    Krzysztof Czaplewski

    2015-06-01

    Full Text Available Over the last decade the Global Positioning System has become a global, multifunctional tool which provides services that are an integral part of U.S. national security as well as the security of other highly developed countries. Economic development, transport security as well as homeland security are important elements of the global economic infrastructure. In 2000 the United States acknowledged the growing significance of GPS for civilian users and stopped intentionally degrading accuracy for non-military signals that are known as “Selective Availability”. Since then, commercial applications of satellite systems have been proliferating even more rapidly, and therefore, their importance in everyday life has greatly increased. Currently, services that depend on information obtained from the Global Positioning System are the driving force behind economic growth, economic development and the improvement in life safety. This economic development would not be possible without the financial and political support of the US government to maintain the operation of the GPS system. Therefore it is important to have knowledge about the intentions of the US government how system GPS will be developed in the future. Decisions taken in the last 3 months are the subject of this article.

  11. Analysis of nonlinearities and effects in direct drive electro-hydraulic position servo system

    Institute of Scientific and Technical Information of China (English)

    WANG Hong-jie; JI Tian-jing; MAO Xin-tao; LIU Quan-zhong

    2005-01-01

    The direct drive electro-hydraulic servo system is a new approach hydraulic system. It is much smaller and easier controlled than traditional systems and is a perfect energy saver. This paper will briefly introduce the popular nonlinearities in the electro-hydraulic system and analyse the effect of nonlinearities in direct drive electro-hydraulic position servo system by means of simulation research. Some valuable conclusions are given.

  12. Direct Current Hopping Conductivity in One-Dimensional Nanometre Systems

    Institute of Scientific and Technical Information of China (English)

    宋祎璞; 徐慧; 罗峰

    2003-01-01

    A one-dimensional random nanocrystalline chain model is established. A dc electron-phonon-field conductance model of electron tunnelling transfer is set up, and a new dc conductance formula in one-dimensional nanometre systems is derived. By calculating the dc conductivity, the relationship among the electric field, temperature and conductivity is analysed, and the effect of the crystalline grain size and the distortion of interfacial atoms on the dc conductance is discussed. The result shows that the nanometre system appears the characteristic of negative differential dependence of resistance and temperature at low temperature. The dc conductivity of nanometre systems varies with the change of electric field and trends to rise as the crystalline grain size increases and to decrease as the distorted degree of interfacial atoms increases.

  13. Romanian health system strategic directions for the next decade

    Directory of Open Access Journals (Sweden)

    Cristian Vlădescu

    2009-03-01

    Full Text Available

    Background: The exceptional changes in the Romanian society after 1989 have strongly influenced the reforms of the health system.

    Methods: Several tools were used for this health policy review: finding the evidence to prove that there are important public health problems in Romania, as well as reviewing the existing legislation and policy papers related to health system, stakeholder analysis, meetings and working groups with key players from the health sector.

    Results: Health indicators for Romania are some of the poorest in the WHO European region, not only at EU level. A mix of specific indicators for developed countries (e.g. high mortality by cardiovascular diseases, increasing incidence of cancer and specific indicators for developing countries (e.g. re-emergence of some communicable diseases, like TB can be noticed. Unfavourable comparisons also exist for the indicators describing the performance of the health system: poor access to basic health services especially in rural area, inadequate human resources management, lack of integration of the health services in order to assure the continuity of care, poor health information management, and reduced intersectoral cooperation. Six major intervention areas have been identified by the Presidential Health Commission set up in 2008, in order to address the dysfunctions of the health system: Health system financing, Health system organization, Drug policy, Primary care, Hospital services and Human resources. In order to address them, 29 specific recommendations were made. Conclusions: If the declared intentions of the new minister of health are implemented in line with the proposed presidential strategy, then coherency might finally have a chance to drive the process forward.

  14. Passive-solar directional-radiating cooling system

    Energy Technology Data Exchange (ETDEWEB)

    Hull, John R. (Hinsdale, IL); Schertz, William W. (Batavia, IL)

    1986-01-01

    A radiative cooling system for use with an ice-making system having a radiating surface aimed at the sky for radiating energy at one or more wavelength bands for which the atmosphere is transparent and a cover thermally isolated from the radiating surface and transparent at least to the selected wavelength or wavelengths, the thermal isolation reducing the formation of condensation on the radiating surface and/or cover and permitting the radiation to continue when the radiating surface is below the dewpoint of the atmosphere, and a housing supporting the radiating surface, cover and heat transfer means to an ice storage reservoir.

  15. Stokes Space in Direct-Detection Data Transmission Systems

    DEFF Research Database (Denmark)

    Estaran Tolosa, Jose Manuel; Lu, Xiaofeng; Zibar, Darko;

    2015-01-01

    Stokes-based processing permits complete and phase-insensitive characterization of the field’s SOP, readily unlocking polarization diversity in transmission systems where DD is desired. We present an overview on Stokes notions and most recent achievements in this context.......Stokes-based processing permits complete and phase-insensitive characterization of the field’s SOP, readily unlocking polarization diversity in transmission systems where DD is desired. We present an overview on Stokes notions and most recent achievements in this context....

  16. Variable Resolution Direction Finding Using the Robust Symmetrical Number System

    Science.gov (United States)

    2006-12-01

    to bond copper foil coatings to a wooden chip board. Measuring 32 inches by 12 inches, it was designed to have a ground plane spacing in excess of...baseband mixer (QMXO, pin 22) from on-board detector input (VDT1, pin 20) VDT1&2 Wired Connects both detector input voltages (pin 20 & 18) to the chip ...adversely affecting the system, a crude shielding was made from metal netting and draped over the entire DF system and connected to ground as shown in

  17. System for controllable magnetic measurement with direct field determination

    Science.gov (United States)

    Stupakov, O.

    2012-02-01

    This work describes a specially designed setup for magnetic hysteresis and Barkhausen noise measurements. The setup combines two main elements: an improved fast algorithm to control the waveform of magnetic induction and simultaneous direct determination of the magnetic field. The digital feedback algorithm uses only the previous measurement cycle to correct the magnetization voltage without any additional correlation parameter; it usually converges after several tens of cycles. The magnetic field is measured at the sample surface using a vertically mounted array of sensitive Hall sensors. Linear extrapolation of the tangential field profile to the sample surface determines the true waveform of the magnetic field. This unique combination of physically based control for both parameters of the magnetization process provides stable and reliable results, which are independent of a specified experimental configuration. This is illustrated for the industrially attractive measurements of non-oriented electrical steels with a 50 Hz sinusoidal induction waveform.

  18. Dual-color reporter switching system to discern dimer formations of G-protein-coupled receptors using Cre/loxP site-specific recombination in yeast.

    Science.gov (United States)

    Nakamura, Yasuyuki; Hashimoto, Takamichi; Ishii, Jun; Kondo, Akihiko

    2016-10-01

    G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that represent major molecular targets in pharmaceutical and medicinal fields. Many GPCRs have been shown to form not only homodimers, but also heterodimers that can confer large functional and physiological diversity and are therefore expected to offer new opportunities for the discovery of new drugs. Yeast is a useful host organism that can be used to investigate the interactions between eukaryotic protein pairs, as demonstrated by the yeast two-hybrid (Y2H) system. Previously, we established reporter gene assay systems to screen for GPCR dimer pairs based on the split-ubiquitin membrane Y2H (mY2H) system. However, conventional systems only induce reporter gene expressions from the OFF to ON states. In this study, we therefore designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout. To invoke reporter switching, we took advantage of Cre/loxP site-specific recombination. Through optimization of Cre-mediated reporter gene recombination using the split-ubiquitin mY2H system, we built a dual-color reporter switching system to discern the formations of GPCR dimers. This system enabled monitoring of the formations of homodimers and heterodimers of human serotonin 1A receptor or β2 -adrenergic receptor as well as homodimers of the yeast endogenous pheromone receptor (Ste2p) in yeast cells. Our reporter switching system may be a useful tool for identifying potential molecular targets among GPCR dimers, and is also applicable to other reporter gene assay systems. Biotechnol. Bioeng. 2016;113: 2178-2190. © 2016 Wiley Periodicals, Inc.

  19. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

    Directory of Open Access Journals (Sweden)

    Trujillo-Roldán Mauricio A

    2010-03-01

    Full Text Available Abstract The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies.

  20. 78 FR 58960 - Airworthiness Directives; BAE SYSTEMS (OPERATIONS) LIMITED Airplanes

    Science.gov (United States)

    2013-09-25

    ... Policies and Procedures (44 FR 11034, February 26, 1979); 3. Will not affect intrastate aviation in Alaska... inspection of certain engine and auxiliary power unit (APU) fire extinguishers to determine if the fire... system to extinguish fires in the engine or APU fire zones, possibly resulting in damage to the...

  1. CRISPR technologies for bacterial systems: Current achievements and future directions

    DEFF Research Database (Denmark)

    Choi, Kyeong Rok; Lee, Sang Yup

    2016-01-01

    Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolution......Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now...... revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial...... microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR...

  2. Power Systems and Energy Storage Modeling for Directed Energy Weapons

    Science.gov (United States)

    2014-06-01

    electron laser kW Kilo-watt LCS Littoral Combat Ship LAWS Laser Weapon System MLD Maritime Laser Demonstration MW Mega -watt NiMH Nickel metal...and various littoral combat ships. Also, an accurate, working model of the capacitor energy bank is being developed and the flywheel model is being

  3. CRISPR system in filamentous fungi: Current achievements and future directions.

    Science.gov (United States)

    Deng, Huaxiang; Gao, Ruijie; Liao, Xiangru; Cai, Yujie

    2017-09-05

    As eukaryotes, filamentous fungi share many features with humans, and they produce numerous active metabolites, some of which are toxic. Traditional genetic approaches are generally inefficient, but the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system that has been widely used for basic research on bacteria, mammals and plants offers a simple, fast, versatile technology for systemic research on filamentous fungi. In this review, we summarized the current knowledge on Cas9 and its variants, various selective markers used to screen positive clones, different ways used to detect off-target mutations, and different approaches used to express and transform the CRISPR complex. We also highlight several methods that improve the nuclease specificity and efficiency, and discuss current and potential applications of CRISPR/Cas9 system in filamentous fungi for pathogenesis decoding, confirmation of the gene and pathway, bioenergy process, drug discovery, and chromatin dynamics. We also describe how the synthetic gene circuit of CRISPR/Cas9 systems has been used in the response to various complex environmental signals to redirect metabolite flux and ensure continuous metabolite biosynthesis. Copyright © 2017. Published by Elsevier B.V.

  4. European Directions for Hypersonic Thermal Protection Systems and Hot Structures

    Science.gov (United States)

    Glass, David E.

    2007-01-01

    This presentation will overview European Thermal Protection Systems (TPS) and Hot Structures activities in Europe. The Europeans have a lot of very good work going on in the area. The presentation will discuss their emphasis on focused technology development for their flight vehicles.

  5. Method of operating a direct dme fuel cell system

    DEFF Research Database (Denmark)

    2011-01-01

    The present invention relates to a method of operating a fuel cell system comprising one or more fuel cells with a proton exchange membrane, wherein the membrane is composed of a polymeric material comprising acid-doped polybenzimidazole (PBI). The method comprises adjusting the operating...

  6. Surface-directed capillary system; theory, experiments and applications.

    Science.gov (United States)

    Bouaidat, Salim; Hansen, Ole; Bruus, Henrik; Berendsen, Christian; Bau-Madsen, Niels Kristian; Thomsen, Peter; Wolff, Anders; Jonsmann, Jacques

    2005-08-01

    We present a capillary flow system for liquid transport in microsystems. Our simple microfluidic system consists of two planar parallel surfaces, separated by spacers. One of the surfaces is entirely hydrophobic, the other mainly hydrophobic, but with hydrophilic pathways defined on it by photolithographic means. By controlling the wetting properties of the surfaces in this manner, the liquid can be confined to certain areas defined by the hydrophilic pathways. This technique eliminates the need for alignment of the two surfaces. Patterned plasma-polymerized hexafluoropropene constitutes the hydrophobic areas, whereas the untreated glass surface constitutes the hydrophilic pathways. We developed a theoretical model of the capillary flow and obtained analytical solutions which are in good agreement with the experimental results. The capillarity-driven microflow system was also used to pattern and immobilize biological material on planar substrates: well-defined 200 microm wide strips of human cells (HeLa) and fluorescence labelled proteins (fluorescein isothiocyanate-labelled bovine serum albumin, i.e., FITC-BSA) were fabricated using the capillary flow system presented here.

