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Sample records for improving baculovirus production

  1. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  2. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  3. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    PubMed Central

    Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  4. Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Chaillet, Maxime; Berger, Imre; Alves, Paula M

    2016-09-10

    The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts.

  5. Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Chaillet, Maxime; Berger, Imre; Alves, Paula M

    2016-09-10

    The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts. PMID:27378622

  6. Baculovirus Insecticide Production in Insect Larvae.

    PubMed

    van Beek, Nikolai; Davis, David C

    2016-01-01

    Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field tests with such recombinant baculoviruses have been done in the past, and more tests are ongoing. However, low-cost production of recombinant baculovirus in larvae poses specific problems, due to the short survival time of the production host.In this chapter, benchtop-scale production of two typical baculoviruses is described. First, we describe the production of wild type Helicoverpa zea nucleopolyhedrovirus in bollworm (H. zea) larvae. H. zea larvae are very aggressive and need to be reared in isolation from each other. Second, we describe the production of a recombinant Autographa californica multiple nucleopolyhedrovirus in the non-cannibalistic cabbage looper, Trichoplusia ni. The recombinant baculovirus encodes the insect-specific scorpion toxin LqhIT2. The tetracycline transactivator system enables the production of wild-type quantity and quality product while toxin expression is repressed since normal toxin production would result in premature death of the production host that would limit progeny virus production.

  7. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  8. Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine.

    PubMed

    Huynh, Hoai T; Chan, Leslie C L; Tran, Trinh T B; Nielsen, Lars K; Reid, Steven

    2012-01-01

    A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in-house low-cost serum-free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex. However, VPM3 is composed of a significant number of undefined components, including five different protein hydrolysates, which introduce a challenging lot-to-lot variability to the production process. In this study, an intensive statistical optimization routine was employed to reduce the number of protein hydrolysates in VPM3 medium. Nearly 300 runs (including replicates) were conducted with great efficiency by using 50 mL TubeSpin® bioreactors to propagate insect cell suspension cultures. Fractional factorial experiments were first used to determine the most important of the five default protein hydrolysates, and to screen for seven potential substitutes for the default meat peptone, Primatone RL. Validation studies informed by the screening tests showed that promising alternative media could be formulated based on just two protein hydrolysates, in particular the YST-AMP (Yeast Extract and Amyl Meat Peptone) and YST-POT (Yeast Extract and Lucratone Potato Peptone) combinations. The YST-AMP (meat-based) and YST-POT (meat-free) variants of VPM3 were optimized using response surface methodology, and were shown to be just as good as the default VPM3 and the commercial Sf-900 II media in supporting baculovirus yields, hence providing a means toward a more reproducible and scalable production process for HaSNPV biopesticides. PMID:22323401

  9. Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine.

    PubMed

    Huynh, Hoai T; Chan, Leslie C L; Tran, Trinh T B; Nielsen, Lars K; Reid, Steven

    2012-01-01

    A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in-house low-cost serum-free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex. However, VPM3 is composed of a significant number of undefined components, including five different protein hydrolysates, which introduce a challenging lot-to-lot variability to the production process. In this study, an intensive statistical optimization routine was employed to reduce the number of protein hydrolysates in VPM3 medium. Nearly 300 runs (including replicates) were conducted with great efficiency by using 50 mL TubeSpin® bioreactors to propagate insect cell suspension cultures. Fractional factorial experiments were first used to determine the most important of the five default protein hydrolysates, and to screen for seven potential substitutes for the default meat peptone, Primatone RL. Validation studies informed by the screening tests showed that promising alternative media could be formulated based on just two protein hydrolysates, in particular the YST-AMP (Yeast Extract and Amyl Meat Peptone) and YST-POT (Yeast Extract and Lucratone Potato Peptone) combinations. The YST-AMP (meat-based) and YST-POT (meat-free) variants of VPM3 were optimized using response surface methodology, and were shown to be just as good as the default VPM3 and the commercial Sf-900 II media in supporting baculovirus yields, hence providing a means toward a more reproducible and scalable production process for HaSNPV biopesticides.

  10. Insect cells-baculovirus system for the production of difficult to express proteins.

    PubMed

    Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

    2015-01-01

    The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

  11. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  12. Maximizing in vivo production of Agrotis ipsilon (Hufnagel) baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The black cutworm, Agrotis ipsilon (Hufnagel), is a pest causing damage to a variety plants of from turf to row crops. A recently discovered baculovirus has the potential to be developed as a biological pesticide to provide targeted control of this insect pest. Initial field trials in turf grass and...

  13. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

    PubMed

    Toth, Ann M; Geisler, Christoph; Aumiller, Jared J; Jarvis, Donald L

    2011-12-01

    In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

  14. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells.

    PubMed

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-03-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.

  15. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  16. The effect of spent medium recycle on cell proliferation, metabolism and baculovirus production by the lepidopteran Se301 cell line infected at very low MOI.

    PubMed

    Beas-Catena, Alba; Sánchez-Mirón, Asterio; Garía-Camacho, Francisco; Contreras-Gómez, Antonio; Molina-Grima, Emilio

    2013-12-01

    The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

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

    PubMed

    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.

  18. Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors.

    PubMed

    van Lier, F L; van der Meijs, W C; Grobben, N G; Olie, R A; Vlak, J M; Tramper, J

    1992-02-01

    Insect cells were exploited to produce bacterial beta-galactosidase by infecting them with a recombinant nuclear polyhedrosis virus (baculovirus) of Autographa californica. The insect cells were cultured in a continuous stirred tank reactor (CSTR) and led to a second CSTR where they were infected with a recombinant virus in which the lacZ gene from Escherichia coli was inserted. In the effluent of the production reactor, maximum activities of 15 units beta-galactosidase per 10(6) cells were measured. For about 25 d beta-galactosidase production remained constant, but then rapidly declined. This drop was due to a decrease in production of active beta-galactosidase rather than to inactivation of this enzyme. It was concluded that the reduced production was due to reduced polyhedrin promoter-driven synthesis.

  19. Baculovirus Stimulates Antiviral Effects in Mammalian Cells

    PubMed Central

    Gronowski, Ann M.; Hilbert, David M.; Sheehan, Kathleen C. F.; Garotta, Gianni; Schreiber, Robert D.

    1999-01-01

    Herein, we report that Autographa californica nucleopolyhedrovirus, a member of the Baculoviridae family, is capable of stimulating antiviral activity in mammalian cells. Baculoviruses are not pathogenic to mammalian cells. Nevertheless, live baculovirus is shown here to induce interferons (IFN) from murine and human cell lines and induces in vivo protection of mice from encephalomyocarditis virus infection. Monoclonal antibodies specific for the baculovirus envelope gp67 neutralize baculovirus-dependent IFN production. Moreover, UV treatment of baculovirus eliminates both infectivity and IFN-inducing activity. In contrast, the IFN-inducing activity of the baculovirus was unaffected by DNase or RNase treatment. These data demonstrate that IFN production can be induced in mammalian cells by baculovirus even though the cells fail to serve as a natural host for an active viral infection. Baculoviruses, therefore, provide a novel model in which to study at least one alternative mechanism for IFN induction in mammalian cells. PMID:10559307

  20. Baculovirus and insect cell gene expression: review of baculovirus biotechnology.

    PubMed

    Patterson, R M; Selkirk, J K; Merrick, B A

    1995-01-01

    The BEVS continues to evolve as a powerful, flexible tool for molecular biology, protein function, and biomedical research. Future developments offer the promise of replacement of hazardous chemical insecticides with environmentally safe biopesticides, construction of baculovirus vectors which encode genes for specific post-translational modifications, and establishment of efficient, stably transformed insect cell lines. FDA approval of BEVS-produced products offer the prospect of new biopharmaceuticals, in particular human therapeutics and vaccines, to improve human health and increase the quality of life for millions of people.

  1. High-level production of a functional immunoglobulin heterodimer in a baculovirus expression system.

    PubMed Central

    Hasemann, C A; Capra, J D

    1990-01-01

    A murine immunoglobulin heterodimer has been expressed in a baculovirus expression system. This was achieved by using both double infection of insect cells with separate heavy- and light-chain-expressing viruses and infection with a double-recombinant virus containing both the immunoglobulin heavy- and light-chain cDNAs. In both cases, the polypeptide chains were correctly processed, glycosylated, and assembled into normal H2L2 (H = heavy, L = light) immunoglobulin monomers. These molecules bound antigen and expressed both polyclonal idiotype and monoclonal idiotopes. Furthermore, the transfer vectors described have been modified to contain the F1 origin of replication for the production of single-stranded DNA, which facilitates site-specific mutations of either the polyhedrin promoter or the inserted foreign gene. Use of this system should significantly advance the analysis of the structural bases for both idiotype expression and antigen binding by immunoglobulin. More importantly, it provides a generic method for the high-level expression of antibodies of diverse interest. Images PMID:2111022

  2. Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin.

    PubMed

    Ali, M P; Kato, Tatsuya; Park, Enoch Y

    2015-12-01

    Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a "head-to-tail" shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.

  3. Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system.

    PubMed

    Mon, Hiroaki; Lee, Jaeman; Fukushima, Mai; Nagata, Yudai; Fujii, Mie; Xu, Jian; Nishi, Oumi; Iiyama, Kazuhiro; Kusakabe, Takahiro

    2013-08-01

    Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca(2+) and Sr(2+) were able to replace Mg(2+) and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.

  4. Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system.

    PubMed

    Muneta, Yoshihiro; Zhao, Hong Kun; Inumaru, Shigeki; Mori, Yasuyuki

    2003-02-01

    In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future. PMID:12655117

  5. Establishment of the BacMam system using silkworm baculovirus.

    PubMed

    Imai, Atsutoshi; Tadokoro, Takashi; Kita, Shunsuke; Horiuchi, Masataka; Fukuhara, Hideo; Maenaka, Katsumi

    2016-09-16

    The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. PMID:27480929

  6. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment

    SciTech Connect

    Yuan Meijin; Wu Wenbi; Liu Chao; Wang Yanjie; Hu Zhaoyang; Yang Kai Pang Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc{sup P48-KO-PH-GFP} was unable to propagate in cell culture, while a 'repair' Bacmid vAc{sup P48-REP-PH-GFP} was able to replicate in a manner similar to a wild-type Bacmid vAc{sup PH-GFP}. Titration assays and Western blotting confirmed that vAc{sup P48-KO-PH-GFP} was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  7. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment.

    PubMed

    Yuan, Meijin; Wu, Wenbi; Liu, Chao; Wang, Yanjie; Hu, Zhaoyang; Yang, Kai; Pang, Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc(P48-KO-PH-GFP) was unable to propagate in cell culture, while a 'repair' Bacmid vAc(P48-REP-PH-GFP) was able to replicate in a manner similar to a wild-type Bacmid vAc(PH-GFP). Titration assays and Western blotting confirmed that vAc(P48-KO-PH-GFP) was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  8. Optimization of the production of triabin, a novel thrombin inhibitor, in High Five™ insect cells infected with a recombinant baculovirus.

    PubMed

    Vallazza, M; Petri, T

    1999-03-01

    The isolation of a new type of thrombin inhibitor, called triabin, from the saliva of the hematophagous bug Triatoma pallidipennis, has recently been described. In the in vitro platelet aggregation inhibition assay triabin has a similar potency as the thrombin inhibitor hirudin now in phase III clinical trials. However, in another in vitro assay using a low molecular weight substrate for thrombin, triabin does not inhibit thrombin completely even at 6 fold higher molar doses in comparison with hirudin. This means that triabin has a novel mode of action towards thrombin making triabin into an interesting candidate as a therapeutic agent. Recently it has been shown that a recombinant baculovirus can be efficiently used for the triabin production in insect cells and that the yields in adherent cultures of High Five™ cells (approx. 20 mg l-1) were about 7 fold higher than in adherent cultures of Sf9 cells (approx. 3 mg l- 1). To optimize the triabin yield from the baculovirus/insect cell expression system, experiments were performed with suspension adapted cultures of High Five™ cells to investigate the effects of the state of the host cell, of the multiplicity of infection, of the cell density at the time of infection and of supplementation of the medium with nutrients and oxygen. Triabin yields of up to 200 mg l-1, as determined by an activity assay, could finally be obtained here. PMID:22359057

  9. Production of DUSP1 protein using the baculovirus insect cell expression system and its in vitro effects on cancer cells.

    PubMed

    Cheng, Peng; Zhu, Shuying; Jun, Li; Huang, Lihua; Hong, Yahui

    2015-06-01

    The aim of the present study was to produce the human dual specificity phosphatase 1 (DUSP1) protein with biological activity and to investigate its in vitro effects on cancer cells. DUSP1 protein was expressed in the baculovirus expression system and purified by Ni-affinity chromatography followed by dialysis in PBS. The purified protein was verified by SDS-PAGE and western blot analysis. Six cancer cell lines were then cultured in the presence of DUSP1 for various periods of time, and the phosphorylated extracellular signal-regulated kinase (p-ERK) content in each cell line was subsequently determined by western blot analysis. Compared to the β-actin level, the amount of p-ERK markedly decreased after 1 h, indicating that DUSP1 suppressed the expression of p-ERK in 6 cancer cell lines examined. Human cervical cancer cells were also collected and counted following co-culture with DUSP1 to examine its effect on the growth rate of cancer cells. A baculovirus expression system for the production of DUSP1 protein was successfully constructed. The p-ERK content was found to be significantly decreased when the cancer cell lines were exposed to DUSP1. The capability of binary fission was reduced when the cells were examined under a microscope. The proliferation of human cervical cancer cells was also inhibited by DUSP1. PMID:25872469

  10. Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

    PubMed

    Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

    2014-05-01

    Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions.

  11. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    PubMed

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

  12. Comparison of strategies for the production of FMDV empty capsids using the baculovirus vector system.

    PubMed

    Ruiz, V; Mignaqui, A C; Nuñez, M C; Reytor, E; Escribano, J M; Wigdorovitz, A

    2014-11-01

    Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.

  13. Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

    PubMed

    Muraki, Michiro; Honda, Shinya

    2011-11-01

    To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.

  14. A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

    PubMed

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M; Jakubowska, Agata K; Herrero, Salvador

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  15. A novel baculovirus-derived promoter with high activity in the baculovirus expression system

    PubMed Central

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M.; Jakubowska, Agata K.

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  16. A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

    PubMed

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M; Jakubowska, Agata K; Herrero, Salvador

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

  17. Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production

    PubMed Central

    Liu, Yang; de Jong, Jondavid; Nagy, Éva; Theilmann, David A.

    2016-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Although me53 is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions, and peptides fused with hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCE Baculovirus me53 is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as

  18. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    PubMed

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  19. Production of hepatitis E virus-like particles presenting multiple foreign epitopes by co-infection of recombinant baculoviruses

    PubMed Central

    Shima, Ryoichi; Li, Tian Cheng; Sendai, Yutaka; Kataoka, Chikako; Mori, Yoshio; Abe, Takayuki; Takeda, Naokazu; Okamoto, Toru; Matsuura, Yoshiharu

    2016-01-01

    Hepatitis E virus (HEV) causes not only endemics via a fecal-oral route but also sporadic cases via zoonotic transmission or blood transfusion. HEV-like particles (HEV-LP) produced by using a baculovirus expression system are considered a candidate for mucosal vaccines for HEV infection. In this study, we attempted to produce a chimeric HEV-LP presenting various foreign epitopes on its surface. Expression of the recombinant capsid proteins carrying a myc- or FLAG-tag inserted between amino acid residues 488 and 489, which are located in the exterior loop on the protruding domain of the HEV capsid, resulted in the production of recombinant HEV-LP. Although expression of the recombinant capsid protein carrying the HA-tag inserted at the same site failed to produce any particles, co-expression with the myc-tagged capsid protein successfully yielded a chimeric HEV-LP consisting of both recombinant capsid proteins. Immunoprecipitation analyses confirmed that the chimeric particles present these foreign epitopes on the surface. Similar results were obtained for the expression of the recombinant capsid proteins carrying neutralizing epitopes of Japanese encephalitis virus. These results suggest the chimeric HEV-LP system provides a novel vaccine carrier that can accommodate multiple neutralizing epitopes on its surface. PMID:26905478

  20. Directional and direct cloning strategy for high-throughput generation of recombinant baculoviruses.

    PubMed

    Ma, Qi; Zhou, Li; Ma, Lixin; Huo, Keke

    2006-10-01

    The baculovirus expression vector system (BEVS) has become one of the most widely used systems for routine protein expression. We have developed an improved strategy to clone foreign genes directionally and directly into the baculovirus genome vector via a one-step procedure to generate recombinant viruses in a week. In this work, we constructed a host strain Escherichia coli DH10BacHB1.1, which contains the modified baculovirus shuttle genome vector pHBMBacmid1.1 for the cloning vector. The treated PCR products of foreign genes were ligated with the Bsu36I-digested vector. Then Spodoptera frugiperda (Sf9) cells were transfected directly with the ligation mixture. Using this method, the DsRed fluorescence protein and mannanase genes have been cloned in the baculovirus genome and expressed in the Sf9 cells. This strategy not only provides a means for high-throughput construction of recombinant baculoviruses, but also offers an idea of constructing other large plasmids and DNA virus-based expression vectors.

  1. Production of human beta interferon in insect cells infected with a Baculovirus expression vector

    SciTech Connect

    Smith, G.E.; Summers, M.D.; Fraser, M.J.

    1983-12-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 x 10/sup 6/ U of interferon activity was produced by 10/sup 6/ infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

  2. Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

    PubMed Central

    Prud'homme, I; Zhou, E M; Traykova, M; Trotter, H; Chan, M; Afshar, A; Harding, M J

    1997-01-01

    The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV. Images Figure 2. Figure 3. Figure 4. PMID:9342453

  3. Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

    PubMed

    Prud'homme, I; Zhou, E M; Traykova, M; Trotter, H; Chan, M; Afshar, A; Harding, M J

    1997-10-01

    The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV.

  4. Baculoviruses and nucleosome management

    SciTech Connect

    Volkman, Loy E.

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  5. Baculoviruses and nucleosome management.

    PubMed

    Volkman, Loy E

    2015-02-01

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management.

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

    PubMed

    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.

  7. Recombinant baculovirus isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2007-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac or BaculoDirect, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  8. Genome Scale Transcriptomics of Baculovirus-Insect Interactions

    PubMed Central

    Nguyen, Quan; Nielsen, Lars K.; Reid, Steven

    2013-01-01

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies. PMID:24226166

  9. Genome scale transcriptomics of baculovirus-insect interactions.

    PubMed

    Nguyen, Quan; Nielsen, Lars K; Reid, Steven

    2013-11-12

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors' and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system' which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  10. The baculovirus uses a captured host phosphatase to induce enhanced locomotory activity in host caterpillars.

    PubMed

    Katsuma, Susumu; Koyano, Yasue; Kang, Wonkyung; Kokusho, Ryuhei; Kamita, Shizuo George; Shimada, Toru

    2012-01-01

    The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme.

  11. The Baculovirus Uses a Captured Host Phosphatase to Induce Enhanced Locomotory Activity in Host Caterpillars

    PubMed Central

    Katsuma, Susumu; Koyano, Yasue; Kang, WonKyung; Kokusho, Ryuhei; Kamita, Shizuo George; Shimada, Toru

    2012-01-01

    The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme. PMID:22496662

  12. Overview of the baculovirus expression system.

    PubMed

    Murphy, C I; Piwnica-Worms, H

    2001-05-01

    Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post-translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system. PMID:18428479

  13. Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    PubMed

    Murakami, K; Uchiyama, A; Kokuho, T; Mori, Y; Sentsui, H; Yada, T; Tanigawa, M; Kuwano, A; Nagaya, H; Ishiyama, S; Kaki, H; Yokomizo, Y; Inumaru, S

    2001-01-01

    The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells. PMID:11145838

  14. Fundamentals of Baculovirus Expression and Applications.

    PubMed

    Kost, Thomas A; Kemp, Christopher W

    2016-01-01

    In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology. PMID:27165326

  15. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene.

    PubMed

    McCarthy, Christina B; Theilmann, David A

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC(ac142)(REP-ac143KO)). Fluorescence and light microscopy showed that infection by AcBAC(ac142)(REP-ac143KO) is limited to a single cell and titration assays confirmed that AcBAC(ac142)(REP-ac143KO) was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC(ac142)(REP-ac143KO) transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  16. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    SciTech Connect

    McCarthy, Christina B.; Theilmann, David A.

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC{sup ac142REP-ac143KO}). Fluorescence and light microscopy showed that infection by AcBAC{sup ac142REP-ac143KO} is limited to a single cell and titration assays confirmed that AcBAC{sup ac142REP-ac143KO} was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC{sup ac142REP-ac143KO} transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  17. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  18. Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

    PubMed

    Harvey, A J; Bidwai, A P; Miller, L K

    1997-05-01

    A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.

  19. Suspension culture titration: A simple method for measuring baculovirus titers.

    PubMed

    Matindoost, Leila; Chan, Leslie C L; Qi, Ying Mei; Nielsen, Lars K; Reid, Steven

    2012-08-01

    The baculovirus-insect cell expression system is an important technology for the production of recombinant proteins and baculovirus-based biopesticides. Budded virus titration is critical when scaling up baculovirus production processes in suspension cultures, to ensure reproducible infections, especially when a low multiplicity of infection (MOI) is applied. In this study, a simple suspension culture titration (SCT) assay was developed that involves accurate measurements of the initial cell densities (ICDs) and peak cell densities (PCDs) of an infected culture, from which the MOI and hence the virus inoculum infectious titer can be estimated, using the established Power-Nielsen baculovirus infection model. The SCT assay was assessed in parallel with two adherent culture-based assays (MTT and AlamarBlue) for the Heliothine baculovirus HaSNPV, and was shown to be more objective, time-efficient and reproducible. The model predicted a linear correlation between log(PCD/ICD) and log(MOI), hence an alternative model-independent SCT assay was also developed, which relies on a well-replicated standard curve relating suspension culture-derived PCD/ICD ratios with plaque or endpoint assay-derived MOIs. Standard curves with excellent linearity were generated for HaSNPV and the industrially significant rAcMNPV, demonstrating the feasibility of this simple titration approach, especially in terms of its applicability to a wide range of virus infection kinetics.

  20. Protein expression in the baculovirus system.

    PubMed

    Bernard, A; Payton, M; Radford, K R

    2001-05-01

    Insect cell-recombinant baculovirus co-cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post-translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large-scale production of recombinant proteins in the baculovirus expression system. A method for large-scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end-point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small-scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS-PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed-batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

  1. Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

    PubMed

    Wilson, L E; Wilkinson, N; Marlow, S A; Possee, R D; King, L A

    1997-04-01

    A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains. PMID:9105619

  2. Overview of the baculovirus expression system.

    PubMed

    Murphy, C I; Piwnica-Worms, H

    2001-05-01

    Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This overview unit describes the baculovirus life cycle and expression system, and also provides information on vectors and protocols for using the baculovirus expression system. PMID:18429185

  3. A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line.

    PubMed

    Hopkins, Ralph; Esposito, Dominic

    2009-09-01

    A new rapid method for titrating baculovirus stocks has been developed using a novel cell line Sf-9 Easy Titer (Sf-9ET). The Sf-9ET cell line has been transfected with plasmid DNA containing the enhanced green fluorescent protein (eGFP) gene under the control of the baculovirus polyhedrin promoter. When used in the titration assay, the Sf-9ET cells turn green when they are infected with baculovirus due to the activation of the polyhedrin promoter/eGFP complex by baculovirus gene products expressed during the infection. Using a 96-well plate format end-point dilution assay, baculovirus titers can be determined in three days using a fluorescence microscope.

  4. Baculovirus-mediated gene delivery and RNAi applications.

    PubMed

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-04-22

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  5. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  6. Improving designer productivity

    NASA Technical Reports Server (NTRS)

    Hill, Gary C.

    1992-01-01

    Designer and design team productivity improves with skill, experience, and the tools available. The design process involves numerous trials and errors, analyses, refinements, and addition of details. Computerized tools have greatly speeded the analysis, and now new theories and methods, emerging under the label Artificial Intelligence (AI), are being used to automate skill and experience. These tools improve designer productivity by capturing experience, emulating recognized skillful designers, and making the essence of complex programs easier to grasp. This paper outlines the aircraft design process in today's technology and business climate, presenting some of the challenges ahead and some of the promising AI methods for meeting those challenges.

  7. Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

    PubMed Central

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-01-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

  8. Baculovirus: an insect-derived vector for diverse gene transfer applications.

    PubMed

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-04-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered.