  7. Method of operating a direct dme fuel cell system

    DEFF Research Database (Denmark)

    2011-01-01

    The present invention relates to a method of operating a fuel cell system comprising one or more fuel cells with a proton exchange membrane, wherein the membrane is composed of a polymeric material comprising acid-doped polybenzimidazole (PBI). The method comprises adjusting the operating...

  8. The Development of A Human Systems Simulation Laboratory: Strategic Direction

    Energy Technology Data Exchange (ETDEWEB)

    Jacques Hugo; Katya le Blanc; David Gertman

    2012-07-01

    The Human System Simulation Laboratory (HSSL) at the Idaho National Laboratory is one of few facilities of its kind that allows human factors researchers to evaluate various aspects of human performance and human system interaction for proposed reactor designs and upgrades. A basic system architecture, physical configuration and simulation capability were established to enable human factors researchers to support multiple, simultaneous simulations and also different power plant technologies. Although still evolving in terms of its technical and functional architecture, the HSSL is already proving its worth in supporting current and future nuclear industry needs for light water reactor sustainability and small modular reactors. The evolution of the HSSL is focused on continual physical and functional refinement to make it a fully equipped, reconfigurable facility where advanced research, testing and validation studies can be conducted on a wider range of reactor technologies. This requires the implementation of additional plant models to produce empirical research data on human performance with emerging human-system interaction technologies. Additional beneficiaries of this information include system designers and HRA practitioners. To ensure that results of control room crew studies will be generalizable to the existing and evolving fleet of US reactors, future expansion of the HSSL may also include other SMR plant models, plant-specific simulators and a generic plant model aligned to the current generation of pressurized water reactors (PWRs) and future advanced reactor designs. Collaboration with industry partners is also proving to be a vital component of the facility as this helps to establish a formal basis for current and future human performance experiments to support nuclear industry objectives. A long-range Program Plan has been developed for the HSSL to ensure that the facility will support not only the Department of Energy’s Light Water Reactor

  9. Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct ItB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacterpylori(Hpylori) strain Y06 and the ItB gene from Escherichiacoli(E coli) strain 44851 were linked into ItB-ureB fusiongene by PCR. The fusion gene sequence was analyzedafter T-A cloning. A prokaryotic recombinant expressionvector pET32a inserted with ItB-ureB fusion gene (pET32aItB-ureB) was constructed. Expression of the recombinantLTB-UreB protein (rLTB-UreB)in E. coliBL21DE3 inducedby isopropylthio-β-D-galactoside (IPTG) at differentconcentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTBUreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant proteinon inducing specific antibody in rabbits. GM1-ELISA wasused to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positivesera from 125 gastric patients was determined by using ELISA. RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ItB-ureB fusion gene were 100%. IPTG withdifferent dosages of 0.1-1.0 mmol/L could efficiently inducepET32a-ItB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E. coli BL21DE3 was approximately 35%of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori andanti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from

  10. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

    Science.gov (United States)

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu

    2016-08-01

    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution.

  11. Mechanistic features of recombination in HIV.

    Science.gov (United States)

    Galetto, Román; Negroni, Matteo

    2005-01-01

    The importance of recombination in retroviral evolution has been acknowledged for several decades. Consequently, after the identification of HIV as the etiological agent of AIDS, it was suspected that recombination could also play a central role in the evolution of this virus. However, only recently, extensive epidemiologic studies of HIV infections worldwide have provided an estimate for the occurrence of recombination in vivo, unveiling recombination frequencies that dwarf those initially expected. Nowadays, recombination is regarded as an integral part of the infectious cycle of this retrovirus, which impacts on diagnosis and treatment of infections, especially when genetically distant viruses have been at the origin of the recombinant forms. Retroviral recombination is observed when two genetically divergent genomic RNA molecules are present in the same viral particle, and arises during the reverse transcription step. This review focuses on the mechanisms that have been proposed to account for the occurrence of recombination in retroviruses, from the strand displacement model, according to which recombination occurs during second DNA strand synthesis; to the description of the factors responsible for copy-choice recombination during first DNA strand synthesis, such as the presence of breaks, pause sites, or secondary structures in the genomic RNA. Most of these models have been supported by experimental data obtained from in vitro reconstituted systems or from cell infection studies using academic model sequences. The situation in vivo is expected to be more complex, since several factors come into play when recombination involves relatively distant isolates, as in the case of inter-subtype recombination. At present, it is clear that further studies are needed in order to evaluate whether a prevailing mechanism exists for in vivo recombination, and these studies will also be essential for understanding how the underlying mechanisms of recombination contribute

  12. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  13. The endocannabinoid system: directing eating behavior and macronutrient metabolism

    Directory of Open Access Journals (Sweden)

    Bruce Alan Watkins

    2015-01-01

    Full Text Available For many years, the brain has been the primary focus for research on eating behavior. More recently, the discovery of the endogenous endocannabinoids (EC and the endocannabinoid system (ECS, as well as the characterization of its actions on appetite and metabolism, has provided greater insight on the brain and food intake. The purpose of this review is to explain the actions of EC in the brain and other organs as well as their precursor polyunsaturated fatty acids (PUFA that are converted to these endogenous ligands. The binding of the EC to the cannabinoid receptors in the brain stimulates food intake, and the ECS participates in systemic macronutrient metabolism where the gastrointestinal system, liver, muscle, and adipose are involved. The EC are biosynthesized from two distinct families of dietary PUFA, namely the n-6 and n-3. Based on their biochemistry, these PUFA are well known to exert considerable physiological and health-promoting actions. However, little is known about how these different families of PUFA compete as precursor ligands of cannabinoid receptors to stimulate appetite or perhaps down-regulate the ECS to amend food intake and prevent or control obesity. The goal of this review is to assess the current available research on ECS and food intake, suggest research that may improve the complications associated with obesity and diabetes by dietary PUFA intervention, and further reveal mechanisms to elucidate the relationships between substrate for EC synthesis, ligand actions on receptors, and the physiological consequences of the ECS. Dietary PUFA are lifestyle factors that could potentially curb eating behavior, which may translate to changes in macronutrient metabolism, systemically and in muscle, benefiting health overall.

  14. The endocannabinoid system: directing eating behavior and macronutrient metabolism

    OpenAIRE

    Bruce Alan Watkins; Jeffrey eKim

    2015-01-01

    For many years, the brain has been the primary focus for research on eating behavior. More recently, the discovery of the endogenous endocannabinoids (EC) and the endocannabinoid system (ECS), as well as the characterization of its actions on appetite and metabolism, has provided greater insight on the brain and food intake. The purpose of this review is to explain the actions of EC in the brain and other organs as well as their precursor polyunsaturated fatty acids (PUFA) that are converte...

  15. The endocannabinoid system: directing eating behavior and macronutrient metabolism.

    Science.gov (United States)

    Watkins, Bruce A; Kim, Jeffrey

    2014-01-01

    For many years, the brain has been the primary focus for research on eating behavior. More recently, the discovery of the endocannabinoids (EC) and the endocannabinoid system (ECS), as well as the characterization of its actions on appetite and metabolism, has provided greater insight on the brain and food intake. The purpose of this review is to explain the actions of EC in the brain and other organs as well as their precursor polyunsaturated fatty acids (PUFA) that are converted to these endogenous ligands. The binding of the EC to the cannabinoid receptors in the brain stimulates food intake, and the ECS participates in systemic macronutrient metabolism where the gastrointestinal system, liver, muscle, and adipose are involved. The EC are biosynthesized from two distinct families of dietary PUFA, namely the n-6 and n-3. Based on their biochemistry, these PUFA are well known to exert considerable physiological and health-promoting actions. However, little is known about how these different families of PUFA compete as precursor ligands of cannabinoid receptors to stimulate appetite or perhaps down-regulate the ECS to amend food intake and prevent or control obesity. The goal of this review is to assess the current available research on ECS and food intake, suggest research that may improve the complications associated with obesity and diabetes by dietary PUFA intervention, and further reveal mechanisms to elucidate the relationships between substrate for EC synthesis, ligand actions on receptors, and the physiological consequences of the ECS. Dietary PUFA are lifestyle factors that could potentially curb eating behavior, which may translate to changes in macronutrient metabolism, systemically and in muscle, benefiting health overall.

  16. The endocannabinoid system: directing eating behavior and macronutrient metabolism

    OpenAIRE

    Watkins, Bruce A.; Kim, Jeffrey

    2015-01-01

    For many years, the brain has been the primary focus for research on eating behavior. More recently, the discovery of the endocannabinoids (EC) and the endocannabinoid system (ECS), as well as the characterization of its actions on appetite and metabolism, has provided greater insight on the brain and food intake. The purpose of this review is to explain the actions of EC in the brain and other organs as well as their precursor polyunsaturated fatty acids (PUFA) that are converted to these en...

  17. Life time test in direct borohydride fuel cell system

    Energy Technology Data Exchange (ETDEWEB)

    Jamard, Romain [Commissariat a l' Energie Atomique (CEA), LITEN-DTNM-LCH, 17 av. des martyrs, 38054 Grenoble Cedex 9 (France); Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique LACCO UMR6503, 40 av. du Recteur Pineau, 86022 Poitiers (France); Salomon, Jeremie; Martinent-Beaumont, Audrey [Commissariat a l' Energie Atomique (CEA), LITEN-DTNM-LCH, 17 av. des martyrs, 38054 Grenoble Cedex 9 (France); Coutanceau, Christophe [Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique LACCO UMR6503, 40 av. du Recteur Pineau, 86022 Poitiers (France)

    2009-09-05

    The electric performances of direct borohydride fuel cells (DBFCs) are evaluated in terms of power density and life time with respect to the NaBH{sub 4} concentration. A DBFC constituted of an anionic membrane, a 0.6 mg{sub Pt} cm{sup -2} anode and a commercial non-platinum based cathode led to performances as high as 200 mW cm{sup -2} at room temperature and with natural convection of air. Electrochemical life time test at 0.55 mA cm{sup -2} with a 5 M NaBH{sub 4}/1 M NaOH solution shows a voltage diminution of 1 mV h{sup -1} and a drastic drop of performances after 250 h. The life time is twice longer with 2 M NaBH{sub 4}/1 M NaOH solution (450 h) and the voltage decrease is 0.5 mV h{sup -1}. Analyses of the components after life time tests indicate that voltage loss is mainly due to the degradation of the cathode performance. Crystallisation of carbonate and borate is observed at the cathode side, although the anionic membrane displays low permeability to borohydride. (author)

  18. Modification of the Clarkson-Kruskal Direct Method for a Coupled System

    Institute of Scientific and Technical Information of China (English)

    QIAN Su-Ping; TIAN Li-Xin

    2007-01-01

    A new idea is put forward to modify the Clarkson-Kruskal (CK) direct method. Using the usual CK direct method to a coupled KdV system, two types of usual similarity reductions can be obtained. However, the application of the modified CK direct method leads to three types of new similarity reductions different from the usual ones.

  19. Recombination drives vertebrate genome contraction.

    Directory of Open Access Journals (Sweden)

    Kiwoong Nam

    Full Text Available Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.

  20. Direct Carbon Fuel Cell System Utilizing Solid Carbonaceous Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Turgut Gur

    2010-04-30

    This 1-year project has achieved most of its objective and successfully demonstrated the viability of the fluidized bed direct carbon fuel cell (FB-DCFC) approach under development by Direct Carbon technologies, LLC, that utilizes solid carbonaceous fuels for power generation. This unique electrochemical technology offers high conversion efficiencies, produces proportionately less CO{sub 2} in capture-ready form, and does not consume or require water for gasification. FB-DCFC employs a specialized solid oxide fuel cell (SOFC) arrangement coupled to a Boudouard gasifier where the solid fuel particles are fluidized and reacted by the anode recycle gas CO{sub 2}. The resulting CO is electrochemically oxidized at the anode. Anode supported SOFC structures employed a porous Ni cermet anode layer, a dense yttria stabilized zirconia membrane, and a mixed conducting porous perovskite cathode film. Several kinds of untreated solid fuels (carbon and coal) were tested in bench scale FBDCFC prototypes for electrochemical performance and stability testing. Single cells of tubular geometry with active areas up to 24 cm{sup 2} were fabricated. The cells achieved high power densities up to 450 mW/cm{sup 2} at 850 C using a low sulfur Alaska coal char. This represents the highest power density reported in the open literature for coal based DCFC. Similarly, power densities up to 175 mW/cm{sup 2} at 850 C were demonstrated with carbon. Electrical conversion efficiencies for coal char were experimentally determined to be 48%. Long-term stability of cell performance was measured under galvanostatic conditions for 375 hours in CO with no degradation whatsoever, indicating that carbon deposition (or coking) does not pose any problems. Similar cell stability results were obtained in coal char tested for 24 hours under galvanostatic conditions with no sign of sulfur poisoning. Moreover, a 50-cell planar stack targeted for 1 kW output was fabricated and tested in 95% CO (balance CO{sub 2

  1. Transcutaneous spinal direct current stimulation modulates human corticospinal system excitability.

    Science.gov (United States)

    Bocci, Tommaso; Marceglia, Sara; Vergari, Maurizio; Cognetto, Valeria; Cogiamanian, Filippo; Sartucci, Ferdinando; Priori, Alberto

    2015-07-01

    This study aimed to assess the effects of thoracic anodal and cathodal transcutaneous spinal direct current stimulation (tsDCS) on upper and lower limb corticospinal excitability. Although there have been studies assessing how thoracic tsDCS influences the spinal ascending tract and reflexes, none has assessed the effects of this technique over upper and lower limb corticomotor neuronal connections. In 14 healthy subjects we recorded motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) from abductor hallucis (AH) and hand abductor digiti minimi (ADM) muscles before (baseline) and at different time points (0 and 30 min) after anodal or cathodal tsDCS (2.5 mA, 20 min, T9-T11 level). In 8 of the 14 subjects we also tested the soleus H reflex and the F waves from AH and ADM before and after tsDCS. Both anodal and cathodal tsDCS left the upper limb MEPs and F wave unchanged. Conversely, while leaving lower limb H reflex unchanged, they oppositely affected lower limb MEPs: whereas anodal tsDCS increased resting motor threshold [(mean ± SE) 107.33 ± 3.3% increase immediately after tsDCS and 108.37 ± 3.2% increase 30 min after tsDCS compared with baseline] and had no effects on MEP area and latency, cathodal tsDCS increased MEP area (139.71 ± 12.9% increase immediately after tsDCS and 132.74 ± 22.0% increase 30 min after tsDCS compared with baseline) without affecting resting motor threshold and MEP latency. Our results show that tsDCS induces polarity-specific changes in corticospinal excitability that last for >30 min after tsDCS offset and selectively affect responses in lower limb muscles innervated by lumbar and sacral motor neurons.

  2. Hotspots for Vitamin-Steroid-Thyroid Hormone Response Elements Within Switch Regions of Immunoglobulin Heavy Chain Loci Predict a Direct Influence of Vitamins and Hormones on B Cell Class Switch Recombination.

    Science.gov (United States)

    Hurwitz, Julia L; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Partridge, Janet F; Maul, Robert W; Gearhart, Patricia J

    2016-03-01

    Vitamin A deficiencies are common throughout the world and have a significant negative influence on immune protection against viral infections. Mouse models demonstrate that the production of IgA, a first line of defense against viruses at mucosal sites, is inhibited in the context of vitamin A deficiency. In vitro, the addition of vitamin A to activated B cells can enhance IgA expression, but downregulate IgE. Previous reports have demonstrated that vitamin A modifies cytokine patterns, and in so doing may influence antibody isotype expression by an indirect mechanism. However, we have now discovered hundreds of potential response elements among Sμ, Sɛ, and Sα switch sites within immunoglobulin heavy chain loci. These hotspots appear in both mouse and human loci and include targets for vitamin receptors and related proteins (e.g., estrogen receptors) in the nuclear receptor superfamily. Full response elements with direct repeats are relatively infrequent or absent in Sγ regions although half-sites are present. Based on these results, we pose a hypothesis that nuclear receptors have a direct effect on the immunoglobulin heavy chain class switch recombination event. We propose that vitamin A may alter S site accessibility to activation-induced deaminase and nonhomologous end-joining machinery, thereby influencing the isotype switch, antibody production, and protection against viral infections at mucosal sites.

  3. Expression of recombinant antibodies.

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  4. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  5. Systemic Immunotherapy for Urothelial Cancer: Current Trends and Future Directions.

    Science.gov (United States)

    Gupta, Shilpa; Gill, David; Poole, Austin; Agarwal, Neeraj

    2017-01-27

    Urothelial cancer of the bladder, renal pelvis, ureter, and other urinary organs is the fifth most common cancer in the United States, and systemic platinum-based chemotherapy remains the standard of care for first-line treatment of advanced/metastatic urothelial carcinoma (UC). Until recently, there were very limited options for patients who are refractory to chemotherapy, or do not tolerate chemotherapy due to toxicities and overall outcomes have remained very poor. While the role of immunotherapy was first established in non-muscle invasive bladder cancer in the 1970s, no systemic immunotherapy was approved for advanced disease until the recent approval of a programmed death ligand-1 (PD-L1) inhibitor, atezolizumab, in patients with advanced/metastatic UC who have progressed on platinum-containing regimens. This represents a significant milestone in this disease after a void of over 30 years. In addition to atezolizumab, a variety of checkpoint inhibitors have shown a significant activity in advanced/metastatic urothelial carcinoma and are expected to gain Food and Drug Administration (FDA) approval in the near future. The introduction of novel immunotherapy agents has led to rapid changes in the field of urothelial carcinoma. Numerous checkpoint inhibitors are being tested alone or in combination in the first and subsequent-line therapies of metastatic disease, as well as neoadjuvant and adjuvant settings. They are also being studied in combination with radiation therapy and for non-muscle invasive bladder cancer refractory to BCG. Furthermore, immunotherapy is being utilized for those ineligible for firstline platinum-based chemotherapy. This review outlines the novel immunotherapy agents which have either been approved, or are currently being investigated in clinical trials in UC.

  6. Systemic Immunotherapy for Urothelial Cancer: Current Trends and Future Directions

    Directory of Open Access Journals (Sweden)

    Shilpa Gupta

    2017-01-01

    Full Text Available Urothelial cancer of the bladder, renal pelvis, ureter, and other urinary organs is the fifth most common cancer in the United States, and systemic platinum-based chemotherapy remains the standard of care for first-line treatment of advanced/metastatic urothelial carcinoma (UC. Until recently, there were very limited options for patients who are refractory to chemotherapy, or do not tolerate chemotherapy due to toxicities and overall outcomes have remained very poor. While the role of immunotherapy was first established in non-muscle invasive bladder cancer in the 1970s, no systemic immunotherapy was approved for advanced disease until the recent approval of a programmed death ligand-1 (PD-L1 inhibitor, atezolizumab, in patients with advanced/metastatic UC who have progressed on platinum-containing regimens. This represents a significant milestone in this disease after a void of over 30 years. In addition to atezolizumab, a variety of checkpoint inhibitors have shown a significant activity in advanced/metastatic urothelial carcinoma and are expected to gain Food and Drug Administration (FDA approval in the near future. The introduction of novel immunotherapy agents has led to rapid changes in the field of urothelial carcinoma. Numerous checkpoint inhibitors are being tested alone or in combination in the first and subsequent-line therapies of metastatic disease, as well as neoadjuvant and adjuvant settings. They are also being studied in combination with radiation therapy and for non-muscle invasive bladder cancer refractory to BCG. Furthermore, immunotherapy is being utilized for those ineligible for firstline platinum-based chemotherapy. This review outlines the novel immunotherapy agents which have either been approved, or are currently being investigated in clinical trials in UC.

  7. Dry low NOx combustion system with pre-mixed direct-injection secondary fuel nozzle

    Science.gov (United States)

    Zuo, Baifang; Johnson, Thomas; Ziminsky, Willy; Khan, Abdul

    2013-12-17

    A combustion system includes a first combustion chamber and a second combustion chamber. The second combustion chamber is positioned downstream of the first combustion chamber. The combustion system also includes a pre-mixed, direct-injection secondary fuel nozzle. The pre-mixed, direct-injection secondary fuel nozzle extends through the first combustion chamber into the second combustion chamber.

  8. Geometry-Of-Fire Tracking Algorithm for Direct-Fire Weapon Systems

    Science.gov (United States)

    2015-09-01

    NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA THESIS Approved for public release; distribution is unlimited GEOMETRY-OF- FIRE ...TRACKING ALGORITHM FOR DIRECT- FIRE WEAPON SYSTEMS by Caleb K. Khan September 2015 Thesis Advisor: Zachary Staples Co-Advisor: Xiaoping...2015 3. REPORT TYPE AND DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE GEOMETRY-OF- FIRE TRACKING ALGORITHM FOR DIRECT- FIRE WEAPON SYSTEMS

  9. Current trends of HIV recombination worldwide

    Directory of Open Access Journals (Sweden)

    Katherine A. Lau

    2013-06-01

    Full Text Available One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O, as well as inter- and intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs and unique recombinant forms (URFs, requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and co-circulation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.

  10. Materials directed to implants for repairing Central Nervous System

    Energy Technology Data Exchange (ETDEWEB)

    Canillas, M.; Moreno-Burriel, B.; Chinarro, E.

    2014-07-01

    Central Nervous System (CNS) can be damaged by a wide range of injuries and disorders which entail permanent disability in some cases. Moreover, CNS repairing process presents some complications. The natural repair mechanism, which consists on the glial scar formation, is triggered by the inflammatory process. Molecules delivered during these processes, inflammation and glial scar formation as well as oxygen and glucose deficiencies due to the injury, create an inhibitory environment for axon regeneration and remyelination which is known as secondary injury. Biomaterials are taking up an even more important role in repairing CNS. Physicochemical properties of some ceramic materials have inspired different applications to repair CNS as substrates, electrodes or molecule vehicles. Based on their biocompatibility, capability to neutralize reactive species involved in the inflammatory processes and their versatile processing to obtain scaffolds with different shapes and sizes, ceramics are a succulent offer in nervous tissue engineering. Furthermore, their possibilities have been increased with polymeric-ceramics composites development, which have given rise to new interesting horizon. (Author)

  11. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    Science.gov (United States)

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.

  12. Practical considerations for a second-order directional hearing aid microphone system

    Science.gov (United States)

    Thompson, Stephen C.

    2003-04-01

    First-order directional microphone systems for hearing aids have been available for several years. Such a system uses two microphones and has a theoretical maximum free-field directivity index (DI) of 6.0 dB. A second-order microphone system using three microphones could provide a theoretical increase in free-field DI to 9.5 dB. These theoretical maximum DI values assume that the microphones have exactly matched sensitivities at all frequencies of interest. In practice, the individual microphones in the hearing aid always have slightly different sensitivities. For the small microphone separation necessary to fit in a hearing aid, these sensitivity matching errors degrade the directivity from the theoretical values, especially at low frequencies. This paper shows that, for first-order systems the directivity degradation due to sensitivity errors is relatively small. However, for second-order systems with practical microphone sensitivity matching specifications, the directivity degradation below 1 kHz is not tolerable. A hybrid order directive system is proposed that uses first-order processing at low frequencies and second-order directive processing at higher frequencies. This hybrid system is suggested as an alternative that could provide improved directivity index in the frequency regions that are important to speech intelligibility.

  13. Atomic excitation and recombination in external fields

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.; Clark, C.W.