  9. Error assessment in recombinant baculovirus titration: evaluation of different methods.

    PubMed

    Roldão, António; Oliveira, Rui; Carrondo, Manuel J T; Alves, Paula M

    2009-07-01

    The success of baculovirus/insect cells system in heterologous protein expression depends on the robustness and efficiency of the production workflow. It is essential that process parameters are controlled and include as little variability as possible. The multiplicity of infection (MOI) is the most critical factor since irreproducible MOIs caused by inaccurate estimation of viral titers hinder batch consistency and process optimization. This lack of accuracy is related to intrinsic characteristics of the method such as the inability to distinguish between infectious and non-infectious baculovirus. In this study, several methods for baculovirus titration were compared. The most critical issues identified were the incubation time and cell concentration at the time of infection. These variables influence strongly the accuracy of titers and must be defined for optimal performance of the titration method. Although the standard errors of the methods varied significantly (7-36%), titers were within the same order of magnitude; thus, viral titers can be considered independent of the method of titration. A cost analysis of the baculovirus titration methods used in this study showed that the alamarblue, real time Q-PCR and plaque assays were the most expensive techniques. The remaining methods cost on average 75% less than the former methods. Based on the cost, time and error analysis undertaken in this study, the end-point dilution assay, microculture tetrazolium assay and flow cytometric assay were found to be the techniques that combine all these three main factors better. Nevertheless, it is always recommended to confirm the accuracy of the titration either by comparison with a well characterized baculovirus reference stock or by titration using two different methods and verification of the variability of results.

  10. Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

    2015-01-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  11. Reaching the melting point: Degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination.

    PubMed

    Ishimwe, Egide; Hodgson, Jeffrey J; Clem, Rollie J; Passarelli, A Lorena

    2015-05-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in "melting" or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process.

  12. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  13. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  14. Budded baculovirus particle structure revisited.

    PubMed

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2016-02-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.

  15. Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    PubMed Central

    Ono, Chikako; Ninomiya, Akinori; Yamamoto, Satomi; Abe, Takayuki; Wen, Xiauyu; Fukuhara, Takasuke; Sasai, Miwa; Yamamoto, Masahiro; Saitoh, Tatsuya; Satoh, Takashi; Kawai, Taro; Ishii, Ken J.; Akira, Shizuo; Okamoto, Toru

    2014-01-01

    The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses. PMID:24335288

  16. Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein.

    PubMed

    Zhu, Xuejiao; Liu, Jie; Bai, Juan; Liu, Panrao; Zhang, Tingjie; Jiang, Ping; Wang, Xianwei

    2016-04-01

    Porcine circovirus type 2 (PCV2) causes significant economic losses to the swine industry worldwide. Heat shock proteins (Hsps) can be used as modulators to enhance both innate and adaptive immune responses. In the present study, recombinant baculoviruses expressing the PCV2Cap protein and the N-terminal 22-370 amino acids of porcine Gp96 (Gp96N), Hsp90, and Hsp70 (rBac-cap/Gp96N, rBac-cap/Hsp90 and rBac-cap/Hsp70, respectively) were constructed and the immune responses were examined in mice and piglets. The mouse experiments showed that rBac-cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies, proliferative responses of peripheral blood mononuclear cells (PBMCs) and IFN-γ levels compared to rBac-cap/Hsp90, rBac-cap/Hsp70, or rBac-cap. The pig experiments showed that the levels of anti-PCV2 antibody, proliferative responses of PBMCs, and IFN-γ in the rBac-cap/Gp96N groups were increased compared to those in rBac-cap group. There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-cap/Gp96N and rBac-cap, and the relative daily weight gains were higher than those in the challenge control (CC) group. The pathological lesions, extent of viremia, and viral loads of the vaccinated groups were milder than those in the CC group. Meanwhile, the extent of viremia and viral load present in the rBac-cap/Gp96N group were significantly lower than those in the rBac-cap group. These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2Cap. Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine. PMID:26826323

  17. Iflavirus increases its infectivity and physical stability in association with baculovirus

    PubMed Central

    Jakubowska, Agata K.; Murillo, Rosa; Carballo, Arkaitz; Williams, Trevor; van Lent, Jan W.M.; Caballero, Primitivo

    2016-01-01

    Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides. PMID:26966651

  18. Iflavirus increases its infectivity and physical stability in association with baculovirus.

    PubMed

    Jakubowska, Agata K; Murillo, Rosa; Carballo, Arkaitz; Williams, Trevor; van Lent, Jan W M; Caballero, Primitivo; Herrero, Salvador

    2016-01-01

    Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus-virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides. PMID:26966651

  19. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  20. Development of recombinant baculoviruses for insect control.

    PubMed

    Bonning, B C; Hammock, B D

    1996-01-01

    In this review, we provide an overview of the current status of recombinant baculoviruses, describe the development of genetically engineered baculoviruses for use as rapid-action biological insecticides, and provide more detailed information on one particular set of recombinant viruses. The advantages and disadvantages of recombinant baculovirus insecticides, and the importance of risk-assessment studies of these genetically modified organisms, are reviewed. Finally the importance of sensible regulatory strategies to the success and future prospects of this technology is discussed. PMID:8546446

  1. [Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector].

    PubMed

    Chen, Qi; Zhang, Shou-Feng; Liu, Ye; Fu, Yun-Hong; Sun, Cheng-Long; Yang, Yang; Gong, Ting; Song, Fei-Fei; Hu, Rong-Liang

    2012-09-01

    To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine.

  2. Arbovirus vaccines; opportunities for the baculovirus-insect cell expression system.

    PubMed

    Metz, Stefan W; Pijlman, Gorben P

    2011-07-01

    The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging group of medically important viral pathogens that are transmitted to humans and animals via arthropod vectors, mostly mosquitoes, ticks or midges. Few arboviral vaccines are currently available, but there is a growing need for safe and effective vaccines against some highly pathogenic arboviruses such as Chikungunya, dengue, West Nile, Rift Valley fever and Bluetongue viruses. This comprehensive review discusses the biology and current state of the art in vaccine development for arboviruses belonging to the families Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae and the potential of the baculovirus-insect cell expression system for vaccine antigen production The members of three of these four arbovirus families have enveloped virions and display immunodominant glycoproteins with a complex structure at their surface. Baculovirus expression of viral antigens often leads to correctly folded and processed (glyco)proteins able to induce protective immunity in animal models and humans. As arboviruses occupy a unique position in the virosphere in that they also actively replicate in arthropod cells, the baculovirus-insect cell expression system is well suited to produce arboviral proteins with correct folding and post-translational processing. The opportunities for recombinant baculoviruses to aid in the development of safe and effective subunit and virus-like particle vaccines against arboviral diseases are discussed.

  3. Penstock productivity improved

    SciTech Connect

    Not Available

    1989-09-01

    The Strawberry Water Users Association recently realized a significant increase in output on their hydroelectric plant near Spanish Fork, Utah. The plant's output was declining steadily and the problem was determined to be caused by build-up in its 11,000 foot long, 18 inch diameter penstock. After considering replacing the penstock or using hydro-blasting, the association decided that the pipe could be cleaned internally with flexible foam pits. Following the cleaning operation, the plant's output increased 22 percent to 345 kW. The new output level represents an increase in the association's annual income from the sale of electricity of $32,850 -enough to cover the costs of the equipment and contractor costs in approximately one year. The association plans to maintain maximum production by pumping an 18 inch pig down the line each year.

  4. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.

    PubMed

    van Oers, Monique M; Pijlman, Gorben P; Vlak, Just M

    2015-01-01

    In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth 'anniversary' of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements.

  5. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  6. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.

    PubMed

    van Oers, Monique M; Pijlman, Gorben P; Vlak, Just M

    2015-01-01

    In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth 'anniversary' of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements. PMID:25246703

  7. Improved productivity through interactive communication

    NASA Technical Reports Server (NTRS)

    Marino, P. P.

    1985-01-01

    New methods and approaches are being tried and evaluated with the goal of increasing productivity and quality. The underlying concept in all of these approaches, methods or processes is that people require interactive communication to maximize the organization's strengths and minimize impediments to productivity improvement. This paper examines Bendix Field Engineering Corporation's organizational structure and experiences with employee involvement programs. The paper focuses on methods Bendix developed and implemented to open lines of communication throughout the organization. The Bendix approach to productivity and quality enhancement shows that interactive communication is critical to the successful implementation of any productivity improvement program. The paper concludes with an examination of the Bendix methodologies which can be adopted by any corporation in any industry.

  8. Improving designer productivity. [artificial intelligence

    NASA Technical Reports Server (NTRS)

    Hill, Gary C.

    1992-01-01

    Designer and design team productivity improves with skill, experience, and the tools available. The design process involves numerous trials and errors, analyses, refinements, and addition of details. Computerized tools have greatly speeded the analysis, and now new theories and methods, emerging under the label Artificial Intelligence (AI), are being used to automate skill and experience. These tools improve designer productivity by capturing experience, emulating recognized skillful designers, and making the essence of complex programs easier to grasp. This paper outlines the aircraft design process in today's technology and business climate, presenting some of the challenges ahead and some of the promising AI methods for meeting these challenges.

  9. Improving efficiency in meat production.

    PubMed

    Brameld, John M; Parr, Tim

    2016-08-01

    Selective breeding and improved nutritional management over the past 20-30 years has resulted in dramatic improvements in growth efficiency for pigs and poultry, particularly lean tissue growth. However, this has been achieved using high-quality feed ingredients, such as wheat and soya that are also used for human consumption and more recently biofuels production. Ruminants on the other hand are less efficient, but are normally fed poorer quality ingredients that cannot be digested by human subjects, such as grass or silage. The challenges therefore are to: (i) maintain the current efficiency of growth of pigs and poultry, but using more ingredients not needed to feed the increasing human population or for the production of biofuels; (ii) improve the efficiency of growth in ruminants; (iii) at the same time produce animal products (meat, milk and eggs) of equal or improved quality. This review will describe the use of: (a) enzyme additives for animal feeds, to improve feed digestibility; (b) known growth promoting agents, such as growth hormone, β-agonists and anabolic steroids, currently banned in the European Union but used in other parts of the world; (c) recent transcriptomic studies into molecular mechanisms for improved growth efficiency via low residual feed intake. In doing so, the use of genetic manipulation in animals will also be discussed.

  10. Improving efficiency in meat production.

    PubMed

    Brameld, John M; Parr, Tim

    2016-08-01

    Selective breeding and improved nutritional management over the past 20-30 years has resulted in dramatic improvements in growth efficiency for pigs and poultry, particularly lean tissue growth. However, this has been achieved using high-quality feed ingredients, such as wheat and soya that are also used for human consumption and more recently biofuels production. Ruminants on the other hand are less efficient, but are normally fed poorer quality ingredients that cannot be digested by human subjects, such as grass or silage. The challenges therefore are to: (i) maintain the current efficiency of growth of pigs and poultry, but using more ingredients not needed to feed the increasing human population or for the production of biofuels; (ii) improve the efficiency of growth in ruminants; (iii) at the same time produce animal products (meat, milk and eggs) of equal or improved quality. This review will describe the use of: (a) enzyme additives for animal feeds, to improve feed digestibility; (b) known growth promoting agents, such as growth hormone, β-agonists and anabolic steroids, currently banned in the European Union but used in other parts of the world; (c) recent transcriptomic studies into molecular mechanisms for improved growth efficiency via low residual feed intake. In doing so, the use of genetic manipulation in animals will also be discussed. PMID:27087253

  11. Purification of infective baculoviruses by monoliths.

    PubMed

    Gerster, Petra; Kopecky, Eva-Maria; Hammerschmidt, Nikolaus; Klausberger, Miriam; Krammer, Florian; Grabherr, Reingard; Mersich, Christa; Urbas, Lidija; Kramberger, Petra; Paril, Tina; Schreiner, Matthias; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

    2013-05-17

    A chromatographic process based on monoliths for purification of infective baculovirus without prior concentration step has been established. Baculovirus produced in Spodoptera frugiperda cells (Sf-9) were harvested by centrifugation, filtered through 0.8 μm filters and directly loaded onto radial 1 mL anion exchange monoliths with a channel size of 1.5-2.0 μm operated at a volumetric flow rate of one bed volume per minute. Optional an epoxy monolith was used as pre-column to reduce interfering compounds and substances influencing the capacity of anion exchange monoliths for baculovirus infectious virus could be eluted with a step gradient at salt concentrations of 440 mM NaCl. Recovery of infectious virus was highly influenced by composition and age of supernatant and ranged from 20 to >99% active baculovirus. Total protein content could be reduced to 1-8% and DNA content to 38-48% in main virus fraction. Infective virus could be 52-fold concentrated within 20.5h and simultaneously an 82-fold volume reduction was possible when loading 1150 mL (2.1×10(8) pfu/mL) onto 1 mL scale support.

  12. Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

    PubMed Central

    Subbaiah, Edupalli V.; Royer, Corinne; Kanginakudru, Sriramana; Satyavathi, Valluri V.; Babu, Adari Sobhan; Sivaprasad, Vankadara; Chavancy, Gérard; DaRocha, Martine; Jalabert, Audrey; Mauchamp, Bernard; Basha, Ibrahim; Couble, Pierre; Nagaraju, Javaregowda

    2013-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture. PMID:23105011

  13. Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

    PubMed Central

    Haase, Santiago; Sciocco-Cap, Alicia; Romanowski, Víctor

    2015-01-01

    Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway. PMID:25941826

  14. Baculovirus insecticides in Latin America: historical overview, current status and future perspectives.

    PubMed

    Haase, Santiago; Sciocco-Cap, Alicia; Romanowski, Víctor

    2015-04-30

    Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

  15. Radioimmunoassay analysis of baculovirus granulins and polyhedrins

    SciTech Connect

    Summers, M.D.; Hoops, P.

    1980-05-01

    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera. Trichoplusia ni, and Spodoptera frugiperda. Antisera were raised against Autographa californica (Ac) polyhedrin and Trichoplusia ni (Tn) granulin and analyzed for homologous and heterologous immunoreactivity by immunodiffusion and radioimmunoassay (RIA). Ac polyhedrin and Tn granulin antisera recognized antigenic determinants on several baculovirus polyhedrin and granulin proteins even though the heterologous proteins had different immunoreactivities when compared by competition radioimmunoassay. Antigenic differences among granulin and polyhedrin proteins were also detected by altered slopes of the competition reaction curves. Antiserum raised against Ac polyhedrin which was purified in the presence of SDS was tested by competition RIA for its ability to detect and react with native polyhedrin produced in the infected TN-368 cells. Ac polyhedrin antiserum had similar if not identical ability to bind to native polyhedrin and to polyhedrin purified in the presence of SDS.

  16. Transcriptome Responses of the Host Trichoplusia ni to Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus

    PubMed Central

    Chen, Yun-Ru; Zhong, Silin; Fei, Zhangjun; Gao, Shan; Zhang, Shiying; Li, Zhaofei; Wang, Ping

    2014-01-01

    ABSTRACT Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ∼156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ∼60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure. IMPORTANCE Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell

  17. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen.

    PubMed

    Hu, Y C; Bentley, W E

    1999-01-01

    Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system. To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system. In this article, several factors were examined to enhance yield. These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors. Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression. A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro. Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold. Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal. Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins. PMID:10585191

  18. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen.

    PubMed

    Hu, Y C; Bentley, W E

    1999-01-01

    Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system. To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system. In this article, several factors were examined to enhance yield. These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors. Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression. A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro. Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold. Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal. Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins.

  19. Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

    PubMed Central

    2012-01-01

    Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule. PMID:22953695

  20. The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics.

    PubMed

    Sari, Duygu; Gupta, Kapil; Thimiri Govinda Raj, Deepak Balaji; Aubert, Alice; Drncová, Petra; Garzoni, Frederic; Fitzgerald, Daniel; Berger, Imre

    2016-01-01

    Multiprotein complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, multiprotein complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce multiprotein complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined. PMID:27165327

  1. Enhanced Expression of Full-Length Human Cytomegalovirus Fusion Protein in Non-Swelling Baculovirus-Infected Cells with a Minimal Fed-Batch Strategy

    PubMed Central

    Patrone, Marco; Carinhas, Nuno; Sousa, Marcos Q.; Peixoto, Cristina; Ciferri, Claudio; Carfì, Andrea; Alves, Paula M.

    2014-01-01

    Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB) has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains. PMID:24595278

  2. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

    PubMed

    Crava, Cristina M; Jakubowska, Agata K; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

  3. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus

    PubMed Central

    Crava, Cristina M.; Jakubowska, Agata K.; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity. PMID:25993013

  4. Radio Relays Improve Wireless Products

    NASA Technical Reports Server (NTRS)

    2009-01-01

    Signal Hill, California-based XCOM Wireless Inc. developed radio frequency micromachine (RF MEMS) relays with a Phase II Small Business Innovation Research (SBIR) contract through NASA?s Jet Propulsion Laboratory. In order to improve satellite communication systems, XCOM produced wireless RF MEMS relays and tunable capacitors that use metal-to-metal contact and have the potential to outperform most semiconductor technologies while using less power. These relays are used in high-frequency test equipment and instrumentation, where increased speed can mean significant cost savings. Applications now also include mainstream wireless applications and greatly improved tactical radios.

  5. Delivery of vaccine peptides by rapid conjugation to baculovirus particles.

    PubMed

    Wilson, Sarah; Baird, Margaret; Ward, Vernon K

    2008-05-12

    Baculoviruses deliver strong activation signals to dendritic cells and can promote potent immune responses. These properties can be harnessed to use baculovirus as an adjuvant and carrier particle for immunogenic peptides. In this study we use a chemical linker to couple peptides to the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Intranasal delivery of baculovirus coupled with immunogenic peptides to mice elicited antigen-specific IgG1 and IgG2a antibody. Furthermore, antigen-specific IgA was detected in the lung, and an IFN-gamma response was observed upon re-stimulation with antigen. We show that chemical coupling enables the rapid modification of AcMNPV, allowing multiple epitopes to be delivered simultaneously on a self-adjuvanting carrier particle. PMID:18417258

  6. Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

    PubMed

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul; Ambudkar, Suresh V

    2012-02-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  7. Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay.

    PubMed

    Wang, Wei; Cheng, Tong; Ma, Ke; Xia, Dezhen; Wang, Yongmei; Liu, Jian; Du, Hailian; Shih, James Wai Kuo; Zhang, Jun; Zhao, Qinjian; Xia, Ningshao

    2013-03-01

    The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, p<0.05) and BmNPV (R(2)=0.9834, p<0.05). In conclusion, a novel, rapid and semi-automated procedure for titrating baculovirus was developed based on the specific immunostaining of infected cells followed by automated spot counting.

  8. Improving functional value of meat products.

    PubMed

    Zhang, Wangang; Xiao, Shan; Samaraweera, Himali; Lee, Eun Joo; Ahn, Dong U

    2010-09-01

    In recent years, much attention has been paid to develop meat and meat products with physiological functions to promote health conditions and prevent the risk of diseases. This review focuses on strategies to improve the functional value of meat and meat products. Value improvement can be realized by adding functional compounds including conjugated linoneleic acid, vitamin E, n3 fatty acids and selenium in animal diets to improve animal production, carcass composition and fresh meat quality. In addition, functional ingredients such as vegetable proteins, dietary fibers, herbs and spices, and lactic acid bacteria can be directly incorporated into meat products during processing to improve their functional value for consumers. Functional compounds, especially peptides, can also be generated from meat and meat products during processing such as fermentation, curing and aging, and enzymatic hydrolysis. This review further discusses the current status, consumer acceptance, and market for functional foods from the global viewpoints. Future prospects for functional meat and meat products are also discussed.

  9. Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

    PubMed

    Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

    2013-01-01

    Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests.

  10. New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus.

    PubMed

    Regev, Avital; Rivkin, Hadassah; Gurevitz, Michael; Chejanovsky, Nor

    2006-12-22

    Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.

  11. Self-renewal: A strategy for quality and productivity improvement

    NASA Technical Reports Server (NTRS)

    Hutchinson, D. H.

    1985-01-01

    Productivity improvement is discussed. The concept of productivity improvement is supplemented with two additional concepts of productivity improvement: effectiveness and innovation. Case studies are provided to illustrate concepts.

  12. Teaching Recognition Skills to Improve Products.

    ERIC Educational Resources Information Center

    Knight, G. William; And Others

    1990-01-01

    First year dental students (n=86) in a conservative restorations course were taught a discrimination learning paradigm to improve production quality. Evaluation of Class 1 amalgam preparations indicates the improved recognition skills corresponded with improved cavity preparation, supporting the use of this teaching model. (Author/MSE)

  13. Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System

    PubMed Central

    Tao, Shiheng; Chen, Hongying

    2015-01-01

    By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells. PMID:26697848

  14. Power plant productivity improvement in New York

    SciTech Connect

    1981-03-01

    The New York Public Service Commission (PSC), under contract with the US Department of Energy (DOE), began a joint program in September 1978 to improve the productivity of coal and nuclear electric generating units in New York State. The project had dual objectives: to ensure that the utilities in New York State have or develop a systematic permanent, cost-effective productivity improvement program based on sound engineering and economic considerations, and to develop a model program for Power Plant Productivity Improvement, which, through DOE, can also be utilized by other regulatory commissions in the country. To accomplish these objectives, the program was organized into the following sequence of activities: compilation and analysis of power plant performance data; evaluation and comparison of utility responses to outage/derating events; power plant productivity improvement project cost-benefit analysis; and evaluation of regulatory procedures and policies for improving productivity. The program that developed for improving the productivity of coal units is substantially different than for nuclear units. Each program is presented, and recommendations are made for activities of both the utilities and regulatory agencies which will promote improved productivity.

  15. Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication.

    PubMed

    Mitchell, Jonathan K; Byers, Nathaniel M; Friesen, Paul D

    2013-12-01

    The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein. PMID:24027328

  16. Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.; Byers, Nathaniel M.

    2013-01-01

    The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein. PMID:24027328

  17. A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

    PubMed Central

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  18. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

    PubMed

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  19. Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

    SciTech Connect

    Changela, Anita; Martin, Alexandra; Shuman, Stewart; Mondragon, Alfonso

    2010-03-05

    Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 {angstrom} resolution. BVP adopts the characteristic cysteine-phosphatase {alpha}/{beta} fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif {sup 118}HCTHGXNRT{sup 126} in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

  20. Baculovirus infection induces heat shock response in vivo and in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are insect pathogens that have been exploited as bio-insecticides for the management of crop pests and for their ability to produce an abundance of heterologous proteins in baculovirus expression systems. Defining the molecular properties of baculovirus strains that broaden host range ...

  1. Gene acquisition convergence between entomopoxviruses and baculoviruses.

    PubMed

    Thézé, Julien; Takatsuka, Jun; Nakai, Madoka; Arif, Basil; Herniou, Elisabeth A

    2015-04-01

    Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host's immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

  2. Performance, Productivity and Continuous Improvement. Symposium.

    ERIC Educational Resources Information Center

    2002

    This document contains four papers from a symposium on performance, productivity, and continuous improvement. "Investigating the Association between Productivity and Quality Performance in Two Manufacturing Settings" (Constantine Kontoghiorghes, Robert Gudgel) summarizes a study that identified the following quality management variables as the…

  3. Productivity improvement and quality enhancement at NASA

    NASA Technical Reports Server (NTRS)

    Braunstein, D. R.

    1985-01-01

    NASA's Productivity Improvement and Quality Enhancement (PIQE) effort has as its objectives the encouragement of greater employee participation in management decision-making and the identification of impediments as well as opportunities for high productivity. Attempts are also made to try out novel management practices, and to evolve productivity trend analysis techniques. Every effort is made to note, reward, and diffuse successfully instituted PIQE approaches throughout the NASA-contractor organization.

  4. Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems

    PubMed Central

    Matindoost, Leila; Nielsen, Lars K.; Reid, Steve

    2015-01-01

    To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as “good” and “bad” BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA was lower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields. PMID:25951488

  5. Productivity improvement in engineering at Rocketdyne

    NASA Technical Reports Server (NTRS)

    Nordlund, R. M.; Vogt, S. T.; Woo, A. K.

    1985-01-01

    The Rocketdyne Division of Rockwell International has embarked on a productivity improvement program in engineering. This effort included participation in the White Collar Productivity Improvement (WCPI) project sponsored by the American Productivity Center. A number of things have been learned through this project. It seems that any productivity improvement project should be employee driven. The Rocketdyne project was essentially started as a result of a grassroots effort to remove some particular hindrances, and employee enthusiasm was a prime factor in the continuing progress of the effort. A significant result was that awareness of problems at all levels increased. Many issues surfaced in the diagnostic phase, and were then noted and discussed. This process added legitimacy to issues that had previously been merely unspoken concerns. The initial feelings of many members of the pilot group was that significant changes would occur relatively quickly. It is now recognized that this will have to be an ongoing, long-term effort.

  6. Barriers to success: How baculoviruses establish efficient systemic infections

    PubMed Central

    Passarelli, A. Lorena

    2011-01-01

    The mechanisms used by baculoviruses to exit the midgut and cause systemic infection of their insect hosts have been debated for decades. After being ingested, baculoviruses reach the midgut, where several host barriers need to be overcome in order to establish successful infection. One of these barriers is the basal lamina, a presumably virus-impermeable extracellular layer secreted by the epithelial cells lining the midgut and trachea. This review discusses new evidence that demonstrates how these viruses breach the basal lamina and establish efficient systemic infections. The biochemical mechanisms involved in dismantling basal lamina during baculovirus infection may also provide new insights into the process of basal lamina remodeling in invertebrate and vertebrate animals. PMID:21300392

  7. Powerplant productivity improvements and regulatory incentives

    SciTech Connect

    Hardy, D; Brown, D

    1980-10-27

    The purpose of this study was to examine the benefits to be gained from increased powerplant productivity and to validate and demonstrate the use of incentives within the regulatory process to promote the improvement of powerplant productivity. The system-wide costs savings to be gained from given productivity improvement scenarios are estimated in both the short and long term. Numerous reports and studies exist which indicate that productivity improvements at the powerplant level are feasible and cost effective. The efforts of this study widen this focus and relate system-wide productivity improvements with system-wide cost savings. The initial thrust of the regulatory section of this study is to validate the existence of reasonable incentive procedures which would enable regulatory agencies to better motivate electric utilities to improve productivity on both the powerplant and system levels. The voluntary incentive format developed in this study was designed to facilitate the link between profit and efficiency which is typically not clear in most regulated market environments. It is concluded that at the present time, many electric utilities in this country could significantly increase the productivity of their base load units, and the adoption of an incentive program of the general type recommended in this study would add to rate of return regulation the needed financial incentives to enable utilities to make such improvements without losing long-run profit. In light of the upcoming oil import target levels and mandatory cutbacks of oil and gas as boiler fuels for electric utilities, the use of incentive programs to encourage more efficient utilization of coal and nuclear base load capacity will become far more inviting over the next two decades.