    1985-01-01

    This volume offers a timely look at Rydberg states of atoms in external fields and dielectronic recombination. Each topic provides authoritative coverage, presents a fresh account of a flourishing field of current atomic physics and introduces new opportunities for discovery and development. Topics considered include electron-atom scattering in external fields; observations of regular and irregular motion as exemplified by the quadratic zeeman effect and other systems; Rydberg atoms in external fields and the Coulomb geometry; crossed-field effects in the absorption spectrum of lithium in a magnetic field; precise studies of static electric field ionization; widths and shapes of stark resonances in sodium above the saddle point; studies of electric field effects and barium autoionizing resonances; autoionization and dielectronic recombination in plasma electric microfields; dielectronic recombination measurements on multicharged ions; merged beam studies of dielectronic recombination; Rydberg atoms and dielectronic recombination in astrophysics; and observations on dielectronic recombination.

  14. Analysis of Direct Samples of Early Solar System Aqueous Fluids

    Science.gov (United States)

    Zolensky, Michael E.; Bodnar, R J.; Fedele, L.; Yurimoto,H.; Itoh, S.; Fries, M.; Steele, A.

    2012-01-01

    Over the past three decades we have become increasingly aware of the fundamental importance of water, and aqueous alteration, on primitive solar-system bodies. Some carbonaceous and ordinary chondrites have been altered by interactions with liquid water within the first 10 million years after formation of their parent asteroids. Millimeter to centimeter-sized aggregates of purple halite containing aqueous fluid inclusions were found in the matrix of two freshly-fallen brecciated H chondrite falls, Monahans (1998, hereafter simply "Monahans") (H5) and Zag (H3-6) (Zolensky et al., 1999; Whitby et al., 2000; Bogard et al., 2001) In order to understand origin and evolution of the aqueous fluids inside these inclusions we much measure the actual fluid composition, and also learn the O and H isotopic composition of the water. It has taken a decade for laboratory analytical techniques to catch up to these particular nanomole-sized aqueous samples. We have recently been successful in (1) measuring the isotopic composition of H and O in the water in a few fluid inclusions from the Zag and Monahans halite, (2) mineralogical characterization of the solid mineral phases associated with the aqueous fluids within the halite, and (3) the first minor element analyses of the fluid itself. A Cameca ims-1270 equipped with a cryo-sample-stage of Hokkaido University was specially prepared for the O and H isotopic measurements. The cryo-sample-stage (Techno. I. S. Corp.) was cooled down to c.a. -190 C using liquid nitrogen at which the aqueous fluid in inclusions was frozen. We excavated the salt crystal surfaces to expose the frozen fluids using a 15 keV Cs+ beam and measured negative secondary ions. The secondary ions from deep craters of approximately 10 m in depth emitted stably but the intensities changed gradually during measurement cycles because of shifting states of charge compensation, resulting in rather poor reproducibility of multiple measurements of standard fluid

  15. Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1.

    Science.gov (United States)

    Kang, H A; Kang, W; Hong, W K; Kim, M W; Kim, J Y; Sohn, J H; Choi, E S; Choe, K B; Rhee, S K

    2001-09-01

    To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.

  16. Materials directed to implants for repairing Central Nervous System

    Directory of Open Access Journals (Sweden)

    Canillas, M.

    2014-12-01

    Full Text Available Central Nervous System (CNS can be damaged by a wide range of injuries and disorders which entail permanent disability in some cases. Moreover, CNS repairing process presents some complications. The natural repair mechanism, which consists on the glial scar formation, is triggered by the inflammatory process. Molecules delivered during these processes, inflammation and glial scar formation as well as oxygen and glucose deficiencies due to the injury, create an inhibitory environment for axon regeneration and remyelination which is known as “secondary injury”. Biomaterials are taking up an even more important role in repairing CNS. Physicochemical properties of some ceramic materials have inspired different applications to repair CNS as substrates, electrodes or molecule vehicles. Based on their biocompatibility, capability to neutralize reactive species involved in the inflammatory processes and their versatile processing to obtain scaffolds with different shapes and sizes, ceramics are a succulent offer in nervous tissue engineering. Furthermore, their possibilities have been increased with polymeric-ceramics composites development, which have given rise to new interesting horizon.Existen diferentes tipos de lesiones o desordenes del Sistema Nervioso Central (SNC que pueden provocar graves secuelas e incluso en algunos casos una discapacidad permanente. Además, el proceso de reparación del SNC tiene algunas complicaciones. El mecanismo natural de reacción a una lesión, el cual consiste en la formación de una cicatriz glial, es desencadenado por un proceso inflamatorio. Las moléculas liberadas durante estos procesos, la inflamación y formación de la cicatriz glial, así como la deficiencia en oxígeno y glucosa debidos a la lesión, crean un ambiente que inhibe la regeneración axonal creando la llamada “lesión secundaria”. Los biomateriales están adquiriendo un papel cada vez más importante en la reparación de SNC. Las

  17. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  18. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  19. Analysis of Bi-directional Effects on the Response of a Seismic Base Isolation System

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyung-Kui; Kim, Jung-Han; Kim, Min Kyu; Choi, In-Kil [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2014-10-15

    The floor response spectrum depends on the height of the floor of the structure. Also FRS depends on the characteristics of the seismic base isolation system such as the natural frequency, damping ratio. In the previous study, the floor response spectrum of the base isolated structure was calculated for each axis without considering bi-directional effect. However, the shear behavior of the seismic base isolation system of two horizontal directions are correlated each other by the bi-directional effects. If the shear behavior of the seismic isolation system changes, it can influence the floor response spectrum and displacement response of isolators. In this study, the analysis of a bi-directional effect on the floor response spectrum was performed. In this study, the response of the seismic base isolation system based on the bi-directional effects was analyzed. By analyzing the time history result, while there is no alteration in the maximum shear force of seismic base isolation system, it is confirmed that the shear force is generally more decreased in a one-directional that in a two-directional in most parts. Due to the overall decreased shear force, the floor response spectrum is more reduced in a two-directional than in a one-directional.

  20. Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract.

    Science.gov (United States)

    Hébrard, Géraldine; Blanquet, Stéphanie; Beyssac, Eric; Remondetto, Gabriel; Subirade, Muriel; Alric, Monique

    2006-12-15

    A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (pwhey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.

  1. Architecture and Hardware Design of Lossless Compression Algorithms for Direct-Write Maskless Lithography Systems

    Science.gov (United States)

    2010-04-29

    system using 22 nm pixels for 45 nm technology, a data rate of 12 Tb/s is required. A recently proposed datapath architecture for direct-write...for 45 nm technology, a data rate of 12 Tb/s is required. A recently pro- posed datapath architecture for direct-write lithography systems shows that...1 1.2 The Architecture of Maskless Lithography Systems . . . . . . . . . . . . . . 2 1.3 Datapath Implementation of

  2. Frequency Properties Research of Elevator Drive System with Direct Torque Control-Pulse with Modulation

    Directory of Open Access Journals (Sweden)

    A. S. Koval

    2008-01-01

    Full Text Available In the article problems of frequency properties research for electric drive system with direct torque control and pulse width modulator are described. The mathematical description of elevator is present. Simplified mathematical description of direct torque control - pulse width modulator electric drive system is shown. Transfer functions for torque and speed loops are determined. Logarithmic frequency characteristics are computed. Damping properties of elevator drive system are estimated.

  3. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

  4. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes.

    Directory of Open Access Journals (Sweden)

    Laura J Marinelli

    Full Text Available Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED, in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

  5. Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

    Directory of Open Access Journals (Sweden)

    Remy Froissart

    2005-03-01

    Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

  6. Direct and Indirect Couplings in Coherent Feedback Control of Linear Quantum Systems

    CERN Document Server

    Zhang, Guofeng

    2010-01-01

    The purpose of this paper is to study and design direct and indirect couplings for use in coherent feedback control of a class of linear quantum stochastic systems. A general physical model for a nominal linear quantum system coupled directly and indirectly to external systems is presented. Fundamental properties of stability, dissipation, passivity, and gain for this class of linear quantum models are presented and characterized using complex Lyapunov equations and linear matrix inequalities (LMIs). Coherent $H^\\infty$ and LQG synthesis methods are extended to accommodate direct couplings using multistep optimization. Examples are given to illustrate the results.

  7. Distribution of the phenotypic effects of random homologous recombination between two virus species.

    Directory of Open Access Journals (Sweden)

    Florence Vuillaume

    2011-05-01

    Full Text Available Recombination has an evident impact on virus evolution and emergence of new pathotypes, and has generated an immense literature. However, the distribution of phenotypic effects caused by genome-wide random homologous recombination has never been formally investigated. Previous data on the subject have promoted the implicit view that most viral recombinant genomes are likely to be deleterious or lethal if the nucleotide identity of parental sequences is below 90%. We decided to challenge this view by creating a bank of near-random recombinants between two viral species of the genus Begomovirus (Family Geminiviridae exhibiting 82% nucleotide identity, and by testing infectivity and in planta accumulation of recombinant clones randomly extracted from this bank. The bank was created by DNA-shuffling-a technology initially applied to the random shuffling of individual genes, and here implemented for the first time to shuffle full-length viral genomes. Together with our previously described system allowing the direct cloning of full-length infectious geminivirus genomes, it provided a unique opportunity to generate hundreds of "mosaic" virus genomes, directly testable for infectivity. A subset of 47 randomly chosen recombinants was sequenced, individually inoculated into tomato plants, and compared with the parental viruses. Surprisingly, our results showed that all recombinants were infectious and accumulated at levels comparable or intermediate to that of the parental clones. This indicates that, in our experimental system, despite the fact that the parental genomes differ by nearly 20%, lethal and/or large deleterious effects of recombination are very rare, in striking contrast to the common view that has emerged from previous studies published on other viruses.

  8. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

    Science.gov (United States)

    In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

  9. A design procedure and handling quality criteria for lateral directional flight control systems

    Science.gov (United States)

    Stein, G.; Henke, A. H.

    1972-01-01

    A practical design procedure for aircraft augmentation systems is described based on quadratic optimal control technology and handling-quality-oriented cost functionals. The procedure is applied to the design of a lateral-directional control system for the F4C aircraft. The design criteria, design procedure, and final control system are validated with a program of formal pilot evaluation experiments.

  10. Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System.

    Science.gov (United States)

    Adamson-Small, Laura; Potter, Mark; Byrne, Barry J; Clément, Nathalie

    2017-02-01

    The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10(14) vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10(15) VG in crude cell harvests and an average of 3.5 × 10(14) total VG of purified rAAV9 material, resulting in greater than 1 × 10(5) VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

  11. Analysis of the System of Direct Subsidy for Grain Production in China

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    This paper introduces the status quo of the system structure of direct subsidy for grain production in China,and analyses the function and essence of direct subsidy for grain production in China as follows:the function of direct subsidy for grain production in China is to protect and promote the development of food industry,ensure food supply and national food security,protect grain growers’ interests,and maintain the dominant position of national food trade;the essence of direct subsidy for grain production is the compensation for grain producers’ reasonable interests,the compensation for positive externality of grain production,and social costs of adjusting equilibrium between food supply and demand.We discuss the problems existing in the system of direct subsidy for grain production in China and put forward corresponding countermeasures as follows:first,adhere to improving synergetic system of direct subsidy for grain production;second,adjust the direct subsidy method of grain production;third,establish long-term mechanism of subsidy for grain production;fourth,promote the information-based service level of the system of direct subsidy for grain production.

  12. Development and Use of the Directives Rating System in Group Therapy.

    Science.gov (United States)

    Stinchfield, Randy D.; Burlingame, Gary M.

    1991-01-01

    Applied the Directives Rating System (DRS), which classifies directives, to transcripts of four expert group therapists. DRS yielded satisfactory estimates of interrater agreement and validity. Findings showed that DRS ratings were related to therapists' theoretical orientation and that therapists tended to use imperatives and requestives when…

  13. A Beta Index to Confirm Causal Directions in a Closed System of Five Variables.

    Science.gov (United States)

    Nigro, George A.