  8. Evaluating Baculovirus Infection Using Green Fluorescent Protein and Variants.

    PubMed

    Wu, Hsuan-Chen; Cha, Hyung Joon; Bentley, William E

    2016-01-01

    By use of a strategy incorporating the green fluorescent protein (GFP), facile and rapid monitoring and visualization of baculovirus infection in insect cells is possible in vivo. This chapter describes two techniques for simple determination of virus titer in the baculovirus expression system using GFP co-expression and rapid monitoring of Sf-9 insect cell infection using a combination of GFP and the early-to-late (ETL) promoter of the virus vector. Because of its early appearance, GFP, when placed under the control of ETL promoter, will facilitate vector construction, virus isolation, and titer determination.

  9. Improved Succinate Production by Metabolic Engineering

    PubMed Central

    Cheng, Ke-Ke; Wang, Gen-Yu; Zeng, Jing; Zhang, Jian-An

    2013-01-01

    Succinate is a promising chemical which has wide applications and can be produced by biological route. The history of the biosuccinate production shows that the joint effort of different metabolic engineering approaches brings successful results. In order to enhance the succinate production, multiple metabolical strategies have been sought. In this review, different overproducers for succinate production, including natural succinate overproducers and metabolic engineered overproducers, are examined and the metabolic engineering strategies and performances are discussed. Modification of the mechanism of substrate transportation, knocking-out genes responsible for by-products accumulation, overexpression of the genes directly involved in the pathway, and improvement of internal NADH and ATP formation are some of the strategies applied. Combination of the appropriate genes from homologous and heterologous hosts, extension of substrate, integrated production of succinate, and other high-value-added products are expected to bring a desired objective of producing succinate from renewable resources economically and efficiently. PMID:23691505

  10. NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide

    PubMed Central

    Shim, Hee Jin; Choi, Jae Young; Wang, Yong; Tao, Xue Ying; Liu, Qin; Roh, Jong Yul; Kim, Jae Su; Kim, Woo Jin; Woo, Soo Dong; Jin, Byung Rae

    2013-01-01

    A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging. PMID:23064343

  11. Improving Pharmaceutical Protein Production in Oryza sativa

    PubMed Central

    Kuo, Yu-Chieh; Tan, Chia-Chun; Ku, Jung-Ting; Hsu, Wei-Cho; Su, Sung-Chieh; Lu, Chung-An; Huang, Li-Fen

    2013-01-01

    Application of plant expression systems in the production of recombinant proteins has several advantages, such as low maintenance cost, absence of human pathogens, and possession of complex post-translational glycosylation capabilities. Plants have been successfully used to produce recombinant cytokines, vaccines, antibodies, and other proteins, and rice (Oryza sativa) is a potential plant used as recombinant protein expression system. After successful transformation, transgenic rice cells can be either regenerated into whole plants or grown as cell cultures that can be upscaled into bioreactors. This review summarizes recent advances in the production of different recombinant protein produced in rice and describes their production methods as well as methods to improve protein yield and quality. Glycosylation and its impact in plant development and protein production are discussed, and several methods of improving yield and quality that have not been incorporated in rice expression systems are also proposed. Finally, different bioreactor options are explored and their advantages are analyzed. PMID:23615467

  12. Improving Organizational Productivity in NASA. Volume 2

    NASA Technical Reports Server (NTRS)

    1986-01-01

    Recognizing that NASA has traditionally been in the forefront of technological change, the NASA Administrator challenged the Agency in 1982 to also become a leader in developing and applying advanced technology and management practices to increase productivity. One of the activities undertaken by the Agency to support this ambitious productivity goal was participation in a 2-year experimental action research project devoted to learning more about improving and assessing the performance of professional organizations. Participating with a dozen private sector organizations, NASA explored the usefulness of a productivity improvement process that addressed all aspects of organizational performance. This experience has given NASA valuable insight into the enhancement of professional productivity. More importantly, it has provided the Agency with a specific management approach that managers and supervisors can effectively use to emphasize and implement continuous improvement. This report documents the experiences of the five different NASA installations participating in the project, describes the improvement process that was applied and refined, and offers recommendations for expanded application of that process. Of particular interest is the conclusion that measuring white collar productivity may be possible, and at a minimum, the measurement process itself is beneficial to management. Volume I of the report provides a project overview, significant findings, and recommendations. Volume II presents individual case studies of the NASA pilot projects that were part of the action research effort.

  13. Origin of Ecdysosteroid UDP-glycosyltransferases of Baculoviruses through Horizontal Gene Transfer from Lepidoptera

    PubMed Central

    Hughes, Austin L.

    2014-01-01

    Baculoviruses infecting Lepidoptera (butterflies and moths) encodes an enzyme known as ecdysosteroid UDP-glycosyltransferase (EGT), which inactivates insect host ecdysosteroid hormones, thereby preventing molt and pupation and permitting a build-up of the viral population within the host. Baculovirus EGT shows evidence of homology to insect UDP-glycosyltransferases, and a phylogenetic analysis supported the closest relative of baculovirus EGT are the UGT33 and UGT34 families of lepidopteran UDP-glycosyltransferases. The phylogenetic analysis thus supported that baculovirus EGT arose by horizontal gene transfer of a UDP-glycosyltransferase from a lepidopteran host, an event that occurred 70 million years ago at the earliest but possibly much more recently. Three amino acid replacements unique to baculovirus EGTs and conserved in all available baculovirus sequences were identified in the N-terminal region of the molecule. Because of their conservation, these amino acids are candidates for playing an important functional role in baculovirus EGT function. PMID:24834437

  14. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  15. Modulation of innate immunity in chickens induced by in vivo administration of baculovirus.

    PubMed

    Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gómez, Evangelina; Gravisaco, María José; Carrillo, Elisa; Berinstein, Analía

    2012-01-15

    Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3(+)CD4(+) T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3(+)CD4(+) T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved. PMID:22142984

  16. Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

    PubMed Central

    Wu, Yueh-Lung; Wu, Carol-P; Huang, Yu-Hui; Huang, Sheng-Ping; Lo, Huei-Ru; Chang, Hao-Shuo; Lin, Pi-Hsiu; Wu, Ming-Cheng; Chang, Chia-Jung

    2014-01-01

    ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells. PMID:25187548

  17. BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

    EPA Science Inventory

    The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

  18. Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems.

    PubMed

    Huang, Zhihong; Li, Ao; Pan, Mengjia; Wu, Wenbi; Yuan, Meijin; Yang, Kai

    2015-10-01

    In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome, and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6. Using the resulting ts helper plasmid pMON7124(ts) and the ts donor plasmid pFB1(ts)-PH-GFP, a recombinant bacmid, bAcWT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For bAcWT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

  19. Improvement of exopolysaccharide production by Porphyridium marinum.

    PubMed

    Soanen, Nastasia; Da Silva, Elise; Gardarin, Christine; Michaud, Philippe; Laroche, Céline

    2016-08-01

    With the aim to optimize the production of exopolysaccharide (EPS) by Porphyridium marinum, cultures in photobioreactors were conducted on a modified Provasoli medium (P) and compared to a new medium (Pm) with an elemental composition of N0.0205S0.0597P0.005. Cultivation on this medium allowed the increase of EPS concentration up to 2.5gL(-1), without modification of the EPS productivity (0.096gL(-1)) and EPS structure. In a second time, photosynthetic activity of the strain was monitored as a function of irradiance and temperature, allowing improvement of kinetic parameters of growth and EPS production. A semi-continuous culture, carried out with the Pm medium, an optimal irradiance and temperature of respectively 360μmolphotonsm(-2)s(-1) and 28°C led to an EPS process productivity of 0.031gh(-1) instead of 0.020gh(-1) in batch culture.

  20. Software productivity improvement through software engineering technology

    NASA Technical Reports Server (NTRS)

    Mcgarry, F. E.

    1985-01-01

    It has been estimated that NASA expends anywhere from 6 to 10 percent of its annual budget on the acquisition, implementation and maintenance of computer software. Although researchers have produced numerous software engineering approaches over the past 5-10 years; each claiming to be more effective than the other, there is very limited quantitative information verifying the measurable impact htat any of these technologies may have in a production environment. At NASA/GSFC, an extended research effort aimed at identifying and measuring software techniques that favorably impact productivity of software development, has been active over the past 8 years. Specific, measurable, software development technologies have been applied and measured in a production environment. Resulting software development approaches have been shown to be effective in both improving quality as well as productivity in this one environment.

  1. Characterization of a baculovirus-encoded RNA 5'-triphosphatase.

    PubMed

    Gross, C H; Shuman, S

    1998-09-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase. PMID:9696798

  2. Generating a host range-expanded recombinant baculovirus

    PubMed Central

    Wu, Chunfeng; Deng, Zihao; Long, Zhao; Cai, Yi; Ying, Zhongfu; Yin, Hanqi; Yuan, Meijin; Clem, Rollie J.; Yang, Kai; Pang, Yi

    2016-01-01

    As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc∆P35) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc∆P35 or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc∆P35, while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV. PMID:27321273

  3. Baculovirus expressed virus-like particles of Pea eation mosaic virus vary in size and encapsidate baculovirus mRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica m...

  4. Greater Green River Basin Production Improvement Project

    SciTech Connect

    DeJarnett, B.B.; Lim, F.H.; Calogero, D.

    1997-10-01

    The Greater Green River Basin (GGRB) of Wyoming has produced abundant oil and gas out of multiple reservoirs for over 60 years, and large quantities of gas remain untapped in tight gas sandstone reservoirs. Even though GGRB production has been established in formations from the Paleozoic to the Tertiary, recent activity has focused on several Cretaceous reservoirs. Two of these formations, the Ahnond and the Frontier Formations, have been classified as tight sands and are prolific producers in the GGRB. The formations typically naturally fractured and have been exploited using conventional well technology. In most cases, hydraulic fracture treatments must be performed when completing these wells to to increase gas production rates to economic levels. The objectives of the GGRB production improvement project were to apply the concept of horizontal and directional drilling to the Second Frontier Formation on the western flank of the Rock Springs Uplift and to compare production improvements by drilling, completing, and testing vertical, horizontal and directionally-drilled wellbores at a common site.

  5. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    PubMed

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  6. Baculoviruses mediate efficient gene expression in a wide range of vertebrate cells.

    PubMed

    Airenne, Kari J; Makkonen, Kaisa-Emilia; Mähönen, Anssi J; Ylä-Herttuala, Seppo

    2011-01-01

    Baculovirus expression vector system (BEVS) is well known as a feasible and safe technology to produce recombinant (re-)proteins in a eukaryotic milieu of insect cells. However, its proven power in gene delivery and gene therapy is still poorly recognized. The basis of BEVS lies in large enveloped DNA viruses derived from insects, the prototype virus being Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Infection of insect cell culture with a virus encoding a desired transgene under powerful baculovirus promoter leads to re-protein production in high quantities. Although the replication of AcMNPV is highly insect specific in nature, it can penetrate and transduce a wide range of cells of other origin. Efficient transduction requires only virus arming with an expression cassette active in the cells under investigation. The inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity and tropism provides many advantages for gene delivery over the other viral vectors typically derived from human pathogens.

  7. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  8. Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication.

    PubMed

    Lynn, D E

    2000-10-01

    Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged

  9. Direct fuel cell product design improvement

    SciTech Connect

    Maru, H.C.; Farooque, M.

    1996-12-31

    Significant milestones have been attained towards the technology development field testing and commercialization of direct fuel cell power plant since the 1994 Fuel Cell Seminar. Under a 5-year cooperative agreement with the Department of Energy signed in December 1994, Energy Research Corporation (ERC) has been developing the design for a MW-scale direct fuel cell power plant with input from previous technology efforts and the Santa Clara Demonstration Project. The effort encompasses product definition in consultation with the Fuel Cell Commercialization Group, potential customers, as well as extensive system design and packaging. Manufacturing process improvements, test facility construction, cell component scale up, performance and endurance improvements, stack engineering, and critical balance-of-plant development are also addressed. Major emphasis of this product design improvement project is on increased efficiency, compactness and cost reduction to establish a competitive place in the market. A 2.85 MW power plant with an efficiency of 58% and a footprint of 420 m{sup 2} has been designed. Component and subsystem testing is being conducted at various levels. Planning and preparation for verification of a full size prototype unit are in progress. This paper presents the results obtained since the last fuel cell seminar.

  10. Transfection of insect cell in suspension for efficient baculovirus generation.

    PubMed

    Roest, S; Kapps-Fouthier, S; Klopp, J; Rieffel, S; Gerhartz, B; Shrestha, B

    2016-01-01

    Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. This method is economical and does not require any special adjustment in existing labs. •An innovative method of transfecting insect cells in suspension using polyethyleneimine (PEI) is described here.•The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation.•The method significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination. PMID:27222826

  11. Unraveling the Entry Mechanism of Baculoviruses and Its Evolutionary Implications

    PubMed Central

    Wang, Manli; Wang, Jue; Yin, Feifei; Tan, Ying; Deng, Fei; Chen, Xinwen; Jehle, Johannes A.; Vlak, Just M.; Hu, Zhihong

    2014-01-01

    The entry of baculovirus budded virus into host cells is mediated by two distinct types of envelope fusion proteins (EFPs), GP64 and F protein. Phylogenetic analysis suggested that F proteins were ancestral baculovirus EFPs, whereas GP64 was acquired by progenitor group I alphabaculovirus more recently and may have stimulated the formation of the group I lineage. This study was designed to experimentally recapitulate a possible major step in the evolution of baculoviruses. We demonstrated that the infectivity of an F-null group II alphabaculovirus (Helicoverpa armigera nucleopolyhedrovirus [HearNPV]) can be functionally rescued by coinsertion of GP64 along with the nonfusogenic Fdef (furin site mutated HaF) from HearNPV. Interestingly, HearNPV enters cells by endocytosis and, less efficiently, by direct membrane fusion at low pH. However, this recombinant HearNPV coexpressing Fdef and GP64 mimicked group I virus not only in its EFP composition but also in its abilities to enter host cells via low-pH-triggered direct fusion pathway. Neutralization assays indicated that the nonfusogenic F proteins contribute mainly to binding to susceptible cells, while GP64 contributes to fusion. Coinsertion of GP64 with an F-like protein (Ac23) from group I virus led to efficient rescue of an F-null group II virus. In summary, these recombinant viruses and their entry modes are considered to resemble an evolutionary event of the acquisition of GP64 by an ancestral group I virus and subsequent adaptive inactivation of the original F protein. The study described here provides the first experimental evidence to support the hypothesis of the evolution of baculovirus EFPs. PMID:24335309

  12. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    PubMed

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  13. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    SciTech Connect

    H.C. Maru; M. Farooque

    2003-03-01

    The program efforts are focused on technology and system optimization for cost reduction, commercial design development, and prototype system field trials. The program is designed to advance the carbonate fuel cell technology from full-size field test to the commercial design. FuelCell Energy, Inc. (FCE) is in the later stage of the multiyear program for development and verification of carbonate fuel cell based power plants supported by DOE/NETL with additional funding from DOD/DARPA and the FuelCell Energy team. FCE has scaled up the technology to full-size and developed DFC{reg_sign} stack and balance-of-plant (BOP) equipment technology to meet product requirements, and acquired high rate manufacturing capabilities to reduce cost. FCE has designed submegawatt (DFC300A) and megawatt (DFC1500 and DFC3000) class fuel cell products for commercialization of its DFC{reg_sign} technology. A significant progress was made during the reporting period. The reforming unit design was optimized using a three-dimensional stack simulation model. Thermal and flow uniformities of the oxidant-In flow in the stack module were improved using computational fluid dynamics based flow simulation model. The manufacturing capacity was increased. The submegawatt stack module overall cost was reduced by {approx}30% on a per kW basis. An integrated deoxidizer-prereformer design was tested successfully at submegawatt scale using fuels simulating digester gas, coal bed methane gas and peak shave (natural) gas.

  14. Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression.

    PubMed

    Chen, Chiu-Ling; Luo, Wen-Yi; Lo, Wen-Hsin; Lin, Kun-Ju; Sung, Li-Yu; Shih, Yung-Shen; Chang, Yu-Han; Hu, Yu-Chen

    2011-12-01

    MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis. PMID:21732325

  15. Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

  16. Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infec...

  17. Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

    PubMed Central

    TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto

    2013-01-01

    ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ. PMID:24212505

  18. Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials

    PubMed Central

    Kwang, Timothy Weixin; Zeng, Xinhui; Wang, Shu

    2016-01-01

    Over the past two decades, baculoviruses have become workhorse research tools for transient transgene expression. Although they have not yet been used directly as a gene therapy vector in the clinical setting, numerous preclinical studies have suggested the highly promising potential of baculovirus as a delivery vector for a variety of therapeutic applications including vaccination, tissue engineering, and cancer treatment. As such, there is growing interest in using baculoviruses as human gene therapy vectors, which has led to advances in baculovirus bioprocessing methods. This review provides an overview of the current approaches for scaled-up amplification, concentration, purification, and formulation of AcMNPV baculoviruses, and highlights the key regulatory requirements that must be met before gene therapy clinical trials can be initiated. PMID:26858963

  19. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    SciTech Connect

    H.C. Maru; M. Farooque

    2004-08-01

    The ongoing program is designed to advance the carbonate fuel cell technology from full-size proof-of-concept field test to the commercial design. DOE has been funding Direct FuelCell{reg_sign} (DFC{reg_sign}) development at FuelCell Energy, Inc. (FCE) for stationary power plant applications. The program efforts are focused on technology and system optimization for cost reduction, leading to commercial design development and prototype system field trials. FCE, Danbury, CT, is a world-recognized leader for the development and commercialization of high efficiency fuel cells that can generate clean electricity at power stations, or at distributed locations near the customers such as hospitals, schools, universities, hotels and other commercial and industrial applications. FCE has designed three different fuel cell power plant models (DFC300A, DFC1500 and DFC3000). FCE's power plants are based on its patented DFC{reg_sign} technology, where the fuel is directly fed to the fuel cell and hydrogen is generated internally. These power plants offer significant advantages compared to the existing power generation technologies--higher fuel efficiency, significantly lower emissions, quieter operation, flexible siting and permitting requirements, scalability and potentially lower operating costs. Also, the exhaust heat by-product can be used for cogeneration applications such as high-pressure steam, district heating and air conditioning. Several FCE sub-megawatt power plants are currently operating in Europe, Japan and the US. Because hydrogen is generated directly within the fuel cell module from readily available fuels such as natural gas and waste water treatment gas, DFC power plants are ready today and do not require the creation of a hydrogen infrastructure. Product improvement progress made during the reporting period in the areas of technology, manufacturing processes, cost reduction and balance of plant equipment designs is discussed in this report.

  20. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    SciTech Connect

    H. C. Maru; M. Farooque

    2003-12-19

    The ongoing program is designed to advance the carbonate fuel cell technology from full-size proof-of-concept field test to the commercial design. DOE has been funding Direct FuelCell{reg_sign} (DFC{reg_sign}) development at FuelCell Energy, Inc. (FCE) for stationary power plant applications. The program efforts are focused on technology and system optimization for cost reduction leading to commercial design development and prototype system field trials. FCE, Danbury, CT, is a world-recognized leader for the development and commercialization of high efficiency fuel cells that can generate clean electricity at power stations or in distributed locations near the customer, including hospitals, schools, universities, hotels and other commercial and industrial applications. FuelCell Energy has designed three different fuel cell power plant models (DFC300, DFC1500 and DFC3000). FCE's power plants are based on its patented Direct FuelCell technology, where the fuel is directly fed to fuel cell and hydrogen is generated internally. These power plants offer significant advantages compared to existing power generation technologies--higher fuel efficiency, significantly lower emissions, quieter operation, flexible siting and permitting requirements, scalability and potentially lower operating costs. Also, the exhaust heat by-product can be used for cogeneration applications such as high-pressure steam, district heating, and air conditioning. Several FCE sub-megawatt power plants are currently operating in Europe, Japan and the US. Because hydrogen is generated directly within the fuel cell module from readily available fuels such as natural gas and waste water treatment gas, DFC power plants are ready today and do not require the creation of a hydrogen infrastructure. Product improvement progress made during the reporting period in the areas of technology, manufacturing processes, cost reduction and balance of plant equipment designs is discussed in this report. FCE's DFC

  1. Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

    PubMed Central

    Rebolledo, Dulce; Guevara, Roger; Murillo, Rosa

    2015-01-01

    Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen. PMID

  2. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.

    PubMed

    Felberbaum, Rachael S

    2015-05-01

    The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge.

  3. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    SciTech Connect

    H.C. Maru; M. Farooque

    2005-03-01

    water treatment gas, DFC power plants are ready today and do not require the creation of a hydrogen infrastructure. Product improvement progress made during the program period in the areas of technology, manufacturing processes, cost reduction and balance-of-plant equipment designs is discussed in this report.

  4. Agricultural Productivity Forecasts for Improved Drought Monitoring

    NASA Technical Reports Server (NTRS)

    Limaye, Ashutosh; McNider, Richard; Moss, Donald; Alhamdan, Mohammad

    2010-01-01

    Water stresses on agricultural crops during critical phases of crop phenology (such as grain filling) has higher impact on the eventual yield than at other times of crop growth. Therefore farmers are more concerned about water stresses in the context of crop phenology than the meteorological droughts. However the drought estimates currently produced do not account for the crop phenology. US Department of Agriculture (USDA) and National Oceanic and Atmospheric Administration (NOAA) have developed a drought monitoring decision support tool: The U.S. Drought Monitor, which currently uses meteorological droughts to delineate and categorize drought severity. Output from the Drought Monitor is used by the States to make disaster declarations. More importantly, USDA uses the Drought Monitor to make estimates of crop yield to help the commodities market. Accurate estimation of corn yield is especially critical given the recent trend towards diversion of corn to produce ethanol. Ethanol is fast becoming a standard 10% ethanol additive to petroleum products, the largest traded commodity. Thus the impact of large-scale drought will have dramatic impact on the petroleum prices as well as on food prices. USDA's World Agricultural Outlook Board (WAOB) serves as a focal point for economic intelligence and the commodity outlook for U.S. WAOB depends on Drought Monitor and has emphatically stated that accurate and timely data are needed in operational agrometeorological services to generate reliable projections for agricultural decision makers. Thus, improvements in the prediction of drought will reflect in early and accurate assessment of crop yields, which in turn will improve commodity projections. We have developed a drought assessment tool, which accounts for the water stress in the context of crop phenology. The crop modeling component is done using various crop modules within Decision Support System for Agrotechnology Transfer (DSSAT). DSSAT is an agricultural crop

  5. Gasification Product Improvement Facility (GPIF). Final report

    SciTech Connect

    1995-09-01

    The gasifier selected for development under this contract is an innovative and patented hybrid technology which combines the best features of both fixed-bed and fluidized-bed types. PyGas{trademark}, meaning Pyrolysis Gasification, is well suited for integration into advanced power cycles such as IGCC. It is also well matched to hot gas clean-up technologies currently in development. Unlike other gasification technologies, PyGas can be designed into both large and small scale systems. It is expected that partial repowering with PyGas could be done at a cost of electricity of only 2.78 cents/kWh, more economical than natural gas repowering. It is extremely unfortunate that Government funding for such a noble cause is becoming reduced to the point where current contracts must be canceled. The Gasification Product Improvement Facility (GPIF) project was initiated to provide a test facility to support early commercialization of advanced fixed-bed coal gasification technology at a cost approaching $1,000 per kilowatt for electric power generation applications. The project was to include an innovative, advanced, air-blown, pressurized, fixed-bed, dry-bottom gasifier and a follow-on hot metal oxide gas desulfurization sub-system. To help defray the cost of testing materials, the facility was to be located at a nearby utility coal fired generating site. The patented PyGas{trademark} technology was selected via a competitive bidding process as the candidate which best fit overall DOE objectives. The paper describes the accomplishments to date.

  6. Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system.

    PubMed

    Carmo, A C V; Giovanni, D N S; Corrêa, T P; Martins, L M; Stocco, R C; Suazo, C A T; Moraes, R H P; Veiga, A B G; Mendonça, R Z

    2012-05-01

    The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress. PMID:22230047

  7. Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system.