    The beta coefficient of an intermediate variable in a causal direction remains relatively constant as other system variables are introduced and controlled in stepped regression, whereas that in the acausal direction changes noticeably. Normalized random numbers (200x5) were generated and substituted in interdependent equations to produce five…

  14. A bi-directional electrochemically driven micro liquid dosing system with integrated sensor/actuator electrodes

    NARCIS (Netherlands)

    Bohm, S.; Olthuis, Wouter; Bergveld, Piet

    2000-01-01

    In this contribution a micro liquid dosing system is presented, which allows bi-directional manipulation of fluids (i.e. pushing out and pulling in of liquids) by the electrochemical generation and removal of gas bubbles. Bi-directionality is obtained by reversal of the actuation current thereby

  15. RFLP analysis in rice by using ECL direct nucleic acid labeling and detection system

    Institute of Scientific and Technical Information of China (English)

    ZHANGShanhong; LIUBin; LUOLin; ZHUXiaoyuan; YANGQiyun; WUShangzhong

    1998-01-01

    ECL (Enhanced Chemiluminescence) direct nu cleic acid labeling and detection syslem were used for RFLP analysis in rice. The system involved directly labeling DNA probe with the enzyme horseradish peroxidase (HRP), and the detection of non-radioactive signal based on chemiluminescence, that was the generation of light via enzyme (HRP)-catalyzed reactions. It was a simple, safe,

  16. Integration of Directional Antennas in an RSS Fingerprinting-Based Indoor Localization System

    National Research Council Canada - National Science Library

    Guzmán-Quirós, Raúl; Martínez-Sala, Alejandro; Gómez-Tornero, José Luis; García-Haro, Joan

    ...) fingerprinting-based indoor localization system (ILS) is studied. The sensor reader (SR), which is in charge of capturing the RSS to infer the tag position, can be attached to an omnidirectional or directional antenna...

  17. Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.

    Science.gov (United States)

    Bučko, Marek; Schenkmayerová, Andrea; Gemeiner, Peter; Vikartovská, Alica; Mihovilovič, Marko D; Lacík, Igor

    2011-08-10

    An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.

  18. Direct and semi-direct aerosol radiative effect on the Mediterranean climate variability using a coupled regional climate system model

    Science.gov (United States)

    Nabat, Pierre; Somot, Samuel; Mallet, Marc; Sevault, Florence; Chiacchio, Marc; Wild, Martin

    2015-02-01

    A fully coupled regional climate system model (CNRM-RCSM4) has been used over the Mediterranean region to investigate the direct and semi-direct effects of aerosols, but also their role in the radiation-atmosphere-ocean interactions through multi-annual ensemble simulations (2003-2009) with and without aerosols and ocean-atmosphere coupling. Aerosols have been taken into account in CNRM-RCSM4 through realistic interannual monthly AOD climatologies. An evaluation of the model has been achieved, against various observations for meteorological parameters, and has shown the ability of CNRM-RCSM4 to reproduce the main patterns of the Mediterranean climate despite some biases in sea surface temperature (SST), radiation and cloud cover. The results concerning the aerosol radiative effects show a negative surface forcing on average because of the absorption and scattering of the incident radiation. The SW surface direct effect is on average -20.9 Wm-2 over the Mediterranean Sea, -14.7 Wm-2 over Europe and -19.7 Wm-2 over northern Africa. The LW surface direct effect is weaker as only dust aerosols contribute (+4.8 Wm-2 over northern Africa). This direct effect is partly counterbalanced by a positive semi-direct radiative effect over the Mediterranean Sea (+5.7 Wm-2 on average) and Europe (+5.0 Wm-2) due to changes in cloud cover and atmospheric circulation. The total aerosol effect is consequently negative at the surface and responsible for a decrease in land (on average -0.4 °C over Europe, and -0.5 °C over northern Africa) and sea surface temperature (on average -0.5 °C for the Mediterranean SST). In addition, the latent heat loss is shown to be weaker (-11.0 Wm-2) in the presence of aerosols, resulting in a decrease in specific humidity in the lower troposphere, and a reduction in cloud cover and precipitation. Simulations also indicate that dust aerosols warm the troposphere by absorbing solar radiation, and prevent radiation from reaching the surface, thus

  19. Recombinant horseradish peroxidase: production and analytical applications.

    Science.gov (United States)

    Grigorenko, V G; Andreeva, I P; Rubtsova, M Yu; Egorov, A M

    2015-04-01

    Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

  20. DSMC modeling of flows with recombination reactions

    Science.gov (United States)

    Gimelshein, Sergey; Wysong, Ingrid

    2017-06-01

    An empirical microscopic recombination model is developed for the direct simulation Monte Carlo method that complements the extended weak vibrational bias model of dissociation. The model maintains the correct equilibrium reaction constant in a wide range of temperatures by using the collision theory to enforce the number of recombination events. It also strictly follows the detailed balance requirement for equilibrium gas. The model and its implementation are verified with oxygen and nitrogen heat bath relaxation and compared with available experimental data on atomic oxygen recombination in argon and molecular nitrogen.

  1. Testing for recombinant erythropoietin.

    Science.gov (United States)

    Delanghe, Joris R; Bollen, Mathieu; Beullens, Monique

    2008-03-01

    Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

  2. Basic Characteristics of New Developed Higher-Voltage Direct-Current Power-Feeding Prototype System

    Science.gov (United States)

    Babasaki, Tadatoshi; Tanaka, Toshimitsu; Tanaka, Toru; Nozaki, Yousuke; Aoki, Tadahito; Kurokawa, Fujio

    High efficiency power feeding systems are effective solutions for reducing the ICT power consumption with reducing power consumption of the ICT equipment and cooling systems. A higher voltage direct current (HVDC) power feeding system prototype was produced. This system is composed of a rectifier equipment, power distribution unit, batteries, and the ICT equipment. The configuration is similar to a -48V DC power supply system. The output of the rectifier equipment is 100kW, and the output voltage is 401.4V. This paper present the configuration of the HVDC power feeding system and discuss its basic characteristics in the prototype system.

  3. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

    Science.gov (United States)

    Lenden Hasse, Hélène; Lescale, Chloé; Bianchi, Joy J; Yu, Wei; Bedora-Faure, Marie; Deriano, Ludovic

    2017-09-04

    Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Direct Load Control (DLC) Considering Nodal Interrupted Energy Assessment Rate (NIEAR) in Restructured Power Systems

    DEFF Research Database (Denmark)

    Wu, Qiuwei; Wang, Peng; Goel, Lalit

    2010-01-01

    A direct load control (DLC) scheme of air conditioning loads (ACL) considering direct monetary compensation to ACL customers for the service interruption caused by the DLC program is proposed in this paper for restructured power systems. The nodal interrupted energy assessment rate (NIEAR), which...... is used as the bids from the ACL customers, is utilized to determine the direct monetary compensation to the ACL customers. The proposed scheme was investigated for the PoolCo electricity market. The optimal DLC scheme is determined based on the minimum system operating cost which is comprised...

  5. The Simulation of Direct Spread Spectrum System based on Transmitted Reference Signal

    Directory of Open Access Journals (Sweden)

    Wu Guoqiang

    2013-01-01

    Full Text Available Code synchronization is indispensable in the direct spread spectrum system because it can influence the incepting capacity directly. Transmitted reference is proposed in this paper to predigest the code synchronization circuit of the incepting machine in order to reduce the cost of time, energy and money for the development of the code synchronization technology. The software named Systemview is employed to simulate the transmitted reference direct spread spectrum system. The simulation results were presented with the condition of gauss noise and temperature. It demonstrates that the proposed simulation has significant effect and benefit in engineering.

  6. Phase system selection with fractional factorial design for purification of recombinant cyanovirin-N from a hydroponic culture medium using centrifugal partition chromatography.

    Science.gov (United States)

    Grudzień, Łukasz; Madeira, Luisa; Fisher, Derek; Ma, Julian; Garrard, Ian

    2013-04-12

    Centrifugal partition chromatography (CPC) with an aqueous two-phase system (ATPS) was used to purify recombinant cyanovirin-N (CV-N) from other proteins which were co-secreted into a hydroponic plant medium in a rhizosecretion process. To achieve satisfactory protein concentration, the purification was preceded by ultrafiltration performed on a 5 kDa filter. ATPS, because of their gentle nature, were selected as the phase system for CPC. A systematic phase system selection was applied. This involved studying the effect of seven parameters of ATPS: polymer type, salt type, the polymer and salt concentration, the polymer molecular weight, pH, and presence of two additional salts; NaCl and NaClO4, which all together gave 320 combinations. design of experiment (DoE) software allowed the reduction of this number to 46. Having tested partitioning of cyanovirin-N and impurities in 46 ATPS, the three best potential phase systems generated by the programme were then tested on the CPC. Out of these three, 13/13% PEG4000 sodium phosphate, pH 3.0, proved to be most effective phase system in the purification of cyanovirin-N, judged by ELISA and SDS-PAGE analysis, as it eliminated most of the impurities from the final cyanovirin-N preparation.

  7. Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems.

    Science.gov (United States)

    Morse, Alison M; Carballo, Valentina; Baldwin, Donald A; Taylor, Christopher G; McIntyre, Lauren M

    2010-09-01

    Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

  8. Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

    Science.gov (United States)

    Lakowitz, Antonia; Godard, Thibault; Biedendieck, Rebekka; Krull, Rainer

    2017-06-09

    Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production. Copyright © 2017. Published by Elsevier B.V.

  9. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in in......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed.......Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved...

  10. Immune regulation in gut and cord : opportunities for directing the immune system

    NARCIS (Netherlands)

    de Roock, S.

    2012-01-01

    The gut is an important organ for the immune system. Microbes and immune cells interact directly or via epithelial cells. Both TH17 and Treg cells mature in this environment. The composition of the microbiota has an important influence on the immune homeostasis. Influencing the immune system via the

  11. Broken symmetries and directed collective energy transport in spatially extended systems

    DEFF Research Database (Denmark)

    Flach, S.; Zolotaryuk, Yaroslav; Miroshnichenko, A. E.;

    2002-01-01

    We study the appearance of directed energy current in homogeneous spatially extended systems coupled to a heat bath in the presence of an external ac field E(t) . The systems are described by nonlinear field equations. By making use of a symmetry analysis, we predict the right choice of E(t) and ...

  12. Evolution of Word-updating Dynamical Systems (WDS) on Directed Graphs

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jie

    2009-01-01

    This paper continues the research on theoretical foundations for computer simulation. We introduce the concept of word-updating dynamical systems (WDS) on directed graphs, which is a kind of generalization of sequential dynamical systems (SDS) on graphs. Some properties on WDS, especially some results on NOR -WDS,which are different from that on NOR -SDS, are obtained.

  13. Direct transition from a stable equilibrium to quasiperiodicity in non-smooth systems

    DEFF Research Database (Denmark)

    Zhusubaliyev, Z.T.; Mosekilde, Erik

    2008-01-01

    The purpose of this Letter is to show how a border-collision bifurcation in a piecewise-smooth dynamical system can produce a direct transition from a stable equilibrium point to a two-dimensional invariant torus. Considering a system of nonautonomous differential equations describing the behavior...

  14. The magnitude and direction movement in Thailand based on Global Positioning System (GPS)

    Science.gov (United States)

    Jamrus, Uthen; Deng, Hui

    2016-10-01

    In this research, we applied the Global Navigation Satellite System (GNSS) with Global Positioning System (GPS) to create new geodetic network, which is referred to ITRF2000. GPS observation data in 2010 and 2012 were used for network adjustment by Least Square Method (Minimally Constrained Adjustment and Fully Constrained Adjustment), then adjusted coordinates were used to determine updated magnitude and direction.

  15. Second-Order Consensus for Multiagent Systems With Directed Topologies and Nonlinear Dynamics

    NARCIS (Netherlands)

    Yu, Wenwu; Chen, Guanrong; Cao, Ming; Kurths, Juergen; Kurths, Jürgen

    This paper considers a second-order consensus problem for multiagent systems with nonlinear dynamics and directed topologies where each agent is governed by both position and velocity consensus terms with a time-varying asymptotic velocity. To describe the system's ability for reaching consensus, a

  16. Optimization Tool for Direct Water Cooling System of High Power IGBT Modules

    DEFF Research Database (Denmark)

    Bahman, Amir Sajjad; Blaabjerg, Frede

    2016-01-01

    important issue for thermal design engineers. This paper aims to present a user friendly optimization tool for direct water cooling system of a high power module which enables the cooling system designer to identify the optimized solution depending on customer load profiles and available pump power. CFD...

  17. Immune regulation in gut and cord : opportunities for directing the immune system

    NARCIS (Netherlands)

    de Roock, S.