    PubMed

    Carmo, A C V; Giovanni, D N S; Corrêa, T P; Martins, L M; Stocco, R C; Suazo, C A T; Moraes, R H P; Veiga, A B G; Mendonça, R Z

    2012-05-01

    The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

  8. Expression and characterization of bovine lactoperoxidase by recombinant baculovirus.

    PubMed

    Tanaka, Tetsuya; Sato, Sanae; Kumura, Haruto; Shimazaki, Kei-ichi

    2003-10-01

    Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of delta-aminolevulinic acid, which is a heme precursor. Differences in the delta-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.

  9. Vocational Education: Its Role in Productivity Improvement and Technological Innovation.

    ERIC Educational Resources Information Center

    Drewes, Donald W.

    This report addresses productivity and technology from the perspective of state vocational education agencies. Chapter 1 explores the meaning and measurement of productivity and the benefits of productivity improvement--profits, a weapon against inflation, success in international trade, increased standard of living, improved quality of life, and…

  10. Improving Productivity in Higher Education: Administration and Support Costs.

    ERIC Educational Resources Information Center

    Massy, William F.

    1991-01-01

    Among the many reasons why college and university costs are rising are factors internal to the institutions which hold down productivity. Most efforts to improve productivity usually fail because they do not introduce new energy or information from the outside. In order to improve productivity, formal, non-quantitative evaluation should include a…

  11. Baculovirus as versatile vectors for protein expression in insect and mammalian cells.

    PubMed

    Kost, Thomas A; Condreay, J Patrick; Jarvis, Donald L

    2005-05-01

    Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

  12. Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

    PubMed

    Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

    2016-01-01

    A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

  13. Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles.

    PubMed

    Hitchman, Richard B; Siaterli, Evangelia A; Nixon, Clare P; King, Linda A

    2007-03-01

    We describe the use of quantitative PCR (QPCR) to titer recombinant baculoviruses. Custom primers and probe were designed to gp64 and used to calculate a standard curve of QPCR derived titers from dilutions of a previously titrated baculovirus stock. Each dilution was titrated by both plaque assay and QPCR, producing a consistent and reproducible inverse relationship between C(T) and plaque forming units per milliliter. No significant difference was observed between titers produced by QPCR and plaque assay for 12 recombinant viruses, confirming the validity of this technique as a rapid and accurate method of baculovirus titration.

  14. Baculovirus-mediated interferon alleviates dimethylnitrosamine-induced liver cirrhosis symptoms in a murine model.

    PubMed

    Nishibe, Y; Kaneko, H; Suzuki, H; Abe, T; Matsuura, Y; Takaku, H

    2008-07-01

    The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis induced by the mutagen dimethylnitrosamine. Intraperitoneal injection of AcMNPV induced an immune response. The baculovirus was taken up by the liver and spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat liver cirrhosis. PMID:18369328

  15. Composition and methods for improved fuel production

    SciTech Connect

    Steele, Philip H.; Tanneru, Sathishkumar; Gajjela, Sanjeev K.

    2015-12-29

    Certain embodiments of the present invention are configured to produce boiler and transportation fuels. A first phase of the method may include oxidation and/or hyper-acidification of bio-oil to produce an intermediate product. A second phase of the method may include catalytic deoxygenation, esterification, or olefination/esterification of the intermediate product under pressurized syngas. The composition of the resulting product--e.g., a boiler fuel--produced by these methods may be used directly or further upgraded to a transportation fuel. Certain embodiments of the present invention also include catalytic compositions configured for use in the method embodiments.

  16. Improvement of biogas production by bioaugmentation.

    PubMed

    Kovács, K L; Ács, N; Kovács, E; Wirth, R; Rákhely, G; Strang, Orsolya; Herbel, Zsófia; Bagi, Z

    2013-01-01

    Biogas production technologies commonly involve the use of natural anaerobic consortia of microbes. The objective of this study was to elucidate the importance of hydrogen in this complex microbial food chain. Novel laboratory biogas reactor prototypes were designed and constructed. The fates of pure hydrogen-producing cultures of Caldicellulosiruptor saccharolyticus and Enterobacter cloacae were followed in time in thermophilic and mesophilic natural biogas-producing communities, respectively. Molecular biological techniques were applied to study the altered ecosystems. A systematic study in 5-litre CSTR digesters revealed that a key fermentation parameter in the maintenance of an altered population balance is the loading rate of total organic solids. Intensification of the biogas production was observed and the results corroborate that the enhanced biogas productivity is associated with the increased abundance of the hydrogen producers. Fermentation parameters did not indicate signs of failure in the biogas production process. Rational construction of more efficient and sustainable biogas-producing microbial consortia is proposed.

  17. Improvement of Biogas Production by Bioaugmentation

    PubMed Central

    Kovács, K. L.; Ács, N.; Kovács, E.; Wirth, R.; Rákhely, G.; Strang, Orsolya; Herbel, Zsófia; Bagi, Z.

    2013-01-01

    Biogas production technologies commonly involve the use of natural anaerobic consortia of microbes. The objective of this study was to elucidate the importance of hydrogen in this complex microbial food chain. Novel laboratory biogas reactor prototypes were designed and constructed. The fates of pure hydrogen-producing cultures of Caldicellulosiruptor saccharolyticus and Enterobacter cloacae were followed in time in thermophilic and mesophilic natural biogas-producing communities, respectively. Molecular biological techniques were applied to study the altered ecosystems. A systematic study in 5-litre CSTR digesters revealed that a key fermentation parameter in the maintenance of an altered population balance is the loading rate of total organic solids. Intensification of the biogas production was observed and the results corroborate that the enhanced biogas productivity is associated with the increased abundance of the hydrogen producers. Fermentation parameters did not indicate signs of failure in the biogas production process. Rational construction of more efficient and sustainable biogas-producing microbial consortia is proposed. PMID:23484123

  18. Eastern Europe: USSR aims to improve production

    SciTech Connect

    Rigassi, D.A.

    1986-08-01

    This article details the perspectives for Eastern European petroleum development. Observations include: Oil output in Russia declined 2.6% last year, a drop more than twice as bad as 1984; Gorbachev cleaned house in 1985 after inspecting production problems in West Siberia; USSR exploratory/appraisal drilling the next five years should exceed 1981-85 by 40%; Gas output in USSR for '85 was 57.5 bcfd. A peak of 64 bcfd was reached last February; Poland hit a major offshore oil and gas find in the Baltic Sea during October 1985; Cash-short Yugoslavia plans to develop Ivana offshore gas field in the Adriatic Sea; Faced with stumbling domestic oil production, Romania turns to the USSR to fill the gap; Gas production in Hungary may drop despite added production from new and old fields.

  19. Strategies for improving biological hydrogen production.

    PubMed

    Hallenbeck, Patrick C; Abo-Hashesh, Mona; Ghosh, Dipankar

    2012-04-01

    Biological hydrogen production presents a possible avenue for the large scale sustainable generation of hydrogen needed to fuel a future hydrogen economy. Amongst the possible approaches that are under active investigation and that will be briefly discussed; biophotolysis, photofermentation, microbial electrolysis, and dark fermentation, dark fermentation has the additional advantages of largely relying on already developed bioprocess technology and of potentially using various waste streams as feedstock. However, the major roadblock to developing a practical process has been the low yields, typically around 25%, well below those achievable for the production of other biofuels from the same feedstocks. Moreover, low yields also lead to the generation of side products whose large scale production would generate a waste disposal problem. Here recent attempts to overcome these barriers are reviewed and recent progress in efforts to increase hydrogen yields through physiological manipulation, metabolic engineering and the use of two-stage systems are described.

  20. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    SciTech Connect

    H.C. Maru; M. Farooque

    2002-02-01

    generation, industrial cogeneration, marine applications and uninterrupted power for military bases. FuelCell Energy operated a 1.8 MW plant at a utility site in 1996-97, the largest fuel cell power plant ever operated in North America. This proof-of-concept power plant demonstrated high efficiency, low emissions, reactive power control, and unattended operation capabilities. Drawing on the manufacture, field test, and post-test experience of the full-size power plant; FuelCell Energy launched the Product Design Improvement (PDI) program sponsored by government and the private-sector cost-share. The PDI efforts are focused on technology and system optimization for cost reduction, commercial design development, and prototype system field trials. The program was initiated in December 1994. Year 2000 program accomplishments are discussed in this report.

  1. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  2. Health and productivity benefits of improved indoor air quality

    SciTech Connect

    Dorgan, C.B.; Dorgan, C.E.; Kanarek, M.S.; Willman, A.J.

    1998-10-01

    This paper is a summary of two studies completed for a national contractor`s association on the health costs and productivity benefits of improved IAQ. The original study documented the general health costs and productivity benefits of improved IAQ. The second study expanded the scope to include medical cost reductions for specific illnesses from improved IAQ. General information on the objectives, assumptions, definitions, and results of the studies are presented, followed by detailed information on research methodology, building inventory and wellness categories, health and medical effects of poor IAQ, health cost benefits, productivity benefits, recommended improvements, and conclusions and future improvements.

  3. Improving Productivity in Dietary Departments Workshop.

    ERIC Educational Resources Information Center

    McGhee, Barbara

    Instructional materials are provided for a workshop to enable participants to explain how productivity can be achieved through the practical use of performance standards and behavior changes. Another purpose is to enable participants to make changes by using methods that involve employees and thereby minimize employee resistance to change.…

  4. Baculovirus: Molecular Insights on Their Diversity and Conservation

    PubMed Central

    Miele, Solange Ana Belen; Garavaglia, Matías Javier; Belaich, Mariano Nicolás; Ghiringhelli, Pablo Daniel

    2011-01-01

    The Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp. In this study, genome sequences from 57 baculoviruses were analyzed to reevaluate the number and identity of core genes and to understand the distribution of the remaining coding sequences. Thirty one core genes with orthologs in all genomes were identified along with other 895 genes differing in their degrees of representation among reported genomes. Many of these latter genes are common to well-defined lineages, whereas others are unique to one or a few of the viruses. Phylogenetic analyses based on core gene sequences and the gene composition of the genomes supported the current division of the Baculoviridae into 4 genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus. PMID:21716740

  5. Cell lines used for the selection of recombinant baculovirus.

    PubMed

    Maruniak, J E; Garcia-Canedo, A; Rodrigues, J J

    1994-04-01

    Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.

  6. Baculovirus-mediated expression of GPCRs in insect cells.

    PubMed

    Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

    2015-01-01

    G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations.

  7. Baculovirus expression system and method for high throughput expression of genetic material

    DOEpatents

    Clark, Robin; Davies, Anthony

    2001-01-01

    The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

  8. The Host Specificities of Baculovirus per os Infectivity Factors

    PubMed Central

    Song, Jingjiao; Wang, Xi; Hou, Dianhai; Huang, Huachao; Liu, Xijia; Deng, Fei; Wang, Hualin; Arif, Basil M.; Hu, Zhihong; Wang, Manli

    2016-01-01

    Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host’s midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses. PMID:27454435

  9. The Host Specificities of Baculovirus per os Infectivity Factors.

    PubMed

    Song, Jingjiao; Wang, Xi; Hou, Dianhai; Huang, Huachao; Liu, Xijia; Deng, Fei; Wang, Hualin; Arif, Basil M; Hu, Zhihong; Wang, Manli

    2016-01-01

    Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host's midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses. PMID:27454435

  10. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A.; Kim, Steve Sungjin

    2014-06-03

    The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1, and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

  11. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A.; Kim, Steve

    2012-07-10

    The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1, and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

  12. Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs▿

    PubMed Central

    Li, Yi; Guarino, Linda A.

    2008-01-01

    The baculovirus Autographa californica nucleopolyhedrovirus encodes two proteins with RNA triphosphatase activity. Late expression factor LEF-4, which is an essential gene, is a component of the RNA polymerase and also encodes the RNA capping enzyme guanylyltransferase. PTP/BVP is also an RNA triphosphatase, but is not essential for viral replication, possibly because its activity is redundant to that of LEF-4. To elucidate the role of these proteins in mRNA cap formation, a mutant virus that lacked both RNA triphosphatase activities was constructed. Infection studies revealed that the double-mutant virus was viable and normal with respect to the production of budded virus. Pulse-labeling studies and immunoblot analyses showed that late gene expression in the double mutant was equivalent to that in the wild type, while polyhedrin expression was slightly reduced. Direct analysis of the mRNA cap structure indicated no alteration of cap processing in the double mutant. Together, these results reveal that baculoviruses replicate and express their late genes at normal levels in the absence of its two different types of RNA triphosphatases. PMID:18385232

  13. Improved fermentative alcohol production. [Patent application

    DOEpatents

    Wilke, C.R.; Maiorella, B.L.; Blanch, H.W.; Cysewski, G.R.

    1980-11-26

    An improved fermentation process is described for producing alcohol which includes the combination of vacuum fermentation and vacuum distillation. Preferably, the vacuum distillation is carried out in two phases, one a fermentor proper operated at atmospheric pressure and a flash phase operated at reduced pressure with recycle of fermentation brew having a reduced alcohol content to the fermentor, using vapor recompression heating of the flash-pot recycle stream to heat the flash-pot or the distillation step, and using water load balancing (i.e., the molar ratio of water in the fermentor feed is the same as the molar ratio of water in the distillation overhead).

  14. Productivity improvement using industrial engineering tools

    NASA Astrophysics Data System (ADS)

    Salaam, H. A.; How, S. B.; Faisae, M. F.

    2012-09-01

    Minimizing the number of defects is important to any company since it influence their outputs and profits. The aim of this paper is to study the implementation of industrial engineering tools in a manufacturing recycle paper box company. This study starts with reading the standard operation procedures and analyzing the process flow to get the whole idea on how to manufacture paper box. At the same time, observations at the production line were made to identify problem occurs in the production line. By using check sheet, the defect data from each station were collected and have been analyzed using Pareto Chart. From the chart, it is found that glue workstation shows the highest number of defects. Based on observation at the glue workstation, the existing method used to glue the box was inappropriate because the operator used a lot of glue. Then, by using cause and effect diagram, the root cause of the problem was identified and solutions to overcome the problem were proposed. There are three suggestions proposed to overcome this problem. Cost reduction for each solution was calculated and the best solution is using three hair drier to dry the sticky glue which produce only 6.4 defects in an hour with cost of RM 0.0224.

  15. NASA's Productivity Improvement and Quality Enhancement Initiatives

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The National Aeronautics and Space Administration celebrated its 25th Anniversary in 1983 at the Air and Space Museum in Washington, DC, with President Reagan in attendance. We look back on the accomplishments of these twenty-five years with pride in our missions and our people. NASA captured the world's imagination during the days of the Apollo mission. So much so, that we now talk about the Apollo era. In the l970s, we moved into the Space Transportation business and in the 199Os, we look forward to having a manned Space Station. Each succeeding mission has presented its own challenge in terms of technology and resources. This is especially true today, when we are being asked to do more with less. To ensure that NASA continues to be a productive and quality conscious agency, one of our highest Agency goals is leadership in the development and application of practices which contribute to high productivity and quality. greatest competitive strength, and this country has a solid scientific and engineering foundation. Traditionally we have spent more money on research and development than Japan and Europe combined, and we are the source of most of this century significant innovations. We should build on this solid base and use it more effectively.

  16. Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity

    PubMed Central

    Ge, Jingping; An, Qi; Gao, Dongni; Liu, Ying; Ping, Wenxiang

    2016-01-01

    Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses. PMID:27063566

  17. Onboard Plasmatron Hydrogen Production for Improved Vehicles

    SciTech Connect

    Daniel R. Cohn; Leslie Bromberg; Kamal Hadidi

    2005-12-31

    A plasmatron fuel reformer has been developed for onboard hydrogen generation for vehicular applications. These applications include hydrogen addition to spark-ignition internal combustion engines, NOx trap and diesel particulate filter (DPF) regeneration, and emissions reduction from spark ignition internal combustion engines First, a thermal plasmatron fuel reformer was developed. This plasmatron used an electric arc with relatively high power to reform fuels such as gasoline, diesel and biofuels at an oxygen to carbon ratio close to 1. The draw back of this device was that it has a high electric consumption and limited electrode lifetime due to the high temperature electric arc. A second generation plasmatron fuel reformer was developed. It used a low-current high-voltage electric discharge with a completely new electrode continuation. This design uses two cylindrical electrodes with a rotating discharge that produced low temperature volumetric cold plasma., The lifetime of the electrodes was no longer an issue and the device was tested on several fuels such as gasoline, diesel, and biofuels at different flow rates and different oxygen to carbon ratios. Hydrogen concentration and yields were measured for both the thermal and non-thermal plasmatron reformers for homogeneous (non-catalytic) and catalytic reforming of several fuels. The technology was licensed to an industrial auto part supplier (ArvinMeritor) and is being implemented for some of the applications listed above. The Plasmatron reformer has been successfully tested on a bus for NOx trap regeneration. The successful development of the plasmatron reformer and its implementation in commercial applications including transportation will bring several benefits to the nation. These benefits include the reduction of NOx emissions, improving engine efficiency and reducing the nation's oil consumption. The objective of this program has been to develop attractive applications of plasmatron fuel reformer

  18. Optimisation of protein expression and establishment of the Wave Bioreactor for Baculovirus/insect cell culture.

    PubMed

    Weber, Wilfried; Weber, Eric; Geisse, Sabine; Memmert, Klaus

    2002-01-01

    As the interest of research is beginning to shift from genomicsto proteomics the number of proteins to be expressed is rapidlyincreasing. To do so, well-established, high-level expressionsystems and rapid, cost-effective production means are needed. For addressing the latter, a novel cultivation system for recombinant cells, the Wave Bioreactortrade mark has recently becomeavailable. We describe the set-up and the optimisation of parameters essential for successful operation and growth of insect cells to high cell densities in the Wave Bioreactor. According to our experience, the Cellbagtrade mark system comparesvery favorably to conventional cultivation vessels such as bioreactors and roller cultures with respect to simplicity ofoperation and cost. Additionally, we developed a rapid and simple protocol for assessing expression and production conditions for the Baculovirus/insect cell system applicable to many different genes/proteins. Important parameters like MOI,TOI, peak cell density (PCD) and expression levels are determinedin pre-experiments on small scale to achieve optimal expressionof a given protein. These conditions are subsequently transformedand applied to large scale cultures grown in nutrient-supplemented medium in the Wave Bioreactor. PMID:19003089

  19. Rapid baculovirus titration assay based on viable cell side scatter (SSC).

    PubMed

    Qi, Jing; Liu, Tao; Pan, Junjie; Miao, Peng; Zhang, Chun

    2015-06-16

    The baculovirus expression system is one of the most powerful tools for the production of recombinant proteins on both laboratory and industrial scales. Multiplicity of infection (MOI) is the crucial parameter for efficient protein expression. To obtain an optimal MOI, it is important to determine titer of virus stock before protein production. Herein, we established a label-free, simple and rapid method for virus titration based on viable cell side scatter (SSC). Generally, the SSC of cells infected with a series of virus dilutions was measured by a flow cytometer at 48 h post-infection, and the probability of infected cells at a given dilution was estimated. For each well with the infection probabilities between 0.20 and 0.80, the range of dilutions was chosen, and virus titer was determined with a statistical method. Log-scale comparison of the results between the SSC based method and a standard plaque assay showed a good correlation (R(2)=0.9853), suggesting the fine accuracy of this proposed method.

  20. Improving microbial biogasoline production in Escherichia coli using tolerance engineering

    DOE PAGES

    Foo, Jee Loon; Jensen, Heather M.; Dahl, Robert H.; George, Kevin; Keasling, Jay D.; Lee, Taek Soon; Leong, Susanna; Mukhopadhyay, Aindrila

    2014-11-04

    Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerancemore » phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production.« less

  1. Improving Microbial Biogasoline Production in Escherichia coli Using Tolerance Engineering

    PubMed Central

    Foo, Jee Loon; Jensen, Heather M.; Dahl, Robert H.; George, Kevin; Keasling, Jay D.; Lee, Taek Soon; Leong, Susanna

    2014-01-01

    ABSTRACT Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. PMID:25370492

  2. Recent Productivity Improvements to the National Transonic Facility

    NASA Technical Reports Server (NTRS)

    Popernack, Thomas G., Jr.; Sydnor, George H.

    1998-01-01

    Productivity gains have recently been made at the National Transonic Facility wind tunnel at NASA Langley Research Center. A team was assigned to assess and set productivity goals to achieve the desired operating cost and output of the facility. Simulations have been developed to show the sensitivity of selected process productivity improvements in critical areas to reduce overall test cycle times. The improvements consist of an expanded liquid nitrogen storage system, a new fan drive, a new tunnel vent stack heater, replacement of programmable logic controllers, an increased data communications speed, automated test sequencing, and a faster model changeout system. Where possible, quantifiable results of these improvements are presented. Results show that in most cases, improvements meet the productivity gains predicted by the simulations.

  3. Service Productivity How to Measure and Improve It?

    NASA Astrophysics Data System (ADS)

    den Hartigh, Erik; Zegveld, Marc

    Productivity is a key performance measure for service businesses and serves as a compass for measuring their innovativeness. In this chapter we present a tool for measuring productivity in service businesses. Improvements in service business productivity do not depend on industry, business size or business growth, but on the specific knowledge and competences of managers. Using case examples we show various ways of how managers can improve the productivity of their service businesses. They can do so by adopting a perspective of standardization, flexibility or individualization. Based on these perspectives, we provide a framework that shows how managers can improve service business productivity by coordinating strategic orientation, value creation and the configuration of business processes.

  4. Prescriptive Package. Improving Patrol Productivity. Volume I. Routine Patrol.

    ERIC Educational Resources Information Center

    Gay, William G.; Schack, Stephen

    Designed to assist police departments in improving the productivity of their patrol operations, this volume on routine patrol and a companion volume on specialized patrol operations are intended for use by various sizes of departments. The volume on routine patrol focuses on the major issues of patrol productivity and recommends a number of…

  5. Exploring Group Communication and Productivity Improvement: Using an Experiential Process.

    ERIC Educational Resources Information Center

    Mandeville, Mary Y.; Mandeville, David E.

    Engineering students at Oklahoma State University used an experiential process (the ACME Basket Exercise) to develop an understanding of how quality and productivity can be improved. The exercise simulates a traditional production organization in the classroom and mirrors the efforts, the successes, and the frustrations of individuals and work…

  6. Workbook for Taguchi Methods for Product Quality Improvement.

    ERIC Educational Resources Information Center

    Zarghami, Ali; Benbow, Don

    Taguchi methods are methods of product quality improvement that analyze major contributions and how they can be controlled to reduce variability of poor performance. In this approach, knowledge is used to shorten testing. Taguchi methods are concerned with process improvement rather than with process measurement. This manual is designed to be used…

  7. Process for improving metal production in steelmaking processes

    DOEpatents

    Pal, Uday B.; Gazula, Gopala K. M.; Hasham, Ali

    1996-01-01

    A process and apparatus for improving metal production in ironmaking and steelmaking processes is disclosed. The use of an inert metallic conductor in the slag containing crucible and the addition of a transition metal oxide to the slag are the disclosed process improvements.

  8. Effective Conveyor Belt Inspection for Improved Mining Productivity

    SciTech Connect

    Chris Fromme

    2006-06-01

    This document details progress on the project entitled ''Effective Conveyor Belt Inspection for Improved Mining Productivity'' during the period from November 15, 2004 to May 14, 2004. Highlights include fabrication of an improved LED lightbar, fabrication of a line-scan sensor head for the Smart-Camera based prototype, and development of prototype vulcanized splice detection algorithms.

  9. Process for improving metal production in steelmaking processes

    DOEpatents

    Pal, U.B.; Gazula, G.K.M.; Hasham, A.

    1996-06-18

    A process and apparatus for improving metal production in ironmaking and steelmaking processes is disclosed. The use of an inert metallic conductor in the slag containing crucible and the addition of a transition metal oxide to the slag are the disclosed process improvements. 6 figs.

  10. Authentic processing and targeting of active maize auxin-binding protein in the baculovirus expression system.

    PubMed Central

    Macdonald, H; Henderson, J; Napier, R M; Venis, M A; Hawes, C; Lazarus, C M

    1994-01-01

    The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells. PMID:7972488

  11. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    PubMed

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

  12. Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

    PubMed Central

    Eldridge, R; O'Reilly, D R; Hammock, B D; Miller, L K

    1992-01-01

    Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses. Images PMID:1622228

  13. Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.

    PubMed

    Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

    2015-01-01

    Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

  14. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids.

    PubMed

    van Loo, N D; Fortunati, E; Ehlert, E; Rabelink, M; Grosveld, F; Scholte, B J

    2001-01-01

    We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

  15. Highly efficient baculovirus-mediated multigene delivery in primary cells.

    PubMed

    Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S; Xie, Ye; Boyce, Frederick M; Davis, M Wayne; Neuhauss, Stephan C F; Taylor, Verdon; Ballmer-Hofer, Kurt; Berger, Imre; Berger, Philipp

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells. PMID:27143231

  16. Highly efficient baculovirus-mediated multigene delivery in primary cells

    PubMed Central

    Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S.; Xie, Ye; Boyce, Frederick M.; Davis, M. Wayne; Neuhauss, Stephan C. F.; Taylor, Verdon; Ballmer-Hofer, Kurt; Berger, Imre; Berger, Philipp

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells. PMID:27143231

  17. Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

    PubMed Central

    Hildebrand, Amanda; Szewczyk, Edyta; Lin, Hui; Kasuga, Takao

    2014-01-01

    We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1. PMID:25381238

  18. Improving heterologous polyketide production in Escherichia coli by transporter engineering.

    PubMed

    Yang, Jingya; Xiong, Zhi-Qiang; Song, Shu-Jie; Wang, Jian-Feng; Lv, Hua-Jun; Wang, Yong

    2015-10-01

    Expelling heterologous compounds out of hosts by transporters is a potential strategy to enhance product titers in microbial cell factories. In this work, to increase heterologous polyketide 6-deoxyerythronolide B (6dEB, erythromycin precursor) production, tripartite multidrug efflux pumps MacAB-TolC, AcrAB-TolC, MdtEF-TolC, and MexAB-OprM were modulated in a 6dEB production strain. Compared with the control, overexpression of a single component of efflux pumps (except oprM) repressed 6dEB production, but modulation of two components MacA and MacB or the complete pumps MacAB-TolC and MdtEF-TolC significantly improved 6dEB titer by 100 ± 11, 118 ± 54, and 98 ± 12 %, respectively. In addition, to avoid the challenging fine-tuning components of pumps, the transcriptional regulators of efflux pumps were modulated to improve the 6dEB production. Overexpression of RpoH (activator of MdtEF-TolC) and EvgA (activator of EmrKY-TolC and AcrAD-TolC) strongly increased 6dEB titer by 152 ± 54 and 142 ± 85 %, respectively. This is the first report of transporter engineering for improving heterologous polyketide production in Escherichia coli. Our results provide an effective strategy for improving the yield of the heterologous products in chassis cell. PMID:26062534

  19. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    PubMed Central

    da Silva, Haroldo Cid; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-01-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  20. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    PubMed

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  1. Development of genetically enhanced baculovirus pesticides (Chapter 5). Book chapter

    SciTech Connect

    Wood, H.A.

    1991-01-01

    The report describes the assessment of the potential environmental impacts of genetically improved viral pesticides, including an evaluation of the properties of the foreign gene product(s) and the biological properties of the altered virus itself. The current field release studies are collecting much of the information which will be needed to assess the environmental safety of these new pesticides. Of primary concern will be the cost-to-benefit ratios as determined by production costs, stability, application, technology, and field efficacy. Despite the improvements afforded through biotechnology, it is clear that viral and other microbial pesticides will only reduce, not eliminate, the agricultural requirements for synthetic pesticides. Even so, biological pesticides are among the best solutions to reducing crop losses in the absence of ecological disturbances and potential health hazards.

  2. Baculovirus cyclobutane pyrimidine dimer photolyases show a close relationship with lepidopteran host homologues.

    PubMed

    Biernat, M A; Ros, V I D; Vlak, J M; van Oers, M M

    2011-08-01

    Cyclobutane pyrimidine dimer (CPD) photolyases repair ultraviolet (UV)-induced DNA damage using blue light. To get insight in the origin of baculovirus CPD photolyase (phr) genes, homologues in the lepidopteran insects Chrysodeixis chalcites, Spodoptera exigua and Trichoplusia ni were identified and characterized. Lepidopteran and baculovirus phr genes each form a monophyletic group, and together form a well-supported clade within the insect photolyases. This suggests that baculoviruses obtained their phr genes from an ancestral lepidopteran insect host. A likely evolutionary scenario is that a granulovirus, Spodoptera litura GV or a direct ancestor, obtained a phr gene. Subsequently, it was horizontally transferred from this granulovirus to several group II nucleopolyhedroviruses (NPVs), including those that infect noctuids of the Plusiinae subfamily. PMID:21477200

  3. A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency

    PubMed Central

    Toth, Ann M.; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L.

    2014-01-01

    Insect cells are often glycoengineered using DNA constructs encoding foreign glyocoenzymes under the transcriptional control of the baculovirus immediate early promoter, ie1. However, we recently found that the delayed early baculovirus promoter, 39K, provides inducible and higher levels of transgene expression than ie1 after baculovirus infection (Lin and Jarvis, 2013). Thus, the purpose of this study was to assess the utility of the 39K promoter for insect cell glycoengineering. We produced two polyclonal transgenic insect cell populations in parallel using DNA constructs encoding foreign glycoenzymes under either ie1 (Sfie1SWT) or 39K (Sf39KSWT) promoter control. The surface of Sfie1SWT cells was constitutively sialylated, whereas the Sf39KSWT cell surface was only strongly sialylated after baculovirus infection, indicating Sf39KSWT cells were inducibly-glycoengineered. All nine glycogene-related transcript levels were induced by baculovirus infection of Sf39KSWT cells and most reached higher levels in Sf39KSWT than in Sfie1SWT cells at early times after infection. Similarly, galactosyltransferase activity, sialyltransferase activity, and sialic acid levels were induced and reached higher levels in baculovirus-infected Sf39KSWT cells. Finally, two different recombinant glycoproteins produced by baculovirus-infected Sf39KSWT cells had lower proportions of paucimannose-type and higher proportions of sialylated, complex-type N-glycans than those produced by baculovirus-infected Sfie1SWT cells. Thus, the 39K promoter provides baculovirus-inducible expression of foreign glycogenes, higher glycoenzyme activity levels, and higher human-type N-glycan processing efficiencies than the ie1 promoter, indicating that this delayed early baculovirus promoter has great utility for insect cell glycoengineering. PMID:24768688

  4. In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

    PubMed

    Wang, Meng; Zheng, Zhenhua; Meng, Jin; Wang, Han; He, Man; Zhang, Fuxian; Liu, Yan; Hu, Bin; He, Zike; Hu, Qinxue; Wang, Hanzhong

    2015-09-01

    Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 μg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios.

  5. Productivity improvement and job satisfaction among public health nutritionists.

    PubMed

    Vermeersch, J A; Feeney, M J; Wesner, K M; Dahl, T

    1979-12-01

    A workshop for public health nutritionists which scrutinized ways to improve productivity and job satisfaction is reported. Participants believed that productivity could be improved most by improving the execution of tasks, followed by better planning of programs, office management, and skills in group education, and by delegation of non-professional activities to lesser-trained personnel. Job satisfaction of public health nutritionists could be increased by reducing stress and discomfort and promoting feelings of personal effectiveness and efficiency through role clarification and by management training. There is a large potential for greater productivity in the profession. Realization of this potential will contribute measurably to the cost-effectiveness of nutritional services. PMID:117042

  6. Version 5 product improvements from the atmospheric infrared sounder (AIRS)

    NASA Astrophysics Data System (ADS)

    Pagano, Thomas S.; Aumann, Hartmut H.; Chahine, Moustafa T.; Manning, Evan; Friedman, Steve; Broberg, Steven E.; Licata, Stephen J.; Elliott, Denis A.; Irion, Fredrick W.; Kahn, Brian H.; Fishbein, Evan; Olsen, Edward; Granger, Stephanie; Susskind, Joel; Keita, Fricky; Blaisdell, John; Strow, Larrabee; DeSouza-Machado, Sergio; Barnet, Chris

    2006-12-01

    The AIRS instrument was launched in May 2002 into a polar sun-synchronous orbit onboard the EOS Aqua Spacecraft. Since then we have released three versions of the AIRS data product to the scientific community. AIRS, in conjunction with the Advanced Microwave Sounding Unit (AMSU), produces temperature profiles with 1K/km accuracy on a global scale, as well as water vapor profiles and trace gas amounts. The first version of software, Version 2.0 was available to scientists shortly after launch with Version 3.0 released to the public in June 2003. Like all AIRS product releases, all products are accessible to the public in order to have the best user feedback on issues that appear in the data. Fortunately the products have had exceptional accuracy and stability. This paper presents the improvement between AIRS Version 4.0 and Version 5.0 products and shows examples of the new products available in Version 5.0.

  7. How can we improve the environmental sustainability of poultry production?

    PubMed

    Leinonen, Ilkka; Kyriazakis, Ilias

    2016-08-01

    The review presents results of recent life cycle assessment studies aiming to quantify and improve the environmental performance of UK poultry production systems, including broiler meat, egg and turkey meat production. Although poultry production has been found to be relatively environmentally friendly compared with the production of other livestock commodities, it still contributes to environmental impacts, such as global warming, eutrophication and acidification. Amongst different sub-processes, feed production and transport contributes about 70 % to the global warming potential of poultry systems, whereas manure management contributes about 40-60 % to their eutrophication potential and acidification potential, respectively. All these impacts can be reduced by improving the feed efficiency, either by changing the birds through genetic selection or by making the feed more digestible (e.g. by using additives such as enzymes). However, although genetic selection has the potential to reduce the resources needed for broiler production (including feed consumption), the changing need of certain feed ingredients, most notably protein sources as a result of changes in bird requirements may limit the benefits of this strategy. The use of alternative feed ingredients, such as locally grown protein crops and agricultural by-products, as a replacement of South American grown soya, can potentially also lead to improvements in several environmental impact categories, as long as such feeding strategies have no negative effect on bird performance. Other management options, such as improving poultry housing and new strategies for manure management have also the potential to further improve the environmental sustainability of the poultry industries in Europe.

  8. How can we improve the environmental sustainability of poultry production?

    PubMed

    Leinonen, Ilkka; Kyriazakis, Ilias

    2016-08-01

    The review presents results of recent life cycle assessment studies aiming to quantify and improve the environmental performance of UK poultry production systems, including broiler meat, egg and turkey meat production. Although poultry production has been found to be relatively environmentally friendly compared with the production of other livestock commodities, it still contributes to environmental impacts, such as global warming, eutrophication and acidification. Amongst different sub-processes, feed production and transport contributes about 70 % to the global warming potential of poultry systems, whereas manure management contributes about 40-60 % to their eutrophication potential and acidification potential, respectively. All these impacts can be reduced by improving the feed efficiency, either by changing the birds through genetic selection or by making the feed more digestible (e.g. by using additives such as enzymes). However, although genetic selection has the potential to reduce the resources needed for broiler production (including feed consumption), the changing need of certain feed ingredients, most notably protein sources as a result of changes in bird requirements may limit the benefits of this strategy. The use of alternative feed ingredients, such as locally grown protein crops and agricultural by-products, as a replacement of South American grown soya, can potentially also lead to improvements in several environmental impact categories, as long as such feeding strategies have no negative effect on bird performance. Other management options, such as improving poultry housing and new strategies for manure management have also the potential to further improve the environmental sustainability of the poultry industries in Europe. PMID:26935025

  9. Improving productivity and firm performance with enterprise resource planning

    NASA Astrophysics Data System (ADS)

    Beheshti, Hooshang M.; Beheshti, Cyrus M.

    2010-11-01

    Productivity is generally considered to be the efficient utilisation of organisational resources and is measured in terms of the efficiency of a worker, company or nation. Focusing on efficiency alone, however, can be harmful to the organisation's long-term success and competitiveness. The full benefits of productivity improvement measures are realised when productivity is examined from two perspectives: operational efficiency (output/input) of an individual worker or a business unit as well as performance (effectiveness) with regard to end user or customer satisfaction. Over the years, corporations have adopted new technology to integrate business activities in order to achieve both effectiveness and efficiency in their operations. In recent years, many firms have invested in enterprise resource planning (ERP) in order to integrate all business activities into a uniform system. The implementation of ERP enables the firm to reduce the transaction costs of the business and improve its productivity, customer satisfaction and profitability.

  10. Improving service provision for patients who are prescribed continence products.

    PubMed

    Mangnall, Joanne; Midgley, Kate; Lakin, Stuart; Beckitt, Liz; Shepherd, Laura

    2010-04-01

    It is estimated that 14 million people experience problems with bladder control and 6.5 million people have a bowel control problem (Bladder and Bowel Foundation, 2008). Many will rely on continence products to manage their problem and as such require products which ensure user dignity and discretion is maintained. Locally, an ever-increasing spend on prescribed continence products promoted a review of current service delivery. The combined results of a clinical audit and patient satisfaction survey highlighted areas in which service delivery needed immediate improvement. Significant service re-design has been undertaken with prescribing responsibility for continence-related products being transferred from GP practices to specialist nurses working within the continence service. Improvements in clinical care have been achieved and cost savings have been re-invested in the service. PMID:20559160

  11. Engineering Escherichia coli for improved ethanol production from gluconate.

    PubMed

    Hildebrand, Amanda; Schlacta, Theresa; Warmack, Rebeccah; Kasuga, Takao; Fan, Zhiliang

    2013-10-10

    We report on engineering Escherichia coli to produce ethanol at high yield from gluconic acid (gluconate). Knocking out genes encoding for the competing pathways (l-lactate dehydrogenase and pyruvate formate lyase A) in E. coli KO11 eliminated lactate production, lowered the carbon flow toward acetate production, and improved the ethanol yield from 87.5% to 97.5% of the theoretical maximum, while the growth rate of the mutant strain was about 70% of the wild type. The corresponding genetic modifications led to a small improvement of ethanol yield from 101.5% to 106.0% on glucose. Deletion of the pyruvate dehydrogenase gene (pdh) alone improved the ethanol yield from 87.5% to 90.4% when gluconate was a substrate. The growth rate of the mutant strain was identical to that of the wild type. The corresponding genetic modification led to no improvements on ethanol yield on glucose.

  12. Effective Conveyor Belt Inspection for Improved Mining Productivity

    SciTech Connect

    David LaRose

    2006-07-01

    This document details progress on the project ''Effective Conveyor Belt Inspection for Improved Mining Productivity'' during the period from November 15, 2005 to May 14, 2006. Highlights include significant improvements in the accuracy and reliability of computer-vision based vulcanized splice detection, deployment of the vulcanized splice detection algorithms for daily use in two working mines, and successful demonstration of an early prototype of a Smart-Camera based system for on-site mechanical splice detection in coal mine installations.

  13. Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

    PubMed

    Guarino, L A; Summers, M D

    1987-07-01

    The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression. PMID:16789264

  14. Assessing customer satisfaction for improving NOAA's climate products and services

    NASA Astrophysics Data System (ADS)

    Meyers, J. C.; Hawkins, M. D.; Timofeyeva, M. M.

    2009-12-01

    NOAA's National Weather Service (NWS) Climate Services Division (CSD) is developing a comprehensive climate user requirements process with the ultimate goal of producing climate services that meet the needs of NWS climate information users. An important part of this effort includes engaging users through periodical surveys conducted by the Claes Fornell International (CFI) Group using the American Customer Satisfaction Index (ACSI). The CFI Group conducted a Climate Services Satisfaction (CSS) Survey in May of 2009 to measure customer satisfaction with current products and services and to gain insight on areas for improvement. The CSS Survey rates customer satisfaction on a range of NWS climate services data and products, including Climate Prediction Center (CPC) outlooks, drought monitoring, and ENSO monitoring and forecasts, as well as NWS local climate data services. In addition, the survey assesses the users of the products to give the NWS insight into its climate customer base. The survey also addresses specific topics such as NWS forecast category names, probabilistic nature of climate products, and interpretation issues. The survey results identify user requirements for improving existing NWS climate services and introducing new ones. CSD will merge the survey recommendations with available scientific methodologies and operational capabilities to develop requirements for improved climate products and services. An overview of the 2009 survey results will be presented, such as users' satisfaction with the accuracy, reliability, display and functionality of products and services.

  15. IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NOVEL LIGNASE PROTEINS FROM TERMITES FOR DEPOLYMERIZATION OF LIGNOCELLULOSE

    SciTech Connect

    SLACK, JEFFREY, M.

    2012-12-06

    Wood is a potential source for biofuels such as ethanol if it can be digested into sugars and fermented by yeast. Biomass derived from wood is a challenging substrate for ethanol production since it is made of lignin and cellulose which cannot be broken down easily into fermentable sugars. Some insects, and termites in particular, are specialized at using enzymes in their guts to digest wood into sugars. If termite gut enzymes could be made abundantly by a recombinant protein expression vector system, they could be applied to an industrial process to make biofuels from wood. In this study, a large cDNA library of relevant termite genes was made using termites fed a normal diet, or a diet with added lignin. A subtracted library yielded genes that were overexpressed in the presence of lignin. Termite gut enzyme genes were identified and cloned into recombinant insect viruses called baculoviruses. Using our PERLXpress system for protein expression, these termite gene recombinant baculoviruses were prepared and used to infect insect larvae, which then expressed abundant recombinant termite enzymes. Many of these expressed enzymes were prepared to very high purity, and the activities were studied in conjunction with collaborators at Purdue University. Recombinant termite enzymes expressed in caterpillars were shown to be able to release sugars from wood. Mixing different combinations of these enzymes increased the amount of sugars released from a model woody biomass substrate. The most economical, fastest and energy conserving way to prepare termite enzymes expressed by recombinant baculoviruses in caterpillars was by making crude liquid homogenates. Making enzymes stable in homogenates therefore was a priority. During the course of these studies, improvements were made to the recombinant baculovirus expression platform so that caterpillar-derived homogenates containing expressed termite enzymes would be more stable. These improvements in the baculoviruses included

  16. Improving animal health and livestock productivity to reduce poverty.

    PubMed

    Pradère, J-P

    2014-12-01

    This study is based on scientific publications, statistics and field observations. It shows the importance of livestock in the economy and in the risk management strategies implemented by poor farming households. A comparison of livestock performance trends with the evolution of rural poverty in developing countries indicates that growth in livestock production alone is not enough to reduce rural poverty. To help reduce poverty, sustainable production should be based on productivity gains. Prerequisites for improving productivity include better public policies, enhanced research and the reduction of animal disease risk. The study draws attention to the economic, social and environmental consequences of inadequate support for animal health and production in the least developed countries, especially those of sub-Saharan Africa. PMID:25812201

  17. Guide for prioritizing power plant productivity improvement projects: handbook of availability improvement methodology

    SciTech Connect

    Not Available

    1981-09-15

    As part of its program to help improve electrical power plant productivity, the Department of Energy (DOE) has developed a methodology for evaluating productivity improvement projects. This handbook presents a simplified version of this methodology called the Availability Improvement Methodology (AIM), which provides a systematic approach for prioritizing plant improvement projects. Also included in this handbook is a description of data taking requirements necessary to support the AIM methodology, benefit/cost analysis, and root cause analysis for tracing persistent power plant problems. In applying the AIM methodology, utility engineers should be mindful that replacement power costs are frequently greater for forced outages than for planned outages. Equivalent availability includes both. A cost-effective ranking of alternative plant improvement projects must discern between those projects which will reduce forced outages and those which might reduce planned outages. As is the case with any analytical procedure, engineering judgement must be exercised with respect to results of purely mathematical calculations.

  18. Management Practices to Improve Productivity of Degraded/Eroded Soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Productivity of degraded/eroded soils can be restored by using organic amendment, such as manure, and improved soil management. A study is being conducted near Hays, KS, to investigate and compare restorative potential of two nitrogen (N) sources. Dried beef manure and urea fertilizer were each appl...

  19. Prescriptive Package. Improving Patrol Productivity. Volume II. Specialized Patrol.

    ERIC Educational Resources Information Center

    Schack, Stephen; Gay, William G.

    Designed to assist police departments in improving the productivity of their patrol operations, this volume on specialized patrol and a companion volume on routine patrol operations are intended for use by various sizes of departments. The volume of specialized patrol focuses upon the appropriate use and effective operation of specialized patrol…

  20. R and D productivity improvement at Honeywell: A case study

    NASA Technical Reports Server (NTRS)

    Lyons, W. E.

    1985-01-01

    The problems encountered when computer-aided-design/documentation was applied to a large design program at Honeywell; how a study team was established to solve the problem; the techniques used by the team and the resulting solutions are described. The techniques used in this instance may be applied to other problem areas in the R&D process to improve productivity.

  1. Creating Shared Instructional Products: An Alternative Approach to Improving Teaching

    ERIC Educational Resources Information Center

    Morris, Anne K.; Hiebert, James

    2011-01-01

    To solve two enduring problems in education--unacceptably large variation in learning opportunities for students across classrooms and little continuing improvement in the quality of instruction--the authors propose a system that centers on the creation of shared instructional products that guide classroom teaching. By examining systems outside…

  2. Effective Conveyor Belt Inspection for Improved Mining Productivity

    SciTech Connect

    Chris Fromme

    2006-06-01

    This document details progress on the project entitled ''Effective Conveyor Belt Inspection for Improved Mining Productivity'' during the period from November 15, 2004 to May 14, 2004. Highlights include fabrication of low-cost prototype hardware, acquisition of infrared thermal data, and initial design of a Smart-Camera based system.

  3. Identification and characterization of vlf-1, a baculovirus gene involved in very late gene expression.

    PubMed Central

    McLachlin, J R; Miller, L K

    1994-01-01

    We have identified a gene required for strong expression of the polyhedrin gene by characterizing a mutant, tsB837, of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which is temperature sensitive (ts) for occluded virus production at the nonpermissive temperature. Marker rescue experiments utilizing an overlapping set of AcMNPV genomic clones revealed that the gene responsible for the ts mutant phenotype mapped to a region between 46 and 48 map units. Fragments (2.2 kb) from both wild-type AcMNPV and tsB837 genomes spanning the mutated region were sequenced, and a single nucleotide difference was observed. This mutation is predicted to substitute a single amino acid within a 44.4-kDa polypeptide. Analysis of protein synthesis in wild-type- and mutant-infected cells at the nonpermissive temperature indicated that polyhedrin synthesis was dramatically reduced in the mutant. Northern (RNA) blot analysis revealed that the mutant had markedly reduced levels of polyhedrin transcripts. Transcripts of another very late gene, p10, were also reduced but to a lesser degree. The transcription of two late genes (603 ORF and vp39) was neither reduced nor temporally delayed. Thus, the gene encoding this very late expression factor, designated vlf-1, regulates the levels of very late gene transcripts, and the tsB837 mutation affects the levels of polyhedrin gene transcripts more strongly than those of p10 transcripts. The product of the newly identified gene has a surprising but significant relationship to a family of integrases and resolvases. Images PMID:7966564

  4. Technical improvements in 19th century Belgian window glass production

    NASA Astrophysics Data System (ADS)

    Lauriks, Leen; Collette, Quentin; Wouters, Ine; Belis, Jan

    Glass was used since the Roman age in the building envelope, but it became widely applied together with iron since the 19th century. Belgium was a major producer of window glass during the nineteenth century and the majority of the produced window glass was exported all over the world. Investigating the literature on the development of 19th century Belgian window glass production is therefore internationally relevant. In the 17th century, wood was replaced as a fuel by coal. In the 19th century, the regenerative tank furnace applied gas as a fuel in a continuous glass production process. The advantages were a clean production, a more constant and higher temperature in the furnace and a fuel saving. The French chemist Nicolas Leblanc (1787-1793) and later the Belgian chemist Ernest Solvay (1863) invented processes to produce alkali out of common salt. The artificial soda ash improved the quality and aesthetics of the glass plates. During the 19th century, the glass production was industrialized, influencing the operation of furnaces, the improvement of raw materials as well as the applied energy sources. Although the production process was industrialized, glassblowing was still the work of an individual. By improving his work tools, he was able to create larger glass plates. The developments in the annealing process followed this evolution. The industry had to wait until the invention of the drawn glass in the beginning of the 20th century to fully industrialise the window glass manufacture process.

  5. Special report. New products that improve officer performance, safety.

    PubMed

    1991-12-01

    The need for products that improve performance of security officers is counterbalanced these days by budgetary constraints. While this may limit major investments in security systems and personnel, less costly improvements or innovations might be worth considering. In this report, we will discuss four advances that may be valuable not only in hospital security, but in other industries as well. One of them, a smoke filter, was originally developed for the hotel industry. Another, a drug detection device, may replace the use of undercover agents or drug-sniffing' dogs in certain circumstances. The third new product is an economical patrol vehicle for parking facilities which might replace more costly vehicles such as golf carts or cars. The fourth product, a roving CCTV camera, is actually being tested at a Midwest medical center and may allow you to monitor areas of parking garages with cameras instead of officers on patrol.

  6. Special report. New products that improve officer performance, safety.

    PubMed

    1991-12-01

    The need for products that improve performance of security officers is counterbalanced these days by budgetary constraints. While this may limit major investments in security systems and personnel, less costly improvements or innovations might be worth considering. In this report, we will discuss four advances that may be valuable not only in hospital security, but in other industries as well. One of them, a smoke filter, was originally developed for the hotel industry. Another, a drug detection device, may replace the use of undercover agents or drug-sniffing' dogs in certain circumstances. The third new product is an economical patrol vehicle for parking facilities which might replace more costly vehicles such as golf carts or cars. The fourth product, a roving CCTV camera, is actually being tested at a Midwest medical center and may allow you to monitor areas of parking garages with cameras instead of officers on patrol. PMID:10116406

  7. Improvement of EUV mix-match overlay for production implementation

    NASA Astrophysics Data System (ADS)

    Park, Sarohan; Lee, ByoungHoon; Lee, Byong-Seog; Lee, Inwhan; Lim, Chang-Moon

    2016-03-01

    The improvement of overlay control in extreme ultra-violet (EUV) lithography is one of critical issues for successful mass production by using it. Especially it is important to improve the mix and match overlay or matched machine overlay (MMO) between EUV and ArF immersion tool, because EUV process will be applied to specific layers that have more competitive cost edge against ArF immersion multiple patterning with the early mass productivity of EUVL. Therefore it is necessary to consider the EUV overlay target with comparing the overlay specification of double patterning technology (DPT) and spacer patterning technology (SPT). This paper will discuss about required overlay controllability and current performance of EUV, and challenges for future improvement.