    2012-01-01

    The gut is an important organ for the immune system. Microbes and immune cells interact directly or via epithelial cells. Both TH17 and Treg cells mature in this environment. The composition of the microbiota has an important influence on the immune homeostasis. Influencing the immune system via the

  18. 78 FR 28005 - System Energy Resources, Inc.; Grand Gulf Nuclear Station; Order Approving Direct and Indirect...

    Science.gov (United States)

    2013-05-13

    ... From the Federal Register Online via the Government Publishing Office NUCLEAR REGULATORY COMMISSION System Energy Resources, Inc.; Grand Gulf Nuclear Station; Order Approving Direct and Indirect Transfers of Early Site Permit and Approving Conforming Amendment I System Energy Resources, Inc. (SERI), is...

  19. Complexity through Recombination: From Chemistry to Biology

    Directory of Open Access Journals (Sweden)

    Carolina Díaz Arenas

    2010-12-01

    Full Text Available Recombination is a common event in nature, with examples in physics, chemistry, and biology. This process is characterized by the spontaneous reorganization of structural units to form new entities. Upon reorganization, the complexity of the overall system can change. In particular the components of the system can now experience a new response to externally applied selection criteria, such that the evolutionary trajectory of the system is altered. In this work we explore the link between chemical and biological forms of recombination. We estimate how the net system complexity changes, through analysis of RNA-RNA recombination and by mathematical modeling. Our results underscore the importance of recombination in the origins of life on the Earth and its subsequent evolutionary divergence.

  20. Ac-dc Bus-interface Bi-directional Converters in Renewable Energy Systems

    OpenAIRE

    Dong, Dong

    2012-01-01

    This dissertation covers several issues related to the ac-dc bus-interface bi-directional converters in renewable energy systems. The dissertation explores a dc-electronic distribution system for residential and commercial applications with a focus on the design of an ac-dc bi-directional converter for such application. This converter is named as the â Energy Control Centerâ due to its unique role in the system. First, the impact of the unbalanced power from the ac grid, especially the ...

  1. A Digital Method for the Detection of Blood Flow Direction in Ultrasonic Doppler Systems

    Directory of Open Access Journals (Sweden)

    P. Acevedo–Contla.

    2010-01-01

    Full Text Available Doppler ultrasound systems are widely used to study blood flow and diagnosis of vascular diseases. An important characteristic of these systems is the ability to detect the direction of the blood flow. Most Doppler ultrasound systems apply a quadrature demodulation technique on the ultrasonic transducer output signal. Therefore additional treatment is necessary to separate forward and reverse flow signals. This work presents a digital method to convert signals in quadrature into directional signals using a Fast Fourier Transform (FFT approach. Validation of the method has been achieved using simulated Doppler ultrasound signals.

  2. RELIABILITY ASSESSMENT OF STRUCTURAL SYSTEMS BASED ON DIRECTIONAL VECTOR SIMULATION TECHNIQUE

    Institute of Scientific and Technical Information of China (English)

    Zhang Liangxin; Hu Yunchang

    2000-01-01

    The new improved directional vector simulation method for analyzing the reliability of struc tural systems failure probability is researched. This paper also points out the defects of general directional vector simulation, and gives rise to a new higher accuracy approximate integral formula of structural systems failure probability. A new geometric meaning of characteristic function is obtained. A new simple method of generating uniformly distributed random vector samples in n-dimensional unit hyper-spherical surface is put forward and strictly proved. This method is easy to put into practice. Numerical examples are given to show the applicability and effectiveness of the suggested approach to structural systems reliability problems.

  3. Global adaptive exponential stabilisation for nonlinear systems with multiple unknown control directions

    Science.gov (United States)

    Sun, Xifang; Chen, Weisheng; Wu, Jian

    2016-12-01

    In this paper, we address the global generalised exponential stabilisation problem for a class of lower-triangular systems with multiple unknown directions. Instead of the well-known Nussbaum-gain adaptive rule, a Lyapunov-based adaptive logic switching rule is proposed to seek the correct control directions for such systems. The main advantage of the proposed controller is that it can guarantee the global generalised exponential stability of closed-loop systems. Simulation examples are given to verify the effectiveness of the developed control approach.

  4. The research on direct-drive wave energy conversion system and performance optimization

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhongxian; YU Haitao; HU Minqiang

    2014-01-01

    A direct-drive wave energy conversion system based on a three-phase permanent magnet tubular linear generator (PMTLG) and a heaving buoy is proposed to convert wave energy into electrical energy. Sufficient experimental methods are adopted to compare the computer simulations, the validity of which is verified by the experiment results from a wave tank laboratory. In the experiment, the motion curves of heaving buoy are with small fluctuations, mainly caused by the PMTLG’s detent force. For the reduction of these small fluctuations and a maximum operational efficiency of the direct-drive wave energy conversion system, the PMTLG’s detent force minimization technique and the heaving buoy optimization will be discussed. It is discovered that the operational efficiency of the direct-drive wave energy conversion system increases dra-matically after optimization. The experiment and optimization results will provide useful reference for the future research on ocean wave energy conversion system.

  5. Antagonistic experimental coevolution with a parasite increases host recombination frequency

    Directory of Open Access Journals (Sweden)

    Kerstes Niels AG

    2012-02-01

    Full Text Available Abstract Background One of the big remaining challenges in evolutionary biology is to understand the evolution and maintenance of meiotic recombination. As recombination breaks down successful genotypes, it should be selected for only under very limited conditions. Yet, recombination is very common and phylogenetically widespread. The Red Queen Hypothesis is one of the most prominent hypotheses for the adaptive value of recombination and sexual reproduction. The Red Queen Hypothesis predicts an advantage of recombination for hosts that are coevolving with their parasites. We tested predictions of the hypothesis with experimental coevolution using the red flour beetle, Tribolium castaneum, and its microsporidian parasite, Nosema whitei. Results By measuring recombination directly in the individuals under selection, we found that recombination in the host population was increased after 11 generations of coevolution. Detailed insights into genotypic and phenotypic changes occurring during the coevolution experiment furthermore helped us to reconstruct the coevolutionary dynamics that were associated with this increase in recombination frequency. As coevolved lines maintained higher genetic diversity than control lines, and because there was no evidence for heterozygote advantage or for a plastic response of recombination to infection, the observed increase in recombination most likely represented an adaptive host response under Red Queen dynamics. Conclusions This study provides direct, experimental evidence for an increase in recombination frequency under host-parasite coevolution in an obligatory outcrossing species. Combined with earlier results, the Red Queen process is the most likely explanation for this observation.

  6. Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

    Directory of Open Access Journals (Sweden)

    Iwashkiw Jeremy A

    2012-01-01

    Full Text Available Abstract Background Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods. Results In this work we expressed the C. jejuni oligosaccharyltansferase (OTase PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera. Conclusion Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

  7. Analytic calculation of formation enthalpies directly from interatomic potentials for binary and ternary refractory metal systems

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    An analytic method is proposed to calculate the formation enthalpy directly from empirical n-body potential and applied to the binary and ternary systems consisting of the refractory metals Mo, Nb, Ta and W. It turns out that the calculated enthalpies are in overall agreement with experimental observations and some other theoretical calculations. Interestingly, it shows that the formation enthalpies of the ternary systems are significantly affected by those of the constituent binary systems.

  8. Mouse Housing System Using Pressurized Cages Intraventilated by Direct-Current Microfans

    OpenAIRE

    Martinewski, Alexandre; Correia, Caio SC; de Souza, Nívea L; Merusse, José LB

    2012-01-01

    We performed the initial assessment of an alternative pressurized intraventilated (PIV) caging system for laboratory mice that uses direct-current microfans to achieve cage pressurization and ventilation. Twenty-nine pairs of female SPF BALB/c mice were used, with 19 experimental pairs kept in PIV cages and 10 control pairs kept in regular filter-top (FT) cages. Both groups were housed in a standard housing room with a conventional atmospheric control system. For both systems, intracage tempe...

  9. A novel speed sensor-less direct torque control system for mining locomotive haulage

    Institute of Scientific and Technical Information of China (English)

    马宪民

    2002-01-01

    A novel speed sensor-less direct torque control induction motor drive system for the mining locomotive haulage is presented in the paper. Rotor speed identification is based on the model reference adaptive control theory with neural network using back propagation algorithm. The system is implemented using a real-time TMS320F240 digital signal processor. The simulation study and experiment results indicate that the suggested system has good performance.

  10. Novel Recombinant Sapovirus

    Science.gov (United States)

    Katayama, Kazuhiko; Miyoshi, Tatsuya; Uchino, Kiyoko; Oka, Tomoichiro; Tanaka, Tomoyuki; Takeda, Naokazu

    2004-01-01

    We determined the complete genome sequences of two sapovirus strains isolated in Thailand and Japan. One of these strains represented a novel, naturally occurring recombinant sapovirus. Evidence suggested the recombination site was at the polymerase-capsid junction within open reading frame one. PMID:15504283

  11. A new multi-motor drive system based on two-stage direct power converter

    OpenAIRE

    Kumar, Dinesh

    2011-01-01

    The two-stage AC to AC direct power converter is an alternative matrix converter topology, which offers the benefits of sinusoidal input currents and output voltages, bidirectional power flow and controllable input power factor. The absence of any energy storage devices, such as electrolytic capacitors, has increased the potential lifetime of the converter. In this research work, a new multi-motor drive system based on a two-stage direct power converter has been proposed, with two motors c...

  12. Combined indirect and direct method for adaptive fuzzy output feedback control of nonlinear system

    Institute of Scientific and Technical Information of China (English)

    Ding Quanxin; Chen Haitong; Jiang Changsheng; Chen Zongji

    2007-01-01

    A novel control method for a general class of nonlinear systems using fuzzy logic systems (FLSs) is presertted.Indirect and direct methods are combined to design the adaptive fuzzy output feedback controller and a high-gain observer is used to estimate the derivatives of the system output. The closed-loop system is proven to be semiglobally uniformly ultimately bounded. In addition, it is shown that if the approximation accuracy of the fuzzy logic system is high enough and the observer gain is chosen sufficiently large, an arbitrarily small tracking error can be achieved. Simulation results verify the effectiveness of the newly designed scheme and the theoretical discussion.

  13. Three-Body Recombination in Cold Atomic Gases

    CERN Document Server

    Sørensen, Peder K

    2013-01-01

    Systems of three particles show a surprising feature in their bound state spectrum: a series of geometrically scaled states, known as Efimov states. These states have not yet been observed directly, but many recent experiments show indirect evidence of their existence via the so-called recombination process. The theories that predict the Efimov states also predicts either resonant enhancement of the recombination process or suppression by destructive interference, depending on the sign of the interaction between the particles. The theories predict universal features for the Efimov states, for instance that the geometric scaling factor is 22.7, meaning that one state is 22.7 times larger than its lower lying neighbour state. This thesis seeks to investigate non-universal effects by incorporating additional information about the physical interactions into the universal theories.

  14. Integration of Directional Antennas in an RSS Fingerprinting-Based Indoor Localization System.

    Science.gov (United States)

    Guzmán-Quirós, Raúl; Martínez-Sala, Alejandro; Gómez-Tornero, José Luis; García-Haro, Joan

    2015-12-23

    In this paper, the integration of directional antennas in a room-level received signal strength (RSS) fingerprinting-based indoor localization system (ILS) is studied. The sensor reader (SR), which is in charge of capturing the RSS to infer the tag position, can be attached to an omnidirectional or directional antenna. Unlike commonly-employed omnidirectional antennas, directional antennas can receive a stronger signal from the direction in which they are pointed, resulting in a different RSS distributions in space and, hence, more distinguishable fingerprints. A simulation tool and a system management software have been also developed to control the system and assist the initial antenna deployment, reducing time-consuming costs. A prototype was mounted in a real scenario, with a number of SRs with omnidirectional and directional antennas properly positioned. Different antenna configurations have been studied, evidencing a promising capability of directional antennas to enhance the performance of RSS fingerprinting-based ILS, reducing the number of required SRs and also increasing the localization success.