  8. Improvement of Stand Jig Sealer and Its Increased Production Capacity

    NASA Astrophysics Data System (ADS)

    Soebandrija, K. E. N.; Astuti, S. W. D.

    2014-03-01

    This paper has the objective to prove that improvement of Stand Jig Sealer can lead to the cycle time target as part of Improvement efforts and its Productivity. Prior researches through prior journals both classics journal such as Quesnay (1766) and Solow (1957) and updated journal such as Reikard (2011) researches, are mentioned and elaborated. Precisely, the research is narrowed down and specified into automotive industry and eventually the software related of SPSS and Structural Equation Modeling ( SEM ). The analysis and its method are conducted through the calculation working time. The mentioned calculation are reinforced with the hypothesis test using SPSS Version 19 and involve parameters of production efficiency, productivity calculation, and the calculation of financial investments. The results obtained are augmented achievement of cycle time target ≤ 80 seconds posterior to improvement stand jig sealer. The result from calculation of SPSS-19 version comprise the following aspects: the one-sided hypothesis test is rejection of Ho:μ≥80 seconds, the correlation rs=0.84, regression y = 0.159+0.642x, validity R table = 0.4438, reliability value of Cronbach's alpha = 0.885>0.70, independence (Chi Square) Asymp. Sig=0.028<0.05, 95% efficiency, increase productivity 11%, financial analysis (NPV 2,340,596>0, PI 2.04>1, IRR 45.56%>i=12.68%, PP=1.86). The Mentioned calculation results support the hypothesis and ultimately align with the objective of this paper to prove that improvement of Stand Jig Sealer and its relation toward the cycle time target. Precisely, the improvement of production capacity of PT. Astra Daihatsu Motor.

  9. Improved Jason-2 altimetry products for coastal ocean: PISTACH

    NASA Astrophysics Data System (ADS)

    Dufau, C.; Mercier, F.; Bouffard, J.; Bronner, E.; Lombard, A.; Picot, N.

    2009-04-01

    As part of Jason-2 project, CNES is conducting a study to improve altimeter products in coastal areas and inland waters. This 18-months-project named PISTACH for « Prototype Innovant de Système de Traitement pour les Applications Côtières et l'Hydrologie » is structured in three phases. In the first months of the project, a study of the user needs and the definition of the products were conducted. The second phase dealt with analysis, selection and development of new fields to be taken into account (retracking of the waveforms, radiometer and model wet troposphere correction, local model for correction of tides and atmospheric forcing, sea state bias, data editing). The third phase consists in prototype implementation, validation and operations during Jason-2 CalVal phases. Since November 2008, the PISTACH prototype is generating coastal dedicated Level 2 (I)GDR altimeter products. These products are freely delivered to users trough an anonymous FTP website ftp://ftpsedr.cls.fr/pub/oceano/pistach/ .The evaluation of the actual improvements and data quality reached near the coasts is now under investigation. The main results of the project (user needs, new algorithms and prototype) as well as the PISTACH products will be presented at the meeting. Early results of comparison with standard products will also be exhibited.

  10. Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

    PubMed

    Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto; Corva, Santiago Gerardo; Mórtola, Eduardo Carlos; Larsen, Alejandra; Jarvis, Donald L; Echeverría, María Gabriela

    2013-07-01

    Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine. PMID:23631926

  11. Evaluation of pig production practices, constraints and opportunities for improvement in smallholder production systems in Kenya.

    PubMed

    Mbuthia, Jackson Mwenda; Rewe, Thomas Odiwuor; Kahi, Alexander Kigunzu

    2015-02-01

    This study evaluated pig production practices by smallholder farmers in two distinct production systems geared towards addressing their constraints and prospects for improvement. The production systems evaluated were semi-intensive and extensive and differed in remoteness, market access, resource availability and pig production intensity. Data were collected using structured questionnaires where a total of 102 pig farmers were interviewed. Qualitative and quantitative research methods were employed to define the socioeconomic characteristics of the production systems, understanding the different roles that pigs play, marketing systems and constraints to production. In both systems, regular cash income and insurance against emergencies were ranked as the main reasons for rearing pigs. Marketing of pigs was mainly driven by the type of production operation. Finances, feeds and housing were identified as the major constraints to production. The study provides important parameters and identifies constraints important for consideration in design of sustainable production improvement strategies. Feeding challenges can be improved through understanding the composition and proper utilization of local feed resources. Provision of adequate housing would improve the stocking rates and control mating.

  12. Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent, cross-clade protection against H5N1 influenza virus in mice.

    PubMed

    Zhang, Jie; Fan, Hui-Ying; Zhang, Zhen; Zhang, Juan; Zhang, Jiao; Huang, Jian-Ni; Ye, Yu; Liao, Ming

    2016-01-27

    H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8(+) T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity. PMID:26724200

  13. THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

    EPA Science Inventory

    Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

  14. DEVELOPMENT OF AN IN SITU TOXICITY ASSAY SYSTEM USING RECOMBINANT BACULOVIRUSES. (R825433)

    EPA Science Inventory

    A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or m...

  15. Tissue specificity of a baculovirus expressed, basement membrane-degrading protease in larvae of Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from flesh fly Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm, Heliothis virescens, significantly faste...

  16. Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): Baculovirus expression, biochemical properties and organophosphate insensitivity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirm...

  17. Delivering Improved Nutrition: Dairy Ingredients in Food Aid Products.

    PubMed

    Schlossman, Nina

    2016-03-01

    The United States has a long history of food assistance for humanitarian need. The Food for Peace Act of 1954 established the United States' permanent food assistance program which has fed over 3 billion people in 150 countries worldwide through thousands of partner organizations. In 60 years, the program has evolved and will continue to do so. Recently, the program has gone from a focus on quantity of food shipped to quality food assistance from improved products, programs, and processes to effectively meet the needs of different vulnerable groups. The current debate focuses on the appropriateness of using fortified blended foods to prevent and treat malnutrition during the first 1000 days of life. Dairy ingredients have been at the center of this debate; they were included initially in fortified blended, removed in the 1980s, and now reincorporated into fortified therapeutic and supplemental foods. Improved quality food baskets and effective nutrition programming to prevent and treat malnutrition were developed through multisectoral collaboration between government and nongovernment organizations. The US Agency for International Development has focused on improving nutrition through development programs often tied to health, education, and agriculture. The years since 2008 have been a particularly intense period for improvement. The Food Aid Quality Review was established to update current food aid programming products, program implementation, cost-effectiveness, and interagency processes. Trials are underway to harmonize the areas of multisectoral nutrition programming and gather more evidence on the effects of dairy ingredients in food aid products.

  18. Improved vanillin production in baker's yeast through in silico design

    PubMed Central

    2010-01-01

    Background Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Conclusion Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories. PMID:21059201

  19. Improving Quality of Seal Leak Test Product using Six Sigma

    NASA Astrophysics Data System (ADS)

    Luthfi Malik, Abdullah; Akbar, Muhammad; Irianto, Dradjad

    2016-02-01

    Seal leak test part is a polyurethane material-based product. Based on past data, defect level of this product was 8%, higher than the target of 5%. Quality improvement effort was done using six sigma method that included phases of define, measure, analyse, improve, and control. In the design phase, a Delphi method was used to identify factors that were critical to quality. In the measure phase, stability and process capability was measured. Fault tree analysis (FTA) and failure mode and effect analysis (FMEA) were used in the next phase to analize the root cause and to determine the priority issues. Improve phase was done by compiling, selecting, and designing alternative repair. Some improvement efforts were identified, i.e. (i) making a checklist for maintenance schedules, (ii) making written reminder form, (iii) modifying the SOP more detail, and (iv) performing a major service to the vacuum machine. To ensure the continuity of improvement efforts, some control activities were executed, i.e. (i) controlling, monitoring, documenting, and setting target frequently, (ii) implementing reward and punishment system, (iii) adding cleaning tool, and (iv) building six sigma organizational structure.

  20. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    PubMed

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins. PMID:26820862

  1. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    PubMed

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

  2. Adhesion improvement of lignocellulosic products by enzymatic pre-treatment.

    PubMed

    Widsten, Petri; Kandelbauer, Andreas

    2008-01-01

    Enzymatic bonding methods, based on laccase or peroxidase enzymes, for lignocellulosic products such as medium-density fiberboard and particleboard are discussed with reference to the increasing costs of presently used petroleum-based adhesives and the health concerns associated with formaldehyde emissions from current composite products. One approach is to improve the self-bonding properties of the particles by oxidation of their surface lignin before they are fabricated into boards. Another method involves using enzymatically pre-treated lignins as adhesives for boards and laminates. The application of this technology to achieve wet strength characteristics in paper is also reviewed.

  3. Effective Conveyer Belt Inspection for Improved Mining Productivity

    SciTech Connect

    David LaRose

    2006-11-14

    This document details progress on the project ''Effective Conveyor Belt Inspection for Improved Mining Productivity'' during the period from May 15, 2006 to November 14, 2006. Progress during this period includes significant advances in development of a Smart Camera based prototype system for on-site mechanical splice detection, and continued deployment of both the mechanical splice detection system and the vulcanized splice detection system in area coal mines.

  4. Identifying improvement potentials in cement production with life cycle assessment.

    PubMed

    Boesch, Michael Elias; Hellweg, Stefanie

    2010-12-01

    Cement production is an environmentally relevant process responsible for 5% of total anthropogenic carbon dioxide emissions and 7% of industrial fuel use. In this study, life cycle assessment is used to evaluate improvement potentials in the cement production process in Europe and the USA. With a current fuel substitution rate of 18% in Europe and 11% in the USA, both regions have a substantial potential to reduce greenhouse gas emissions and save virgin resources by further increasing the coprocessing of waste fuels. Upgrading production technology would be particularly effective in the USA where many kiln systems with very low energy efficiency are still in operation. Using best available technology and a thermal substitution rate of 50% for fuels, greenhouse gas emissions could be reduced by 9% for Europe and 18% for the USA per tonne of cement. Since clinker production is the dominant pollution producing step in cement production, the substitution of clinker with mineral components such as ground granulated blast furnace slag or fly ash is an efficient measure to reduce the environmental impact. Blended cements exhibit substantially lower environmental footprints than Portland cement, even if the substitutes feature lower grindability and require additional drying and large transport distances. The highest savings in CO(2) emissions and resource consumption are achieved with a combination of measures in clinker production and cement blending. PMID:21047057

  5. Improvement of biohydrogen production using a reduced pressure fermentation.

    PubMed

    Kisielewska, M; Dębowski, M; Zieliński, M

    2015-10-01

    This study investigated the effect of reduced pressure on biohydrogen production in an upflow anaerobic sludge blanket (UASB) reactor from whey permeate. The results showed that the reduced pressure fermentation was more effective in enhancing biohydrogen production than dark fermentative hydrogen production at atmospheric pressure. Mesophilic fermentative biohydrogen production was investigated at a constant hydraulic retention time (HRT) of 24 h and increasing organic loading rates (OLRs) of 20, 25, 30, 35 kg COD/m(3) day. The reduced pressure fermentation was successfully operated at all OLRs tested. The maximum proportion of hydrogen in biogas of 47.7 %, volumetric hydrogen production rate (VHPR) of 7.10 L H2/day and hydrogen yield of 4.55 mol H2/kg COD removed occurred at the highest OLR. Increase in OLR affected the hydrogen production in UASB reactor exploited at atmospheric pressure. The reduced pressure process was able to remarkably improve the biohydrogen performance at high OLRs. PMID:26111633

  6. Production improvements plug water effluent by 90 percent

    SciTech Connect

    Wett, T.

    1993-03-01

    Ciba-Geigy Corp. is a wholly owned subsidary of Ciba-Geigy Ltd., a publicly owned company headquartered in Basel, Switzerland. The subsidary's St. Gabriel, LA, production site contains operations of the Agricultural and Textile Products divisions. The latter division has been involved in the dyestuff industry for more than 200 years. In late 1991, dyestuff production was initiated in the St. Gabriel Delta manufacturing facility. Here, a series of environmentally sound products - dyes for use in nylon carpeting, automotive products, towels, paper napkins, and cotton sportswear and other fabrics - are made using only water-soluble materials. Ciba Corp. actively participates in the Chemical Manufacturers Association's Responsible Care[reg sign] Initiative. In practice, the company strives to actively reflect the goals of the initiative for continuous improvement in environmental performance. Under its operating permit, the Delta plant is permitted a daily discharge averaging 125 lbs. per day of total organic carbon. The goal is to remain continuously below the permit level in long-term operation. Currently, discharge levels are averaging between 5 and 15 lbs. per day. Thanks to process automation and aggressive treatment, many materials are recycled or reused in the production process, particularly water. The volume of treated water effluent has been reduced to 80,000 gal./day, down from 1 million gal./day in older dyestuff plants - a reduction of more than 90%.

  7. Improvement of biohydrogen production using a reduced pressure fermentation.

    PubMed

    Kisielewska, M; Dębowski, M; Zieliński, M

    2015-10-01

    This study investigated the effect of reduced pressure on biohydrogen production in an upflow anaerobic sludge blanket (UASB) reactor from whey permeate. The results showed that the reduced pressure fermentation was more effective in enhancing biohydrogen production than dark fermentative hydrogen production at atmospheric pressure. Mesophilic fermentative biohydrogen production was investigated at a constant hydraulic retention time (HRT) of 24 h and increasing organic loading rates (OLRs) of 20, 25, 30, 35 kg COD/m(3) day. The reduced pressure fermentation was successfully operated at all OLRs tested. The maximum proportion of hydrogen in biogas of 47.7 %, volumetric hydrogen production rate (VHPR) of 7.10 L H2/day and hydrogen yield of 4.55 mol H2/kg COD removed occurred at the highest OLR. Increase in OLR affected the hydrogen production in UASB reactor exploited at atmospheric pressure. The reduced pressure process was able to remarkably improve the biohydrogen performance at high OLRs.

  8. Identifying improvement potentials in cement production with life cycle assessment.

    PubMed

    Boesch, Michael Elias; Hellweg, Stefanie

    2010-12-01

    Cement production is an environmentally relevant process responsible for 5% of total anthropogenic carbon dioxide emissions and 7% of industrial fuel use. In this study, life cycle assessment is used to evaluate improvement potentials in the cement production process in Europe and the USA. With a current fuel substitution rate of 18% in Europe and 11% in the USA, both regions have a substantial potential to reduce greenhouse gas emissions and save virgin resources by further increasing the coprocessing of waste fuels. Upgrading production technology would be particularly effective in the USA where many kiln systems with very low energy efficiency are still in operation. Using best available technology and a thermal substitution rate of 50% for fuels, greenhouse gas emissions could be reduced by 9% for Europe and 18% for the USA per tonne of cement. Since clinker production is the dominant pollution producing step in cement production, the substitution of clinker with mineral components such as ground granulated blast furnace slag or fly ash is an efficient measure to reduce the environmental impact. Blended cements exhibit substantially lower environmental footprints than Portland cement, even if the substitutes feature lower grindability and require additional drying and large transport distances. The highest savings in CO(2) emissions and resource consumption are achieved with a combination of measures in clinker production and cement blending.

  9. Highlights of contractor initiatives in quality enhancement and productivity improvement

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The NASA/Contractor Team efforts are presented as part of NASA's continuing effort to facilitate the sharing of quality and productivity improvement ideas among its contractors. This complilation is not meant to be a comprehensive review of contractor initiative nor does it necessarily express NASA's views. The submissions represent samples from a general survey, and were not edited by NASA. The efforts are examples of quality and productivity programs in private industry, and as such, highlight company efforts in individual areas. Topics range from modernization of equipment, hardware, and technology to management of human resources. Of particular interest are contractor initiatives which deal with measurement and evaluation data pertaining to quality and productivity performance.

  10. Metabolic engineering strategies for improving xylitol production from hemicellulosic sugars.

    PubMed

    Su, Buli; Wu, Mianbin; Lin, Jianping; Yang, Lirong

    2013-11-01

    Xylitol is a five-carbon sugar alcohol with potential for use as a sweetener. Industrially, xylitol is currently produced by chemical hydrogenation of D-xylose using Raney nickel catalysts and this requires expensive separation and purification steps as well as high pressure and temperature that lead to environmental pollution. Highly efficient biotechnological production of xylitol using microorganisms is gaining more attention and has been proposed as an alternative process. Although the biotechnological method has not yet surpassed the advantages of chemical reduction in terms of yield and cost, various strategies offer promise for the biotechnological production of xylitol. In this review, the focus is on the most recent developments of the main metabolic engineering strategies for improving the production of xylitol.

  11. Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production.

    PubMed

    Feng, Xueyang; Lian, Jiazhang; Zhao, Huimin

    2015-01-01

    Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45mg/L to 71mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71mg/L to 140mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140mg/L to 330mg/L). Through fed-batch fermentation using resting cells, over 1.1g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date. PMID:25466225

  12. Fertility management of bulls to improve beef cattle productivity.

    PubMed

    Thundathil, Jacob C; Dance, Alysha L; Kastelic, John P

    2016-07-01

    Global demand for animal proteins is increasing, necessitating increased efficiency of global food production. Improving reproductive efficiency of beef cattle, especially bull fertility, is particularly critical, as one bull can breed thousands of females (by artificial insemination). Identifying the genetic basis of male reproductive traits that influence male and female fertility, and using this information for selection, would improve herd fertility. Early-life selection of elite bulls by genomic approaches and feeding them to optimize postpubertal reproductive potential are essential for maximizing profitability. Traditional bull breeding soundness evaluation, or systematic analysis of frozen semen, eliminates bulls or semen samples that are grossly abnormal. However, semen samples classified as satisfactory on the basis of traditional approaches differ in fertility. Advanced sperm function assays developed for assessing compensatory and noncompensatory (submicroscopic) sperm traits can predict such variations in bull fertility. New knowledge on epigenetic modulations of sperm DNA, messenger RNA, and proteins is fundamental to refine and expand sperm function assays. Sexed semen, plus advanced reproductive technologies (e.g., ovum pickup and in vitro production of embryos) can maximize the efficiency of beef cattle production. This review is focused on genetic considerations for bull selection, physiology of reproductive development, breeding soundness evaluation, recent advances in assessing frozen semen, and existing and emerging uses of sexed semen in beef cattle production. PMID:27173954

  13. Product Quality Improvement Using FMEA for Electric Parking Brake (EPB)

    NASA Astrophysics Data System (ADS)

    Dumitrescu, C. D.; Gruber, G. C.; Tişcă, I. A.

    2016-08-01

    One of the most frequently used methods to improve product quality is complex FMEA. (Failure Modes and Effects Analyses). In the literature various FMEA is known, depending on the mode and depending on the targets; we mention here some of these names: Failure Modes and Effects Analysis Process, or analysis Failure Mode and Effects Reported (FMECA). Whatever option is supported by the work team, the goal of the method is the same: optimize product design activities in research, design processes, implementation of manufacturing processes, optimization of mining product to beneficiaries. According to a market survey conducted on parts suppliers to vehicle manufacturers FMEA method is used in 75%. One purpose of the application is that after the research and product development is considered resolved, any errors which may be detected; another purpose of applying the method is initiating appropriate measures to avoid mistakes. Achieving these two goals leads to a high level distribution in applying, to avoid errors already in the design phase of the product, thereby avoiding the emergence and development of additional costs in later stages of product manufacturing. During application of FMEA method using standardized forms; with their help will establish the initial assemblies of product structure, in which all components will be viewed without error. The work is an application of the method FMEA quality components to optimize the structure of the electrical parking brake (Electric Parching Brake - E.P.B). This is a component attached to the roller system which ensures automotive replacement of conventional mechanical parking brake while ensuring its comfort, functionality, durability and saves space in the passenger compartment. The paper describes the levels at which they appealed in applying FMEA, working arrangements in the 4 distinct levels of analysis, and how to determine the number of risk (Risk Priority Number); the analysis of risk factors and established

  14. CHARACTERIZATION OF THE GLYCOSYLATED ECDYSTEROIDS IN THE HEMOLYMPH OF BACULOVIRUS-INFECTED GYPSY MOTH LARVAE AND CELLS IN CULTURE

    EPA Science Inventory

    Fourth-instar gypsy moth (Lymantria dispar; Lepidoptera: Lymantriidae) larvae, infected with the gypsy moth baculovirus (LdNPV), show an elevated and prolonged extension of the hemolymph ecdysteroid titer peak associated with molting. The ecdysteroid immunoreactivity associated w...

  15. Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

    PubMed

    Tan, Zaigao; Yoon, Jong Moon; Nielsen, David R; Shanks, Jacqueline V; Jarboe, Laura R

    2016-05-01

    Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness. PMID:26875445

  16. Metabolic engineering of Escherichia coli to improve recombinant protein production.

    PubMed

    Liu, Min; Feng, Xinjun; Ding, Yamei; Zhao, Guang; Liu, Huizhou; Xian, Mo

    2015-12-01

    Escherichia coli is one of the most widely used strains for recombinant protein production. However, obstacles also exist in both academic researches and industrial applications, such as the metabolic burden, the carbon source waste, and the cells' physiological deterioration. This article reviews recent approaches for improving recombinant protein production in metabolic engineering, including workhorse selection, stress factor application, and carbon flux regulation. Selecting a suitable host is the first key point for recombinant protein production. In general, it all depends on characteristics of the strains and the target proteins. It will be triggered cells physiological deterioration when the medium is significantly different from the cell's natural environment. Coexpression of stress factors can help proteins to fold into their native conformation. Carbon flux regulation is a direct approach for redirecting more carbon flux toward the desirable pathways and products. However, some undesirable consequences are usually found in metabolic engineering, such as glucose transport inhibition, cell growth retardation, and useless metabolite accumulation. More efficient regulators and platform cell factories should be explored to meet a variety of production demands.

  17. Improving products of anaerobic sludge digestion by microaeration.

    PubMed

    Jenicek, P; Celis, C A; Krayzelova, L; Anferova, N; Pokorna, D

    2014-01-01

    Biogas, digested sludge and sludge liquor are the main products of anaerobic sludge digestion. Each of the products is influenced significantly by specific conditions of the digestion process. Therefore, any upgrade of the digestion technology must be considered with regard to quality changes in all products. Microaeration is one of the methods used for the improvement of biogas quality. Recently, microaeration has been proved to be a relatively simple and highly efficient biological method of sulfide removal in the anaerobic digestion of biosolids, but little attention has been paid to comparing the quality of digested sludge and sludge liquor in the anaerobic and microaerobic digestion and that is why this paper primarily deals with this area of research. The results of the long-term monitoring of digested sludge quality and sludge liquor quality in the anaerobic and microaerobic digesters suggest that products of both technologies are comparable. However, there are several parameters in which the 'microaerobic' products have a significantly better quality such as: sulfide (68% lower) and soluble chemical oxygen demand (COD) (33% lower) concentrations in the sludge liquor and the lower foaming potential of the digested sludge. PMID:24569280

  18. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    PubMed Central

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. PMID:26090442

  19. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

    PubMed

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Vaca, Luis; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  20. Improved photobiological H2 production in engineered green algal cells.

    PubMed

    Kruse, Olaf; Rupprecht, Jens; Bader, Klaus-Peter; Thomas-Hall, Skye; Schenk, Peer Martin; Finazzi, Giovanni; Hankamer, Ben

    2005-10-01

    Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, +/- uncoupler). Typically, approximately 540 ml of H2 liter(-1) culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) (efficiency = approximately 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems. PMID:16100118

  1. Pretreatment of microalgae to improve biogas production: a review.

    PubMed

    Passos, Fabiana; Uggetti, Enrica; Carrère, Hélène; Ferrer, Ivet

    2014-11-01

    Microalgae have been intensively studied as a source of biomass for replacing conventional fossil fuels in the last decade. The optimization of biomass production, harvesting and downstream processing is necessary for enabling its full-scale application. Regarding biofuels, biogas production is limited by the characteristics of microalgae, in particular the complex cell wall structure of most algae species. Therefore, pretreatment methods have been investigated for microalgae cell wall disruption and biomass solubilization before undergoing anaerobic digestion. This paper summarises the state of the art of different pretreatment techniques used for improving microalgae anaerobic biodegradability. Pretreatments were divided into 4 categories: (i) thermal; (ii) mechanical; (iii) chemical and (iv) biological methods. According to experimental results, all of them are effective at increasing biomass solubilization and methane yield, pretreatment effect being species dependent. Pilot-scale research is still missing and would help evaluating the feasibility of full-scale implementation.

  2. Pretreatment of microalgae to improve biogas production: a review.

    PubMed

    Passos, Fabiana; Uggetti, Enrica; Carrère, Hélène; Ferrer, Ivet

    2014-11-01

    Microalgae have been intensively studied as a source of biomass for replacing conventional fossil fuels in the last decade. The optimization of biomass production, harvesting and downstream processing is necessary for enabling its full-scale application. Regarding biofuels, biogas production is limited by the characteristics of microalgae, in particular the complex cell wall structure of most algae species. Therefore, pretreatment methods have been investigated for microalgae cell wall disruption and biomass solubilization before undergoing anaerobic digestion. This paper summarises the state of the art of different pretreatment techniques used for improving microalgae anaerobic biodegradability. Pretreatments were divided into 4 categories: (i) thermal; (ii) mechanical; (iii) chemical and (iv) biological methods. According to experimental results, all of them are effective at increasing biomass solubilization and methane yield, pretreatment effect being species dependent. Pilot-scale research is still missing and would help evaluating the feasibility of full-scale implementation. PMID:25257071

  3. Application of the suggestion system in the improvement of the production process and product quality control

    NASA Astrophysics Data System (ADS)

    Gołaś, H.; Mazur, A.; Gruszka, J.; Szafer, P.

    2016-08-01

    The elaboration is a case study and the research was carried out in the company Alco-Mot Ltd., which employs 120 people. The company specializes in the production of lead poles for industrial and traction batteries using gravity casting. The elements embedded in the cast are manufactured on a machining centre, which provides the stability of the process and of the dimensions of the product as well as a very short production time. As a result of observation and analysis the authors have developed a concept for the implementation of a dynamic suggestion system in ALCO-MOT, including, among others, a standard for actions in the implementation of the suggestion system, as well as clear guidelines for the processing and presentation of the activities undertaken in the time between the establishment of the concept (suggestions) and the benefits analysis after the proposed solutions have been implemented. The authors also present how suggestions proposed by ALCO-MOT staff contributed to the improvement of the processes of production and quality control. Employees offered more than 30 suggestions, of which more than a half are being implemented now and further actions are being prepared for implementation. The authors will present the results of improvements in, for example, tool replacement time, scrap reduction. The authors will present how kaizen can improve the production and quality control processes. They will present how the production and quality control processes looked before and after the implementation of employee suggestions.

  4. Genetic improvement for root growth angle to enhance crop production

    PubMed Central

    Uga, Yusaku; Kitomi, Yuka; Ishikawa, Satoru; Yano, Masahiro

    2015-01-01

    The root system is an essential organ for taking up water and nutrients and anchoring shoots to the ground. On the other hand, the root system has rarely been regarded as breeding target, possibly because it is more laborious and time-consuming to evaluate roots (which require excavation) in a large number of plants than aboveground tissues. The root growth angle (RGA), which determines the direction of root elongation in the soil, affects the area in which roots capture water and nutrients. In this review, we describe the significance of RGA as a potential trait to improve crop production, and the physiological and molecular mechanisms that regulate RGA. We discuss the prospects for breeding to improve RGA based on current knowledge of quantitative trait loci for RGA in rice. PMID:26069440

  5. Genetic improvement for root growth angle to enhance crop production.

    PubMed

    Uga, Yusaku; Kitomi, Yuka; Ishikawa, Satoru; Yano, Masahiro

    2015-03-01

    The root system is an essential organ for taking up water and nutrients and anchoring shoots to the ground. On the other hand, the root system has rarely been regarded as breeding target, possibly because it is more laborious and time-consuming to evaluate roots (which require excavation) in a large number of plants than aboveground tissues. The root growth angle (RGA), which determines the direction of root elongation in the soil, affects the area in which roots capture water and nutrients. In this review, we describe the significance of RGA as a potential trait to improve crop production, and the physiological and molecular mechanisms that regulate RGA. We discuss the prospects for breeding to improve RGA based on current knowledge of quantitative trait loci for RGA in rice.

  6. Biohydrogen production: strategies to improve process efficiency through microbial routes.

    PubMed

    Chandrasekhar, Kuppam; Lee, Yong-Jik; Lee, Dong-Woo

    2015-01-01

    The current fossil fuel-based generation of energy has led to large-scale industrial development. However, the reliance on fossil fuels leads to the significant depletion of natural resources of buried combustible geologic deposits and to negative effects on the global climate with emissions of greenhouse gases. Accordingly, enormous efforts are directed to transition from fossil fuels to nonpolluting and renewable energy sources. One potential alternative is biohydrogen (H2), a clean energy carrier with high-energy yields; upon the combustion of H2, H2O is the only major by-product. In recent decades, the attractive and renewable characteristics of H2 led us to develop a variety of biological routes for the production of H2. Based on the mode of H2 generation, the biological routes for H2 production are categorized into four groups: photobiological fermentation, anaerobic fermentation, enzymatic and microbial electrolysis, and a combination of these processes. Thus, this review primarily focuses on the evaluation of the biological routes for the production of H2. In particular, we assess the efficiency and feasibility of these bioprocesses with respect to the factors that affect operations, and we delineate the limitations. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed to address industrial-level applications.

  7. Glycosylation: impact, control and improvement during therapeutic protein production.

    PubMed

    Costa, Ana Rita; Rodrigues, Maria Elisa; Henriques, Mariana; Oliveira, Rosário; Azeredo, Joana

    2014-12-01

    The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.

  8. Biohydrogen production: strategies to improve process efficiency through microbial routes.

    PubMed

    Chandrasekhar, Kuppam; Lee, Yong-Jik; Lee, Dong-Woo

    2015-01-01

    The current fossil fuel-based generation of energy has led to large-scale industrial development. However, the reliance on fossil fuels leads to the significant depletion of natural resources of buried combustible geologic deposits and to negative effects on the global climate with emissions of greenhouse gases. Accordingly, enormous efforts are directed to transition from fossil fuels to nonpolluting and renewable energy sources. One potential alternative is biohydrogen (H2), a clean energy carrier with high-energy yields; upon the combustion of H2, H2O is the only major by-product. In recent decades, the attractive and renewable characteristics of H2 led us to develop a variety of biological routes for the production of H2. Based on the mode of H2 generation, the biological routes for H2 production are categorized into four groups: photobiological fermentation, anaerobic fermentation, enzymatic and microbial electrolysis, and a combination of these processes. Thus, this review primarily focuses on the evaluation of the biological routes for the production of H2. In particular, we assess the efficiency and feasibility of these bioprocesses with respect to the factors that affect operations, and we delineate the limitations. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed to address industrial-level applications. PMID:25874756

  9. Biohydrogen Production: Strategies to Improve Process Efficiency through Microbial Routes

    PubMed Central

    Chandrasekhar, Kuppam; Lee, Yong-Jik; Lee, Dong-Woo

    2015-01-01

    The current fossil fuel-based generation of energy has led to large-scale industrial development. However, the reliance on fossil fuels leads to the significant depletion of natural resources of buried combustible geologic deposits and to negative effects on the global climate with emissions of greenhouse gases. Accordingly, enormous efforts are directed to transition from fossil fuels to nonpolluting and renewable energy sources. One potential alternative is biohydrogen (H2), a clean energy carrier with high-energy yields; upon the combustion of H2, H2O is the only major by-product. In recent decades, the attractive and renewable characteristics of H2 led us to develop a variety of biological routes for the production of H2. Based on the mode of H2 generation, the biological routes for H2 production are categorized into four groups: photobiological fermentation, anaerobic fermentation, enzymatic and microbial electrolysis, and a combination of these processes. Thus, this review primarily focuses on the evaluation of the biological routes for the production of H2. In particular, we assess the efficiency and feasibility of these bioprocesses with respect to the factors that affect operations, and we delineate the limitations. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed to address industrial-level applications. PMID:25874756

  10. Reservoir management options for improving water productivity in Africa

    NASA Astrophysics Data System (ADS)

    Niasse, M.

    2003-04-01

    Although there seems to be a global consensus on the need for improving the productivity of water, little progress has been made, especially in Africa, toward applying this concept in approaches to water resources planning and management. The notion of water productivity refers to the types, amount and value of material and non material products generated from water use, and embraces therefore a broad spectrum outputs, including crop and hydropower production, livestock and fishery yields, as well as outputs from other water-based activities such as domestic and industrial water consumption. In contexts of water scarcity, like in many regions in Africa, it becomes more and more important to allocate available freshwater resources among competing sectors as efficiently as possible, while taking into account the needs of aquatic ecosystems and the concerns of equity and poverty reduction. A minimum level of control of water is often necessary for the implementation of these water allocation principles. On the basis of the water budget of the Senegal River -- a basin where a significant share of the river flow is controlled by dams since the late 1980s—and other river basins in Africa, the proposed communication analyses the merits and limitations of different reservoir management options. On this basis the paper discuss approaches to optimal and sustainable water allocation which takes into consideration multiple uses and the need for greater equity in access and reduced vulnerability of the poor.

  11. Informativeness Improvement of Hardness Test Methods for Metal Product Assessment

    NASA Astrophysics Data System (ADS)

    Osipov, S.; Podshivalov, I.; Osipov, O.; Zhantybaev, A.

    2016-06-01

    The paper presents a combination of theoretical suggestions, results, and observations allowing to improve the informativeness of hardness testing process in solving problems of metal product assessment while in operation. The hardness value of metal surface obtained by a single measurement is considered to be random. Various measures of location and scattering of the random variable were experimentally estimated for a number of test samples using the correlation analysis, and their close interaction was studied. It was stated that in metal assessment, the main informative characteristics of hardness testing process are its average value and mean-square deviation for measures of location and scattering, respectively.

  12. Independent component analysis for improving the quality of interferometric products

    NASA Astrophysics Data System (ADS)

    Saqellari Likoka, A.; Vafeiadi-Bila, E.; Karathanassi, V.

    2016-05-01

    The accuracy of InSAR DEMs is affected by the temporal decorrelation of SAR images which is due to atmosphere, land use/cover, soil moisture, and roughness changes. Elimination of the temporal decorrelation of the master and slave image improves the DEMs accuracy. In this study, the Independent Component Analysis was applied before interferometric process. It was observed that using three ICA entries, ICA independent sources can be interpreted as background and changed images. ICA when performed on the master and slave images using the same couple of additional images produces two background images which enable the production of high quality DEMs. However, limitations exist in the proposed approach.

  13. A New Medium for Improving Spinosad Production by Saccharopolyspora spinosa

    PubMed Central

    Guojun, Yang; Yuping, He; Yan, Jiang; Kaichun, Lin; Haiyang, Xia

    2016-01-01

    Background Spinosad (a mixture of spinosyns A and D) is a unique natural pesticide produced by Saccharopolyspora spinosa. With regard to attempts to improve S. spinosa by classical mutagenesis, we propose that the bottleneck of screening out high-spinosad-production strains is probably caused by the fermentation media. Objectives The current study aimed to identify a new medium to extensively investigate the potential of S. spinosa strains to produce spinosad. Materials and Methods Statistical and regressive modeling methods were used to investigate the effects of the carbon source and to optimize the production media. Results The spinosad production of S. spinosa Co121 increased 77.13%, from 310.44 ± 21.84 μg/mL in the initial fermentation medium (with glucose as the main carbon source) to 549.89 ± 38.59 μg/mL in a new optimized fermentation medium (98.0 g of mannitol, 43.0 g of cottonseed flour, 12.9 g of corn steep liquor, 0.5 g of KH2PO4, and 3.0 g of CaCO3 in 1 L of H2O; pH was adjusted to 7.0 before autoclaving). After screening 4,000 strains, an overall 3.33-fold increase was observed in spinosad titers, starting from the parental strain Co121 in the original fermentation medium and ending with the mutant strain J78 (1035 ± 34 μg/mL) in the optimized medium. Conclusions The optimized fermentation medium developed in this study can probably be used to improve spinosad production in screening industrial strains of S. spinosa. PMID:27635207

  14. A New Medium for Improving Spinosad Production by Saccharopolyspora spinosa

    PubMed Central

    Guojun, Yang; Yuping, He; Yan, Jiang; Kaichun, Lin; Haiyang, Xia

    2016-01-01

    Background Spinosad (a mixture of spinosyns A and D) is a unique natural pesticide produced by Saccharopolyspora spinosa. With regard to attempts to improve S. spinosa by classical mutagenesis, we propose that the bottleneck of screening out high-spinosad-production strains is probably caused by the fermentation media. Objectives The current study aimed to identify a new medium to extensively investigate the potential of S. spinosa strains to produce spinosad. Materials and Methods Statistical and regressive modeling methods were used to investigate the effects of the carbon source and to optimize the production media. Results The spinosad production of S. spinosa Co121 increased 77.13%, from 310.44 ± 21.84 μg/mL in the initial fermentation medium (with glucose as the main carbon source) to 549.89 ± 38.59 μg/mL in a new optimized fermentation medium (98.0 g of mannitol, 43.0 g of cottonseed flour, 12.9 g of corn steep liquor, 0.5 g of KH2PO4, and 3.0 g of CaCO3 in 1 L of H2O; pH was adjusted to 7.0 before autoclaving). After screening 4,000 strains, an overall 3.33-fold increase was observed in spinosad titers, starting from the parental strain Co121 in the original fermentation medium and ending with the mutant strain J78 (1035 ± 34 μg/mL) in the optimized medium. Conclusions The optimized fermentation medium developed in this study can probably be used to improve spinosad production in screening industrial strains of S. spinosa.

  15. Improvements in Production of Single-Walled Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Balzano, Leandro; Resasco, Daniel E.

    2009-01-01

    A continuing program of research and development has been directed toward improvement of a prior batch process in which single-walled carbon nanotubes are formed by catalytic disproportionation of carbon monoxide in a fluidized-bed reactor. The overall effect of the improvements has been to make progress toward converting the process from a batch mode to a continuous mode and to scaling of production to larger quantities. Efforts have also been made to optimize associated purification and dispersion post processes to make them effective at large scales and to investigate means of incorporating the purified products into composite materials. The ultimate purpose of the program is to enable the production of high-quality single-walled carbon nanotubes in quantities large enough and at costs low enough to foster the further development of practical applications. The fluidized bed used in this process contains mixed-metal catalyst particles. The choice of the catalyst and the operating conditions is such that the yield of single-walled carbon nanotubes, relative to all forms of carbon (including carbon fibers, multi-walled carbon nanotubes, and graphite) produced in the disproportionation reaction is more than 90 weight percent. After the reaction, the nanotubes are dispersed in various solvents in preparation for end use, which typically involves blending into a plastic, ceramic, or other matrix to form a composite material. Notwithstanding the batch nature of the unmodified prior fluidized-bed process, the fluidized-bed reactor operates in a continuous mode during the process. The operation is almost entirely automated, utilizing mass flow controllers, a control computer running software specific to the process, and other equipment. Moreover, an important inherent advantage of fluidized- bed reactors in general is that solid particles can be added to and removed from fluidized beds during operation. For these reasons, the process and equipment were amenable to

  16. Improving of Aura Microwave Limb Sounder Data Products

    NASA Astrophysics Data System (ADS)

    Cuddy, D.; Wagner, P.; Read, W.; Livesey, N. J.; Martinez, E.

    2011-12-01

    The Microwave Limb Sounder (MLS) on NASA's Aura satellite began collecting atmospheric data in August of 2004, and the MLS Science Investigator-led Processing System (SIPS) processes the raw data to calibrated radiances and the 20 different geophysical parameters. Currently, SIPS provides two versions (V2 and V3) of these data products, and Goddard Earth Science Data and Information Service Centers (GES-DISC) archives and provides them to the user community. This paper will describe the current plans by the MLS Science Team (MST) to improve the V2 and V3 algorithms, and at the top of the list are how to ameliorate the issue with oscillations in the upper troposphere/lower stratosphere (UT/LS) ozone and improve behavior of UT/LS species in thick cloud. Other improvements include: removing adverse cloud interactions in some products (e.g. CO) that now occur in V3, ideally better still with the new cloud forward model; work to further reduce biases in 640 GHz species; extend species to lower altitude (including potentially those at 190 GHz); consider joint retrievals spanning multiple radiometers (e.g., joint 190/640 GHZ ClO to get methanol independently); better HCN lower down using a separate phase (q.v. 190 GHz goal above); and gain better understanding of hydrostatic / pressure inconsistency in Band 1. This paper will also discuss usability improvement such as TAI93 at 0Z of granule, day boundary discontinuities, and extending the data format to be compatible with NetCDF (network Common Data Form) that supports a machine-independent format for representing scientific data and is widely used in the community.

  17. Implementing quality/productivity improvement initiatives in an engineering environment

    NASA Technical Reports Server (NTRS)

    Ruda, R. R.

    1985-01-01

    Quality/Productivity Improvement (QPI) initiatives in the engineering environment at McDonnell Douglas-Houston include several different, distinct activities, each having its own application, yet all targeted toward one common goal - making continuous improvement a way of life. The chief executive and the next two levels of management demonstrate their commitment to QPI with hands-on involvement in several activities. Each is a member of a QPI Council which consists of six panels - Participative Management, Communications, Training, Performance/Productivity, Human Resources Management and Strategic Management. In addition, each manager conducts Workplace Visits and Bosstalks, to enhance communications with employees and to provide a forum for the identification of problems - both real and perceived. Quality Circles and Project Teams are well established within McConnel Douglas as useful and desirable employee involvement teams. The continued growth of voluntary membership in the circles program is strong evidence of the employee interest and management support that have developed within the organization.

  18. Baculovirus infection triggers a positive phototactic response in caterpillars to induce 'tree-top' disease.

    PubMed

    van Houte, Stineke; van Oers, Monique M; Han, Yue; Vlak, Just M; Ros, Vera I D

    2014-12-01

    Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions ('tree-top' disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.

  19. Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

    PubMed

    Nadala, E C; Tapay, L M; Cao, S; Loh, P C

    1997-12-01

    The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

  20. Hesperidinase encapsulation towards hesperitin production targeting improved bioavailability.

    PubMed

    Furtado, Andreia F M; Nunes, Mario A P; Ribeiro, Maria H L

    2012-11-01

    Hesperidin (hesperitin-7-O-rutinoside) and hesperitin (hesperitin-7-O-glucoside) show anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic effects and prevent bone loss. However, hesperidin has a low bioavailability compared to hesperitin due to the rutinoside moiety attached to the flavonoid. The removal of the rhamnose group to yield the corresponding flavonoid glucoside (hesperetin-7-glucoside) improved the bioavailability of the aglycone, hesperetin, in humans. In line with these assumptions, the aim of this work was the enzymatic production of hesperitin from hesperidin with hesperidinase. Despite the low hesperidin solubility in the reaction medium, the enzymatic bioconversion was carried with hesperidin soluble at lower concentrations (≤0.05 mg ml(-1)) and insoluble for high concentrations (>0.1-50 mg ml(-1)). A twofold increase in maximum reaction rates overtook the expected values, pointing to the enzyme ability to degrade insoluble hesperidin. To improve the bioprocess, hesperidinase was tested soluble and immobilized in calcium alginate (2%), k-carrageenan (2%), and chitosan (2%) beads. The immobilization was carried out by adsorption and encapsulation. Chitosan was cross-linked with glutaraldehyde (1% and 2%) and sodium sulfate (13.5% and 15%) in acetate buffer (0.02 M, pH 4.0). The relation between bioprocessing conditions and hesperidinase stability was studied. A residual activity of 193% was obtained with immobilized hesperidinase compared to the soluble form. A half-life of 770 min was attained with hesperidinase encapsulated in calcium alginate beads. The results presented in this work highlight the potential of hesperidinase encapsulation towards hesperitin production with insoluble substrate. To our knowledge, this work presents for the first time the potential of hesperidinase encapsulation on hydrogels for hesperitin production. This is an important achievement for pharmaceutical and nutraceutical applications of hesperitin

  1. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    SciTech Connect

    Koener, J.F.; Leong, J.A.C. )

    1990-01-01

    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  2. Improving Access to MODIS Biophysical Science Products for NACP Investigators

    NASA Technical Reports Server (NTRS)

    Wolfe, Robert E.; Gao, Feng; Morisette, Jeffrey T.; Ederer, Gregory A.; Pedelty, Jeffrey A.

    2007-01-01

    MODIS 4 NACP is a NASA-funded project supporting the North American Carbon Program (NACP). The purpose of this Advancing Collaborative Connections for Earth-Sun System Science (ACCESS) project is to provide researchers with Moderate Resolution Imaging Spectroradiometer (MODIS) biophysical data products that are custom tailored for use in NACP model studies. Standard MODIS biophysical products provide used to improve our understanding on the climate and ecosystem changes. However, direct uses of the MODIS biophysical parameters are constrained by retrieval quality and cloud contamination. Another challenge that NACP users face is acquiring MODIS data in formats and at spatial-temporal resolutions consistent with other data sets they use. We have been working closely with key NACP users to tailor the MODIS products to fit their needs. First, we provide new temporally smoothed and spatially continuous MODIS biophysical data sets. Second, we are distributing MODIS data at suitable spatial-temporal resolutions and in formats consistent with other data integration into model studies.

  3. Fisher Pierce products for improving distribution system reliability

    SciTech Connect

    1994-12-31

    The challenges facing the electric power utility today in the 1990s has changed significantly from those of even 10 years ago. The proliferation of automation and the personnel computer have heightened the requirements and demands put on the electric distribution system. Today`s customers, fighting to compete in a world market, demand quality, uninterrupted power service. Privatization and the concept of unregulated competition require utilities to streamline to minimize system support costs and optimize power delivery efficiency. Fisher Pierce, serving the electric utility industry for over 50 years, offers a line of products to assist utilities in meeting these challenges. The Fisher Pierce Family of products provide tools for the electric utility to exceed customer service demands. A full line of fault indicating devices are offered to expedite system power restoration both locally and in conjunction with SCADA systems. Fisher Pierce is the largest supplier of roadway lighting controls, manufacturing on a 6 million dollar automated line assuring the highest quality in the world. The distribution system capacitor control line offers intelligent local or radio linked switching control to maintain system voltage and Var levels for quality and cost efficient power delivery under varying customer loads. Additional products, designed to authenticate revenue metering calibration and verify on sight metering service wiring, help optimize the profitability of the utility assuring continuous system service improvements for their customers.

  4. Improved sustainability of feedstock production with sludge and interacting mycorrhiza.

    PubMed

    Seleiman, Mahmoud F; Santanen, Arja; Kleemola, Jouko; Stoddard, Frederick L; Mäkelä, Pirjo S A

    2013-05-01

    Recycling nutrients saves energy and improves agricultural sustainability. Sewage sludge contains 2.6% P and 3.1% N, so the availability of these nutrients was investigated using four crops grown in either soil or sand. Further attention was paid to the role of mycorrhiza in improvement of nutrient availability. The content of heavy metals and metalloids in the feedstock was analyzed. Sewage sludge application resulted in greater biomass accumulation in ryegrass than comparable single applications of either synthetic fertilizer or digested sludge. Sewage sludge application resulted in more numerous mycorrhizal spores in soil and increased root colonization in comparison to synthetic fertilizer. All plants studied had mycorrhizal colonized roots, with the highest colonization rate in maize, followed by hemp. Sewage sludge application resulted in the highest P uptake in all soil-grown plants. In conclusion, sewage sludge application increased feedstock yield, provided beneficial use for organic wastes, and contributed to the sustainability of bioenergy feedstock production systems. It also improves the soil conditions and plant nutrition through colonization by mycorrhizal fungi as well as reducing leaching and need of synthetic fertilizers.

  5. Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1

    PubMed Central

    Seshagiri, Somasekar; Miller, Lois K.

    1997-01-01

    We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1. PMID:9391073

  6. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  7. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    PubMed Central

    Chateigner, Aurélien; Bézier, Annie; Labrousse, Carole; Jiolle, Davy; Barbe, Valérie; Herniou, Elisabeth A.

    2015-01-01

    Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential. PMID:26198241

  8. Quality and productivity improvement program (PPKP) from alumni perspective

    NASA Astrophysics Data System (ADS)

    Ruza, Nadiah; Mustafa, Zainol

    2013-04-01

    Defining the quality of the university education system is not easy. Institutions of higher education, through curriculum are hoped to provide the knowledge, wisdom and personality of students. It is questionable of how far Quality and Productivity Improvement Program (PPKP) are capable to ensure the courses offered relevant and effective in preparing the students for job market. The effectiveness of a university to undertake responsibilities and the impact given to students even after they graduate can be a measure of education quality at university. So, the quality of education can be enhanced and improved from time to time. In general, this study is aims to determine the effectiveness of PPKP's education system from the perspective of their alumni as well as their satisfaction and the importance level based on how PPKP be able to meet their needs. In overall, summary of open-ended questions from the questionnaire, Importance-Performance analysis and correlation analysis were conducted for this study. Based on result, it appears that there are still some deficiencies that can be improve, particularly in terms of teaching skills and PPKP's relationships with external organizations to enable knowledge be channel effectively. Importance-Performance analysis highlights some topics or courses that should be offered by PPKP based on their importance in industrial practice. Summary of the results of correlation analysis was found that women are more positive and not too demanding compared to men. In addition, it is found that the responsibilities and workload of the older generations, higher income and a high level of experience demands them to use and practice what they have learned during their studies at PPKP. Results of this study are hoped could be used to improve the quality of education system at PPKP.