  15. Seven-channel digital telemetry system for monitoring and direct computer capturing of biological data.

    Science.gov (United States)

    Drewes, A M; Andreasen, A; Assentoft, J E; Nagel, O

    1993-09-01

    A seven-channel telemetry system for collection and display of biological data is presented. The system can amplify bioelectrical signals in the range of 2 microV to 200 mV and has a bandwidth of 0.1-80 Hz. After multiplexing, the signals are digitized with a resolution of 8 bits. The data are frequency modulated directly on a VHF transmitter. After receiving the data on a VHF receiver, they are routed directly to the RS232 input connector on the PC. Thereby the advantage of direct communication between the transmitter and the PC can be utilized. Expensive analog equipment is avoided and display of the signals on the PC screen as well as signal analysis can be performed. The system has been tested and was found to be stable and highly reliable.

  16. Recommended Research Directions for Improving the Validation of Complex Systems Models.

    Energy Technology Data Exchange (ETDEWEB)

    Vugrin, Eric D. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Trucano, Timothy G. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Swiler, Laura Painton [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Finley, Patrick D. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Flanagan, Tatiana Paz [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Naugle, Asmeret Bier [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Tsao, Jeffrey Y. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Verzi, Stephen Joseph [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2017-02-01

    Improved validation for models of complex systems has been a primary focus over the past year for the Resilience in Complex Systems Research Challenge. This document describes a set of research directions that are the result of distilling those ideas into three categories of research -- epistemic uncertainty, strong tests, and value of information. The content of this document can be used to transmit valuable information to future research activities, update the Resilience in Complex Systems Research Challenge's roadmap, inform the upcoming FY18 Laboratory Directed Research and Development (LDRD) call and research proposals, and facilitate collaborations between Sandia and external organizations. The recommended research directions can provide topics for collaborative research, development of proposals, workshops, and other opportunities.

  17. The efficiency of homologous recombination and non-homologous end joining systems in repairing double-strand breaks during cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Leonardo Bee

    Full Text Available This study investigated the efficiency of Non-Homologous End Joining (NHEJ and Homologous Recombination (HR repair systems in rejoining DNA double-strand breaks (DSB induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density parameter and counting foci number in the time-interval 0.5-24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.

  18. Direct imaging discovery of a Jovian exoplanet within a triple-star system.

    Science.gov (United States)

    Wagner, Kevin; Apai, Dániel; Kasper, Markus; Kratter, Kaitlin; McClure, Melissa; Robberto, Massimo; Beuzit, Jean-Luc

    2016-08-12

    Direct imaging allows for the detection and characterization of exoplanets via their thermal emission. We report the discovery via imaging of a young Jovian planet in a triple-star system and characterize its atmospheric properties through near-infrared spectroscopy. The semimajor axis of the planet is closer relative to that of its hierarchical triple-star system than for any known exoplanet within a stellar binary or triple, making HD 131399 dynamically unlike any other known system. The location of HD 131399Ab on a wide orbit in a triple system demonstrates that massive planets may be found on long and possibly unstable orbits in multistar systems. HD 131399Ab is one of the lowest mass (4 ± 1 Jupiter masses) and coldest (850 ± 50 kelvin) exoplanets to have been directly imaged.

  19. Systematic description of direct push sensor systems: A conceptual framework for system decomposition as a basis for the optimal sensor system design

    Science.gov (United States)

    Bumberger, Jan; Paasche, Hendrik; Dietrich, Peter

    2015-11-01

    Systematic decomposition and evaluation of existing sensor systems as well as the optimal design of future generations of direct push probes are of high importance for optimized geophysical experiments since the employed equipment is a constrain on the data space. Direct push technologies became established methods in the field of geophysical, geotechnical, hydrogeological, and environmental sciences for the investigation of the near subsurface. By using direct push sensor systems it is possible to measure in-situ parameters with high vertical resolution. Such information is frequently used for quantitative geophysical model calibration of interpretation of geotechnical and hydrological subsurface conditions. Most of the available direct push sensor systems are largely based on empirical testing and consecutively evaluated under field conditions. Approaches suitable to identify specific characteristics and problems of direct push sensor systems have not been established, yet. We develop a general systematic approach for the classification, analysis, and optimization of direct push sensor systems. First, a classification is presented for different existing sensor systems. The following systematic description, which is based on the conceptual decomposition of an existing sensor system into subsystems, is a suitable way to analyze and explore the transfer behavior of the system components and therefore of the complete system. Also, this approach may serve as guideline for the synthesis and the design of new and optimized direct push sensor systems.

  20. Changes in direct and indirect taxation in the process of the EU tax system reform

    Directory of Open Access Journals (Sweden)

    Miletić Vesna

    2015-01-01

    Full Text Available Proceeding from the changes in direct and indirect taxation in the process of the EU tax system reform and the fact that new trends bring about vital social changes, the research in this area could add to the understanding of economic development of these countries. In the EU with its decentralized tax policy, changes in direct and indirect taxation are permanently implemented in the process of tax system reform. Harmonization of direct and indirect taxation in the EU is conducted by means of the adopted community law regulations. In the harmonization of direct taxation there is a tendency towards an increase of the level of fiscal coordination and elimination of barriers to the common market. Thus direct taxation reforms are limited to achieving higher level of coordination and preventing evasion and double taxation. The current policy is the result of the principle according to which fiscal competition has positive effects on reducing consumption and on the EU economic development. In the harmonization process the systems of indirect taxation are aligned particularly with excise duty and value added tax. Fiscal strategies in the reform process are mostly aimed at fiscal coordination, modernization, informative cooperation, evasion reduction and elimination of malpractices in fulfilling a VAT payment liability. Within the excise tax system minimal excise rates are determined and an environmentally-friendly policy is pursued. The main priorities of taxation changes within the harmonization process are in respect of evasion reduction and irrational fiscal competition on the EU market. A study of direct and indirect taxation changes in the process of the tax system reform is highly significant both from the aspect of efficient tax-system functioning, which results in achieving EU macroeconomic goals, and from the aspect of single states' interests in the process of transition and accession.

  1. Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

    Science.gov (United States)

    1987-09-01

    Report No. 4 If MOLECULAR CLONING OF O HUMAN GENE(S) DIRECTING qTHE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASES cc Annual/Final Report 0 N November...62734A I734A875 IAl 451 MOLECULAR CLONING OF HUMAN GEME(S) DIRECTING THE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASE 12. PERSONAL AUTHOR(S) Hermona Soreq...important roles in regulating the pace and mode of function of particular types of synapses. For example, molecular cloning of the nicotinic (44-46) and the

  2. Geometric detection of coupling directions by means of inter-system recurrence networks

    CERN Document Server

    Feldhoff, Jan H; Donges, Jonathan F; Marwan, N; Kurths, J; 10.1016/j.physleta.2012.10.008

    2013-01-01

    We introduce a geometric method for identifying the coupling direction between two dynamical systems based on a bivariate extension of recurrence network analysis. Global characteristics of the resulting inter-system recurrence networks provide a correct discrimination for weakly coupled R\\"ossler oscillators not yet displaying generalised synchronisation. Investigating two real-world palaeoclimate time series representing the variability of the Asian monsoon over the last 10,000 years, we observe indications for a considerable influence of the Indian summer monsoon on climate in Eastern China rather than vice versa. The proposed approach can be directly extended to studying $K>2$ coupled subsystems.

  3. Geometric detection of coupling directions by means of inter-system recurrence networks

    Energy Technology Data Exchange (ETDEWEB)

    Feldhoff, Jan H. [Potsdam Institute for Climate Impact Research, P.O. Box 60 12 03, 14412 Potsdam (Germany); Department of Physics, Humboldt University, Newtonstr. 15, 12489 Berlin (Germany); Donner, Reik V., E-mail: reik.donner@pik-potsdam.de [Potsdam Institute for Climate Impact Research, P.O. Box 60 12 03, 14412 Potsdam (Germany); Donges, Jonathan F. [Potsdam Institute for Climate Impact Research, P.O. Box 60 12 03, 14412 Potsdam (Germany); Department of Physics, Humboldt University, Newtonstr. 15, 12489 Berlin (Germany); Marwan, Norbert [Potsdam Institute for Climate Impact Research, P.O. Box 60 12 03, 14412 Potsdam (Germany); Kurths, Jürgen [Potsdam Institute for Climate Impact Research, P.O. Box 60 12 03, 14412 Potsdam (Germany); Department of Physics, Humboldt University, Newtonstr. 15, 12489 Berlin (Germany); Institute for Complex Systems and Mathematical Biology, University of Aberdeen, Aberdeen AB243UE (United Kingdom)

    2012-10-15

    We introduce a geometric method for identifying the coupling direction between two dynamical systems based on a bivariate extension of recurrence network analysis. Global characteristics of the resulting inter-system recurrence networks provide a correct discrimination for weakly coupled Rössler oscillators not yet displaying generalised synchronisation. Investigating two real-world palaeoclimate time series representing the variability of the Asian monsoon over the last 10,000 years, we observe indications for a considerable influence of the Indian summer monsoon on climate in Eastern China rather than vice versa. The proposed approach can be directly extended to studying K>2 coupled subsystems.

  4. Consensus of Multiagent Systems with Directed Topology and Communication Time Delay Bases on the Laplace Transform

    Directory of Open Access Journals (Sweden)

    Bo Liu

    2014-01-01

    Full Text Available This paper investigates the consensus problem of multiagent systems with directed topologies. Different from the literatures, a new method, the Laplace transform, to study the consensus of multiagent systems with directed topology and communication time delay is proposed. The accurate state of the consensus center and the upper bound of the communication delay to make the agents reach consensus are given. It is proved that all the agents could aggregate and eventually form a cohesive cluster in finite time under certain conditions, and the consensus center is only determined by the initial states and the communication configuration among the agents. Finally, simulations are given to illustrate the theoretical results.

  5. Production of recombinant proteins by filamentous fungi.

    Science.gov (United States)

    Ward, Owen P

    2012-01-01

    The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the

  6. An examination of the vibration transmissibility of the hand-arm system in three orthogonal directions.

    Science.gov (United States)

    Welcome, Daniel E; Dong, Ren G; Xu, Xueyan S; Warren, Christopher; McDowell, Thomas W; Wu, John Z

    2015-02-01

    The objective of this study is to enhance the understanding of the vibration transmission in the hand-arm system in three orthogonal directions (X, Y, and Z). For the first time, the transmitted vibrations distributed on the entire hand-arm system exposed in the three orthogonal directions via a 3-D vibration test system were measured using a 3-D laser vibrometer. Seven adult male subjects participated in the experiment. This study confirms that the vibration transmissibility generally decreased with the increase in distance from the hand and it varied with the vibration direction. Specifically, to the upper arm and shoulder, only moderate vibration transmission was measured in the test frequency range (16 to 500 Hz), and virtually no transmission was measured in the frequency range higher than 50 Hz. The resonance vibration on the forearm was primarily in the range of 16-30 Hz with the peak amplitude of approximately 1.5 times of the input vibration amplitude. The major resonance on the dorsal surfaces of the hand and wrist occurred at around 30-40 Hz and, in the Y direction, with peak amplitude of more than 2.5 times of the input amplitude. At higher than 50 Hz, vibration transmission was effectively limited to the hand and fingers. A major finger resonance was observed at around 100 Hz in the X and Y directions and around 200 Hz in the Z direction. In the fingers, the resonance magnitude in the Z direction was generally the lowest, and the resonance magnitude in the Y direction was generally the highest with the resonance amplitude of 3 times the input vibration, which was similar to the transmissibility at the wrist and hand dorsum. The implications of the results are discussed.

  7. Design of a Solar Motor Drive System Fed by a Direct-Connected Photovoltaic Array

    Directory of Open Access Journals (Sweden)

    AYDOGMUS, O.

    2012-08-01

    Full Text Available A solar motor pump drive system is modeled and simulated. The proposed drive system does not require any kind of energy storage system and dc-dc converter. The system is connected directly to a photovoltaic (PV array. Thus, a low cost solar system can be achieved. A vector controlled Permanent Magnet Synchronous Motor (PMSM is used as a solar motor to increase the efficiency of system. The motor is designed for a low rated voltage level about 24V. The hill climbing MPPT method is used for balanced the motor power and PV power to obtain a high efficiency. The results are performed by using MATLAB/SimPowerSystem blocks. In addition, the PV array is modeled to allow for the possibility of running as on-line adjustable in simulation environment without using lookup table. The performances of motor, MPPT and drive system are analyzed in different conditions as temperature and irradiation of PV array.

  8. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

    Directory of Open Access Journals (Sweden)

    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  9. Fusion-Expressed CTB Improves Both Systemic and Mucosal T-Cell Responses Elicited by an Intranasal DNA Priming/Intramuscular Recombinant Vaccinia Boosting Regimen

    Directory of Open Access Journals (Sweden)

    Sugan Qiu

    2014-01-01

    Full Text Available Previous study showed that CTB (Cholera toxin subunit B can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting, pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562±567 SFCs/106 splenocytes versus 330±182 SFCs/106 splenocytes, P<0.01, mesenteric LN (96±83 SFCs/106 lymphocytes versus 1±2 SFCs/106 lymphocytes, P<0.05, draining LNs of respiratory tract (109±60 SFCs/106 lymphocytes versus 2±2 SFCs/106 lymphocytes, P<0.01 and female genital tract (89±48 SFCs/106 lymphocytes versus 23±21 SFCs/106 lymphocytes, P<0.01. These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

  10. Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system.

    Science.gov (United States)

    Zhang, Zhi-Jun; Pan, Jiang; Liu, Jun-Feng; Xu, Jian-He; He, Yu-Cai; Liu, You-Yan

    2011-03-10

    The enantioselective hydrolysis of mandelonitrile with whole cells of a recombinant Escherichia coli expressing nitrilase activity was severely inhibited by the substrate at high concentrations (>300mM), which resulted in a low yield of the target product (R)-(-)-mandelic acid. To relieve the substrate inhibition and to enhance the (R)-(-)-mandelic acid productivity, eight water-organic solvent biphasic systems were attempted in this work. Toluene was found to be the most suitable solvent as the organic phase among the solvents tested. Various parameters were systematically examined and optimized in shake flasks. The phase volume ratio, buffer pH and reaction temperature were shown to be sensitive parameters affecting both the yield and the enantiopurity of product in the biphasic system. Under the optimized conditions, significant enhancement of substrate tolerance from 200mM to 500mM and average productivity from 179.6gl(-1)d(-1) to 352.6gl(-1)d(-1) were achieved. Subsequently, the biocatalytic hydrolysis of mandelonitrile was successfully carried out in a stirred reactor (2-l scale) by repeated use of the calcium alginate entrapped cells for 5 batches, affording 110.7g (R)-(-)-mandelic acid in 98.0% ee (enantiomeric excess) and a specific production of 13.8g (mandelic acid) g(-1) (cell), respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. [Enzymatic regulatory processes in gene recombination].

    Science.gov (United States)

    Kovarskiĭ, V A; Profir, A V

    1988-01-01

    Recombination bistability in the system of genetic regulation in pro- and eucaryots is analysed on the basis of sigmoid kinetics of regulatory enzymes. It is shown that under an increase of either exogenic factors (temperature) or endogenic factors (concentration of molecules, which activate the enzymes) of crucial values, bistability solutions for recombination frequencies are possible. Histeresic character of the dependence of this value on the external parameters is pointed out. The role of fluctuation processes in distortion of the memory effects is discussed. On the basis of monostable solutions molecular account for the empiric Plau law is given for U-shaped dependence of recombination frequency on temperature.

  12. Homologous recombination in Sulfolobus acidocaldarius: genetic assays and functional properties.

    Science.gov (United States)

    Grogan, Dennis W

    2009-02-01

    HR (homologous recombination) is expected to play important roles in the molecular biology and genetics of archaea, but, so far, few functional properties of archaeal HR have been measured in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables quantitative analysis of HR between chromosomal markers. Early studies of this system indicated that HR occurred frequently between closely spaced mutations within the pyrE gene, and this result was later supported by various analyses involving defined point mutations and deletions. These properties of intragenic HR suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Because fragmentation of donor DNA during cell-to-cell transfer could not be excluded from contributing to this result, subsequent analyses have focused on electroporation of selectable donor DNA directly into recipient strains. For example, S. acidocaldarius was found to incorporate synthetic ssDNA (single-stranded DNA) of more than approximately 20 nt readily into its genome. With respect to various molecular properties of the ssDNA substrates, the process resembled bacteriophage lambdaRed-mediated 'recombineering' in Escherichia coli. Another approach used electroporation of a multiply marked pyrE gene to measure donor sequence tracts transferred to the recipient genome in individual recombination events. Initial results indicate multiple discontinuous tracts in the majority of recombinants, representing a relatively broad distribution of tract lengths. This pattern suggests that properties of the HR process could, in principle, account for many of the apparent peculiarities of intragenic recombination initiated by S. acidocaldarius conjugation.

  13. Design of folded holographic spectrum-splitting photovoltaic system for direct and diffuse illumination conditions

    Science.gov (United States)

    Wu, Yuechen; Vorndran, Shelby D.; Russo, Juan M.; Ayala, Silvana; Kostuk, Raymond K.

    2014-10-01

    Spectrum-splitting is a beneficial technique to increase the efficiency and reduce the cost of photovoltaic (PV) systems. This method divides the incident solar spectrum into spectral components that are spatially separated and directed to PV cells with matching spectral responsivity characteristics. This approach eliminates problems associated with current and lattice matching that must be maintained in tandem multi-junction systems. In this paper, a two-junction holographic spectrum-splitting photovoltaic system is demonstrated with a folded PV geometry. The system is designed to use both direct and diffuse solar irradiation. It consists of holographic elements, a wedge-shaped optical guide, and PV substrates with back reflectors. The holographic elements and back reflectors spatially separate the incident solar spectrum and project spectral components onto matching PV cell types. In addition, the wedge-shaped optical guide traps diffuse illumination inside the system to increase absorption. In this paper, the wedge spectrum splitting system is analyzed using tabulated data for InGaP2/GaAs cells with direct illumination combined with experimental data for reflection volume holograms. A system efficiency of 31.42% is obtained with experimental reflection hologram data. This efficiency is a 21.42% improvement over a similar system that uses one PV cell with the highest efficiency (GaAs). Simulation results show large acceptance angle for both in-plane and out-of plane directions. Simulation of the output power of the system with different configurations at different times of the year are also presented.

  14. Direct Heat-Flux Measurement System (MDF) for Solar central Receiver Evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Ballestrin, J.

    2001-07-01

    A direct flux measurement system, MDF, has been designed, constructed and mounted on top of the SSPS-CRS tower at the Plataforma Solar de Almeria (PSA) in addition to an indirect flux measurement system based on a CCD camera. It's one of the main future objectives to compare systematically both measurements of the concentrated solar power, increasing in this way the confidence in the estimate of this quantity. Today everything is prepared to perform the direct flux measurement on the aperture of solar receivers: calorimeter array, data acquisition system and software. the geometry of the receiver determines the operation and analysis procedures to obtain the indecent power onto the defined area. The study of previous experiences with direct flux measurement systems ha been useful to define a new simpler and more accurate system. A description of each component of the MDF system is included, focusing on the heat-flux sensors or calorimeters, which enables these measurements to be done in a few seconds without water-cooling. The incident solar power and the spatial flux distribution on the aperture of the volumetric receiver Hitrec II are supplied by the above-mentioned MDF system. The first results obtained during the evaluation of this solar receiver are presented including a sunrise-sunset test. All these measurements have been concentrated in one coefficient that describes the global behavior of the Solar Power Plant. (Author) 18 refs.

  15. ORNL Direct Purchase Information System (DPIS) user's manual. [For PDP-10

    Energy Technology Data Exchange (ETDEWEB)

    Grubb, J.W.; Lovin, J.K.; Smith, M.B.; Haese, R.L.; Needham, D.B.; Wilson, J.K.

    1980-08-01

    The ORNL Management Information System (MIS) Direct Purchase Information System (DPIS) is an on-line interactive system of computer programs. The system can provide a manager with commitment and delivery schedule information on current direct purchase requisitions. The commitment data accounts for the orders that have been placed and those requisitions yet to be placed with a vendor. Information can be summarized at many different levels, and individuals can quickly determine the status of their requisitions. DPIS contains data only on active outside direct purchases, but has the capability to access historical data. It provides sufficient flexibility to be used to answer many questions pertinent to the status of these direct purchases and their obligating costs. Even an inexperienced computer user should have little difficulty in learning to use DPIS. The User Module prompts the user on what type of response it is expecting. If the user has doubts as to the response, or if the meaning of the response is not clear, the module will give a detailed list of the options available at that level. The user has control of what data are to be considered, how they are to be grouped, and what format the output will take. As the user selects the options available at a given level, the module proceeds to the next lower level until sufficient input has been supplied to provide the requested information. A major benefit of this interactive, user-oriented system is that the manager can specify the information requirements and does not have to spend time going through a great deal of other data to locate what is needed. Because it is interactive, a search can begin at a summary level and then resort to a more detailed level if needed. DPIS allows the user direct control for selecting the type of commitment data, output, funds, and direct purchase.

  16. Stimulation of pneumovirus-specific CD8(+) T-cells using a non-toxic recombinant ricin delivery system

    NARCIS (Netherlands)

    Grimaldi, E.; Claassen, E.A.W.

    2007-01-01

    Internalisation of the plant toxin ricin occurs by retrograde transport which delivers the toxin to the ER where it intersects with the MHC class I system for peptide antigen display. Here, we describe the generation of an inactivated, non-toxic, ricin molecule fused to a peptide which elicits a CD8

  17. Extracellular secretion of a recombinant therapeutic peptide by Bacillus halodurans utilizing a modified flagellin type III secretion system

    CSIR Research Space (South Africa)

    Berger, E

    2011-08-01

    Full Text Available utilizing 106 chemical steps. The authors used Enfuvirtide as a model system in an effort to develop a more cost-effective biological process for therapeutic peptide production. Results: An attempt was made to increase the levels of the fusion peptide by two...

  18. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  19. Direct medical costs and their predictors in South Korean patients with systemic lupus erythematosus.

    Science.gov (United States)

    Park, So-Yeon; Joo, Young Bin; Shim, Jeeseon; Sung, Yoon-Kyoung; Bae, Sang-Cheol

    2015-11-01

    We aimed to estimate the annual direct medical costs of South Korean systemic lupus erythematosus (SLE) patients, and their predictors. The 2010 annual direct medical costs of SLE patients in the Hanyang BAE Lupus cohort in South Korea were assessed. The information was taken directly from the hospital database and medical records, and included clinical characteristics, disease activity, organ damage, and healthcare utilization. Cost predictors were estimated with a multivariate linear regression model. A total of 749 SLE patients (92.7 % female, mean age 35.7 ± 11.3 years, mean disease duration 9.6 ± 4.9 years) were studied. Their mean annual direct medical costs amounted to USD 3305. The largest component of these costs was the cost of medication (USD 1269, 38.4 %), followed by those of diagnostic procedures and tests (USD 1177, 35.6 %). Regression analysis showed that adjusted mean SLE disease activity index score (p systemic damage index (p < 0.0001), and renal (p = 0.0039) and hematologic (p = 0.0353) involvement were associated with increased direct medical costs, whereas longer disease duration was associated with lower direct medical costs. Greater disease activity and greater organ damage predict higher costs for South Korean SLE patients. Major organ involvement such as renal disorder and hematologic involvement also predicts higher costs, whereas longer duration of disease predicts lower costs.

  20. Stabilisation of a class of 2-DOF underactuated mechanical systems via direct Lyapunov approach

    Science.gov (United States)

    Turker, Turker; Gorgun, Haluk; Cansever, Galip

    2013-06-01

    This paper represents an alternative stabilisation procedure for a class of two degree-of-freedom underactuated mechanical systems based on a set of transformations and a Lyapunov function. After simplifying dynamic equations of the system via partial feedback linearisation and coordinate changes, the stability of the system is provided with Lyapunov's direct method. Proposed control scheme is used on two different examples and asymptotic convergence for each system is proven by means of La Salle's invariance principle. The designed controller is successfully illustrated through numerical simulations for each example.