  9. Improvements of soil quality for increased food production in Norway

    NASA Astrophysics Data System (ADS)

    Øygarden, Lillian; Klakegg, Ove; Børresen, Trond; Krogstad, Tore; Kjersti Uhlen, Anne

    2016-04-01

    Since the 1990ties, agricultural land in use in Norway has diminished and yields per hectare for cereals and forages have stagnated. An expert panel appointed to advice on how to increase Norwegian grain production emphasizes low profitability and poor soil quality as limiting factors. A White Paper from the Norwegian Government, Report No.9 (2011-2012), stated that the main goal for the agricultural sector is to increase food production proportional to the expected increase in population (20 % by 2030) in order to maintain self-sufficiency at the present level. This is the background for the interdisciplinary project AGROPRO "Agronomy for increased food production - Challenges and solutions" (2013 - 2017)" financed by the Norwegian research council. A mail goal is seeking possibilities for improvements in agronomic practices for increased and sustainable food production and to identify drivers and challenges for their implementation. Are the key to higher yields hidden in the soil? The paper present an overview of the research activities in the project and some results of the improvements of soil quality to minimize yield gap in cereal and forage production. Detailed new soil maps provide soil information on field scale of soil quality and the suitability for growing different crops like cereal production or vegetables. The detailed soil information is also beeing used for development and adaptation of the planning tool «Terranimo» to reduce risk of soil compaction.The farmer get available soil information for each field, provide information about the maschinery in use- tractors and equipment, tyres, pressure. The decision tool evaluate when the soil is suitable for tillage, calculate the risk of compaction for dry, moist and wet soil. New research data for compaction on Norwegian clay and silt soil are included. Climate change with wetter conditions gives challenges for growing cereals. The project is testing genetic variation in cereals for tolerance to water

  10. Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

    PubMed

    Yee, C J; DeFrances, M C; Bell, A; Bowen, W; Petersen, B; Michalopoulos, G K; Zarnegar, R

    1993-08-10

    A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by

  11. Improved Element Production Networks for Type Ia Supernova Simulations

    NASA Astrophysics Data System (ADS)

    Chupryna, Viktor; Budiardja, Reuben; Guidry, Mike

    2004-11-01

    The cosmological implications of Type Ia supernovae depend crucially on their assumed standardizable candle properties. Therefore it is highly desirable to understand the detailed mechanism of the Ia supernova explosion from a fundamental point of view. There is some consensus that Type Ia supernovae result when a white dwarf in a binary star system is driven to the Chandrasekhar limit by accretion from a companion star, with the resulting instability triggering a thermonuclear runaway that burns most of the white dwarf to iron and nickel. However, the details of this mechanism are very poorly understood. The energy released in the supernovae comes primarily from the element and energy production network that powers the thermonuclear flash, but in most simulations of Ia explosions this network and its coupling to the hydrodynamics are treated only in an approximate fashion. In this presentation we shall discuss our current efforts to incorporate an improved description of energy generation networks coupled to hydrodynamics in Type Ia supernova simulations.

  12. Natural Product Discovery through Improved Functional Metagenomics in Streptomyces.

    PubMed

    Iqbal, Hala A; Low-Beinart, Lila; Obiajulu, Joseph U; Brady, Sean F

    2016-08-01

    Because the majority of environmental bacteria are not easily culturable, access to many bacterially encoded secondary metabolites will be dependent on the development of improved functional metagenomic screening methods. In this study, we examined a collection of diverse Streptomyces species for the best innate ability to heterologously express biosynthetic gene clusters. We then optimized methods for constructing high quality metagenomic cosmid libraries in the best Streptomyces host. An initial screen of a 1.5 million-membered metagenomic library constructed in Streptomyces albus, the species that exhibited the highest propensity for heterologous expression of gene clusters, led to the identification of the novel natural product metatricycloene (1). Metatricycloene is a tricyclic polyene encoded by a reductive, iterative polyketide-like gene cluster. Related gene clusters found in sequenced genomes appear to encode a largely unexplored collection of structurally diverse, polyene-based metabolites. PMID:27447056

  13. Productivity improvements in longhole stoping with repumpable emulsions

    SciTech Connect

    Thorpe, R.

    1994-12-31

    Bulk emulsion explosives have been used at surface mines and quarries for many years. Recently, this technology has been adapted for underground use in relatively confined work areas and in smaller diameter blastholes. From November 1992 to April 1993, approximately 20 tons of emulsion was successfully pumped and blasted in longhole stopes at Westmin`s HW Mine. This demonstration was undertaken to determine if the use of repumpable emulsions could improve productivity compared to conventional loading. Hole diameters were 54 mm, 76 mm and 89 mm, up to 24 m in length, and oriented from vertically up to vertically down. In each ring of holes, the collars were staggered to maintain a constant powder factor. Compared to gravity or pneumatically loading ANFO or cartridged explosives, repumpable emulsions have been loaded two to four times faster, in stoping operations. Other factors considered in this program were operating costs, physical effort required of the loading crew, efficiency, waste control and safety.

  14. CAD/CAM approach to improving industry productivity gathers momentum

    NASA Technical Reports Server (NTRS)

    Fulton, R. E.

    1982-01-01

    Recent results and planning for the NASA/industry Integrated Programs for Aerospace-Vehicle Design (IPAD) program for improving productivity with CAD/CAM methods are outlined. The industrial group work is being mainly done by Boeing, and progress has been made in defining the designer work environment, developing requirements and a preliminary design for a future CAD/CAM system, and developing CAD/CAM technology. The work environment was defined by conducting a detailed study of a reference design process, and key software elements for a CAD/CAM system have been defined, specifically for interactive design or experiment control processes. Further work is proceeding on executive, data management, geometry and graphics, and general utility software, and dynamic aspects of the programs being developed are outlined

  15. Lean production of taste improved lipidic sodium benzoate formulations.

    PubMed

    Eckert, C; Pein, M; Breitkreutz, J

    2014-10-01

    Sodium benzoate is a highly soluble orphan drug with unpleasant taste and high daily dose. The aim of this study was to develop a child appropriate, individually dosable, and taste masked dosage form utilizing lipids in melt granulation process and tableting. A saliva resistant coated lipid granule produced by extrusion served as reference product. Low melting hard fat was found to be appropriate as lipid binder in high-shear granulation. The resulting granules were compressed to minitablets without addition of other excipients. Compression to 2mm minitablets decreased the dissolved API amount within the first 2 min of dissolution from 33% to 23%. The Euclidean distances, calculated from electronic tongue measurements, were reduced, indicating an improved taste. The reference product showed a lag time in dissolution, which is desirable for taste masking. Although a lag time was not achieved for the lipidic minitablets, drug release in various food materials was reduced to 2%, assuming a suitable taste masking for oral sodium benzoate administration. PMID:24859305

  16. Genetic approaches to improvement of alcohol production by Zymomonas mobilis

    SciTech Connect

    Buchholz, S.E.

    1987-01-01

    A single spontaneous mutant of Z. mobilis was isolated which was capable of feeble growth on mannitol as the sole carbohydrate source. Several months of continuous culture, including addition of a mutagen to a chemostat, led to the isolation of a sequential series of mutants, each with improved growth rates on mannitol. Metabolism of mannitol is oxygen-dependent, resulting in limited production of ethanol and increased production of lactic acid. The conversion of mannitol to fructose is apparently via an altered alcohol dehydrogenase. Analogously, for development of another mutant series, very limited growth of Z. mobilis has been obtained on raffinose after extended incubation in shake flasks. Z. mobilis containing the lactose operon fails to grow on lactose. A single plasmid carrying both the lactose and galactose operons was constructed and introduced into Z. mobilis CP4.45, followed by mutation to yield a culture with slow growth on lactose. Z. mobilis SB6 is capable of producing 0.25% ethanol from 5% lactose in 15 days.

  17. Innovative applications of technology for nuclear power plant productivity improvements

    SciTech Connect

    Naser, J. A.

    2012-07-01

    The nuclear power industry in several countries is concerned about the ability to maintain high plant performance levels due to aging and obsolescence, knowledge drain, fewer plant staff, and new requirements and commitments. Current plant operations are labor-intensive due to the vast number of operational and support activities required by commonly used technology in most plants. These concerns increase as plants extend their operating life. In addition, there is the goal to further improve performance while reducing human errors and increasingly focus on reducing operations and maintenance costs. New plants are expected to perform more productively than current plants. In order to achieve and increase high productivity, it is necessary to look at innovative applications of modern technologies and new concepts of operation. The Electric Power Research Inst. is exploring and demonstrating modern technologies that enable cost-effectively maintaining current performance levels and shifts to even higher performance levels, as well as provide tools for high performance in new plants. Several modern technologies being explored can provide multiple benefits for a wide range of applications. Examples of these technologies include simulation, visualization, automation, human cognitive engineering, and information and communications technologies. Some applications using modern technologies are described. (authors)

  18. Improved oxides for production of lead/acid battery plates

    NASA Astrophysics Data System (ADS)

    Boden, D. P.

    For many years, the plates of lead/acid batteries have been produced from leady oxide, a mixture of finely divided lead (`free-lead') and lead monoxide. Although this material is generally satisfactory, it suffers from the disadvantages that it is variable in composition and requires complicated and lengthy processing after pasting to remove the residual free-lead. Plates made from leady oxide also require cycling before they achieve their full performance, and this can result in either depressed initial capacity or additional processing cost. There is a growing trend towards the use of pure lead monoxide ( β-PbO) for the production of positive plates. This material is particularly valuable in valve-regulated batteries where cell-to-cell uniformity is essential for proper control of battery performance. It also reduces processing cost since it does not require time-consuming curing to remove free-lead. Red lead (Pb 3O 4) is also being more widely used in industrial batteries since it reduces formation time, and improves initial and high-rate performance. The methods of production of leady oxide, β-PbO and red lead are briefly reviewed and the characteristics of battery-grade materials are described. Particular emphasis is placed on optimum particle-size distribution, and how this can affect the battery performance. The benefits in processing and performance are described together with information on how pure litharge and red lead are used in battery plates.

  19. Improving biohydrogen production using Clostridium beijerinckii immobilized with magnetite nanoparticles.

    PubMed

    Seelert, Trevor; Ghosh, Dipankar; Yargeau, Viviane

    2015-05-01

    In order to supplement the need for alternative energy resources within the near future, enhancing the production of biohydrogen with immobilized Clostridium beijerinckii NCIMB8052 was investigated. Magnetite nanoparticles were functionalized, with chitosan and alginic acid polyelectrolytes using a layer-by-layer method, to promote bacterial attachment. Cultivating C. beijerinckii with these nanoparticles resulted in a shorter lag growth phase and increased total biohydrogen production within 100-ml, 250-ml and 3.6-L reactors compared with freely suspended organisms. The greatest hydrogen yield was obtained in the 250-ml reactor with a value of 2.1 ± 0.7 mol H2/mol glucose, corresponding to substrate conversion and energy conversion efficiencies of 52 ± 18 and 10 ± 3 %, respectively. The hydrogen yields obtained using the immobilized bacteria are comparable to values found in literature. However, to make this process viable, further improvements are required to increase the substrate and energy conversion efficiencies.

  20. Serial CSTR digester configuration for improving biogas production from manure.

    PubMed

    Boe, Kanokwan; Angelidaki, Irini

    2009-01-01

    A new configuration of manure digesters for improving biogas production has been investigated in laboratory scale. A single thermophilic continuous-flow stirred tank reactor (CSTR) operated with a hydraulic retention time (HRT) of 15 days was compared to a serial CSTR configuration with volume distribution ratio of 80/20 and 90/10, and total HRT of 15 days. The results showed that the serial CSTR could obtain 11% higher biogas yield compared to the single CSTR. The increased biogas yield in the serial CSTR was mainly from the second reactor, which accounted for 16% and 12% of total biogas yield in the 90/10 and 80/20 configuration, respectively. VFA concentration in the serial CSTR was high in the first reactor but very low in the second reactor. The results from organic pulse load test showed that the second reactor in serial CSTR helped utilizing VFA produced from overloading in the first reactor, which improved the effluent quality and conversion efficiency of the serial CSTR.

  1. Improved production of fatty alcohols in cyanobacteria by metabolic engineering

    PubMed Central

    2014-01-01

    Background Fatty alcohols are widely used in industrial chemicals. The biosynthetic pathways for fatty alcohols are diverse and widely existing in nature. They display a high capacity to produce fatty alcohols by the metabolic engineering of different microbe hosts. Direct recycling of carbon dioxide to fatty alcohols can be achieved by introducing a fatty alcohol-producing pathway into photosynthetic cyanobacteria. According to our precious reports, a relatively low yield of fatty alcohols was obtained in the genetically engineered cyanobacterium Synechocystis sp. PCC 6803. Results The photosynthetic production of fatty alcohols in Synechocystis sp. PCC 6803 was improved through heterologously expressing fatty acyl-Coenzyme A (acyl-CoA) reductase gene maqu_2220 from the marine bacterium Marinobacter aquaeolei VT8. Maqu_2220 has been proved to catalyze both the four-electron reduction of fatty acyl-CoA or acyl-Acyl Carrier Protein (acyl-ACP) and the two-electron reduction of fatty aldehyde to fatty alcohol. The knockout of the aldehyde-deformylating oxygenase gene (sll0208) efficiently blocked the hydrocarbon accumulation and redirected the carbon flux into the fatty alcohol-producing pathway. By knocking-out both sll0208 and sll0209 (encoding an acyl-ACP reductase), the productivity of fatty alcohols was further increased to 2.87 mg/g dry weight. Conclusions The highest yield of fatty alcohols was achieved in cyanobacteria by expressing the prokaryotic fatty acyl-CoA reductase Maqu_2220 and knocking-out the two key genes (sll0208 and sll0209) that are involved in the alka(e)ne biosynthesis pathway. Maqu_2220 was demonstrated as a robust enzyme for producing fatty alcohols in cyanobacteria. The production of fatty alcohols could be significantly increased by blocking the hydrocarbon biosynthesis pathway. PMID:25024742

  2. Environmental report for the Gasification Product Improvement Facility (GPIF)

    SciTech Connect

    Sadowski, R.S.; Skinner, W.H.; Norris, E.S.; Duck, R.R.; Hass, R.B.; Morgan, M.E.; Helble, J.J.; Johnson, S.A.

    1993-01-01

    The Fossil Energy Program has a mission to develop energy systems that utilize national coal resources in power systems with increased efficiency and environmental compatibility. Coal gasification technology is a versatile candidate that meets this goal. This two phased project consists primarily of the design, construction and operation of a 5-foot inside diameter (minimum) fixed-bed gasifier called PyGas{trademark} and supporting infrastructure (Phase I), and an additional follow on phase consisting of the design, construction and operation of a hot fuel gas cleanup unit (Phase II). Issues expected to be successfully overcome by PyGas{trademark} through its application in this test facility include the processing of high-swelling coals, which causes agglomeration in conventional fixed-bed gasifiers. Such coals comprise 87% of all eastern coals. Other issues expected to be eliminated or significantly reduced include: production of ash clinkers, production of ammonia, the presence of significant tars and fines, and the volatilization of alkalinity in the product fuel gas. A second portion of the NEPA report is concerned with the emission of toxic metal compounds by the gasification process improvement facility (GPIF). The GPIF facility will be located on site at the Fort Martin facility of Allegheny Power Company, and the energy produced (steam) will be directly used by Fort Martin to produce electricity. The coal used at the GPIF facility will be the same coal used by the utility. Therefore, the emissions of the GPIF will be put in context of the entire facility. The GPIF assessment will be divided into four sections: Estimation of the toxic metals content of the raw coal; calculation of the emissions from Fort Martin normally; an estimate of the emission from the GPIF; and a comparison of the two flows.

  3. Improvements of AIMS D2DB matching for product patterns

    NASA Astrophysics Data System (ADS)

    Nishiguchi, Masaharu; Kanno, Koichi; Miyashita, Hiroyuki; Ohara, Kana; Son, Donghwan; Tolani, Vikram; Satake, Masaki

    2015-07-01

    AIMSTM is mainly used in photomask industry for verifying the print impact of mask defects on wafer CD in DUV lithography process. AIMS verification is typically used in D2D configuration, wherein two AIMS images, reference and defect, are captured and compared. Criticality of defects is then analyzed off these images using a number of criteria. As photomasks with aggressive OPC, sub-resolution assist features (SRAFs), and single-die are being routinely manufactured in production environment, it is required to improve cycle time through the AIMS step by saving time in searching for and capturing an adequate reference AIMS image. One solution is to use AIMS D2DB methodology which compares AIMS defect image with a reference image simulated from the corresponding mask design data. In general, such simulation needs calibration with the native images captured on the AIMS tool. In our previous paper we evaluated a calibration procedure directly using the defect AIMS image and compared the analysis results with a D2D capture using AIA (Aerial Image Analyzer) software product from Luminescent Technologies (now part of KLA-Tencor Corporation). The results showed that calibration using defect AIMS image does not influence AIMS judgment as long as the defect size is less than 100nm in case of typical basic patterns. When applying this methodology to product patterns, it was found that there were differences between reference AIMS image and simulation image. These differences influenced AIMS verification. Then new method to compensate would be needed. Our approach to compensate the difference between AIMS image and simulated image is examination with some factors likely to cause the difference.

  4. Bacon Production: Evaluating Potential Processing and Management Practices to Improve Product Quality of Industrial Sliced Bacon

    ERIC Educational Resources Information Center

    Scramlin, Stacy Maurine

    2009-01-01

    The objective of this research was to determine areas of improvement to bacon production. The first trial was conducted to determine differences in belly and bacon quality traits in pigs fed ractopamine (RAC) for various durations during finishing. A 2x3x2 factorial arrangement was used with barrows and gilts, fed RAC levels of 0.0, 5.0, or 7.4…

  5. Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

    PubMed

    Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gravisaco, María José; Lucero, María Soledad; Richetta, Matías; Gómez, Evangelina; Berinstein, Analía

    2016-07-01

    Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases. PMID:27063861

  6. M-C Power`s product design and improvement

    SciTech Connect

    Laurens, R.M.; Petraglia, V.J.

    1995-08-01

    The sole mission of M-C Power is the development and subsequent commercialization of molten carbonate fuel cell (MCFC) stacks. These MCFC stacks are based on the Internally Manifolded Heat EXchanger plate design developed by the Institute of Gas Technology. Integration of the MCFC stack into a commercially viable power plant is the mission of the IMHEX{reg_sign} team. The team is composed of leaders in the packaging and design of power generation equipment, including fuel cell technology, and includes Stewart & Stevenson, Bechtel, The Institute of Gas Technology and M-C Power. In an effort to succeed in their respective missions, M-C Power and the IMHEX{reg_sign} team have developed a commercialization program. At the present time the team is making the transition from Phase I (Technology Development) to Phase II (Product Design & Improvement) of the program. Phase II`s objective is a commercially viable (cost effective and technologically reliable) MCFC power plant ready for market by the turn of the century.

  7. Digital Radiography and Computed Tomography (DRCT) Product Improvement Plan (PIP)

    SciTech Connect

    Tim Roney; Bob Pink; Karen Wendt; Robert Seifert; Mike Smith

    2010-12-01

    The Idaho National Laboratory (INL) has been developing and deploying x-ray inspection systems for chemical weapons containers for the past 12 years under the direction of the Project Manager for Non-Stockpile Chemical Materiel (PMNSCM). In FY-10 funding was provided to advance the capabilities of these systems through the DRCT (Digital Radiography and Computed Tomography) Product Improvement Plan (PIP), funded by the PMNSCM. The DRCT PIP identified three research tasks; end user study, detector evaluation and DRCT/PINS integration. Work commenced in February, 2010. Due to the late start and the schedule for field inspection of munitions at various sites, it was not possible to spend sufficient field time with operators to develop a complete end user study. We were able to interact with several operators, principally Mr. Mike Rowan who provided substantial useful input through several discussions and development of a set of field notes from the Pueblo, CO field mission. We will be pursuing ongoing interactions with field personnel as opportunities arise in FY-11.

  8. Vitreoscilla hemoglobin gene ( vgb) improves lutein production in Chlorella vulgaris

    NASA Astrophysics Data System (ADS)

    Ma, Ruijuan; Lin, Xiangzhi

    2014-03-01

    Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene ( vgb) into Chlorella vulgaris by Agrobacterium tumefaciens -mediated transformation (ATMT). PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.

  9. Chrysanthemum Cutting Productivity and Rooting Ability Are Improved by Grafting

    PubMed Central

    Zhang, Jing; Chen, Sumei; Liu, Ruixia; Jiang, Jiafu; Chen, Fadi; Fang, Weimin

    2013-01-01

    Chrysanthemum has been commercially propagated by rooting of cuttings, whereas the quality will decline over multiple collections from a single plant. Therefore, we compared the vigour, rooting ability, and some physiological parameters between cuttings harvested from nongrafted “Jinba” (non-grafted cuttings) with those collected from grafted “Jinba” plants onto Artemisia scoparia as a rootstock (grafted cuttings). The yield, length, node number, stem diameter, fresh weight, and dry weight of the grafted cuttings were superior to the non-grafted cuttings. Also grafted cuttings “Jinba” rooted 1 day earlier, but showing enhanced rooting quality including number, length, diameter, and dry weight of roots, where compared to the non-grafted. The physiological parameters that indicated contents of soluble protein, peroxidase activity, soluble sugar, and starch, ratios of soluble sugar/nitrogen ratio, and carbohydrate/nitrogen (C/N), as well as contents of indole-3-acetic acid (IAA) and abscisic acid (ABA), and IAA/ABA ratio were significantly increased in the grafted cuttings. This suggested their important parts in mediating rooting ability. Results from this study showed that grafting improved productivity and rooting ability related to an altered physiology, which provide a means to meet the increasing demand. PMID:23878523

  10. Attached cultivation for improving the biomass productivity of Spirulina platensis.

    PubMed

    Zhang, Lanlan; Chen, Lin; Wang, Junfeng; Chen, Yu; Gao, Xin; Zhang, Zhaohui; Liu, Tianzhong

    2015-04-01

    To improve cultivation efficiency for microalgae Spirulina platensis is related to increase its potential use as food source and as an effective alternative for CO2 fixation. The present work attempted to establish a technique, namely attached cultivation, for S. platensis. Laboratory experiments were made firstly to investigate optimal conditions on attached cultivation. The optimal conditions were found: 25 g m(-2) for initial inoculum density using electrostatic flocking cloth as substrata, light intensity lower than 200 μmol m(-2) s(-1), CO2 enriched air flow (0.5%) at a superficial aeration rate of 0.0056 m s(-1) in a NaHCO3-free Zarrouk medium. An outdoor attached cultivation bench-scale bioreactor was built and a 10d culture of S. platensis was carried out with daily harvesting. A high footprint areal biomass productivity of 60 g m(-2) d(-1) was obtained. The nutrition of S. platensis with attached cultivation is identical to that with conventional liquid cultivation. PMID:25647023

  11. Method and apparatus for improving oil production in oil wells

    SciTech Connect

    Vogen, W.V.

    1987-06-23

    This patent describes a method for improving the production of oil from appropriate wells, comprising: attaching the lower end of an elastic steel column to the upper end of a liner; the upper end of the column extending to the top of the well and above; attaching the upper end of the column to a reaction mass vertically above through vertically mounted compression spring means and, in parallel with a vertically mounted servo-controlled hydraulic cylinder-piston assembly; applying a substantially constant upward load to the reactions mass; reciprocating the piston of the hydraulic cylinder under servo control to apply vertical vibration to the upper end of the column with resultant vertical displacement of the upper end of the column; developing a displacement signal while developing an electrical, pressure-differential signal corresponding to the pressure across the cylinder-piston assembly; adjusting the vertical vibration through the servo control in accordance with the displacement signal and the pressure differential signal: to seek and find an appropriate resonant frequency for the column in the range of 5 Hz to 25 Hz; and maintaining the frequency at resonance.

  12. Chrysanthemum cutting productivity and rooting ability are improved by grafting.

    PubMed

    Zhang, Jing; Chen, Sumei; Liu, Ruixia; Jiang, Jiafu; Chen, Fadi; Fang, Weimin

    2013-01-01

    Chrysanthemum has been commercially propagated by rooting of cuttings, whereas the quality will decline over multiple collections from a single plant. Therefore, we compared the vigour, rooting ability, and some physiological parameters between cuttings harvested from nongrafted "Jinba" (non-grafted cuttings) with those collected from grafted "Jinba" plants onto Artemisia scoparia as a rootstock (grafted cuttings). The yield, length, node number, stem diameter, fresh weight, and dry weight of the grafted cuttings were superior to the non-grafted cuttings. Also grafted cuttings "Jinba" rooted 1 day earlier, but showing enhanced rooting quality including number, length, diameter, and dry weight of roots, where compared to the non-grafted. The physiological parameters that indicated contents of soluble protein, peroxidase activity, soluble sugar, and starch, ratios of soluble sugar/nitrogen ratio, and carbohydrate/nitrogen (C/N), as well as contents of indole-3-acetic acid (IAA) and abscisic acid (ABA), and IAA/ABA ratio were significantly increased in the grafted cuttings. This suggested their important parts in mediating rooting ability. Results from this study showed that grafting improved productivity and rooting ability related to an altered physiology, which provide a means to meet the increasing demand. PMID:23878523

  13. Conceptual design report -- Gasification Product Improvement Facility (GPIF)

    SciTech Connect

    Sadowski, R.S.; Skinner, W.H.; House, L.S.; Duck, R.R.; Lisauskas, R.A.; Dixit, V.J.; Morgan, M.E.; Johnson, S.A.; Boni, A.A.

    1994-09-01

    The problems heretofore with coal gasification and IGCC concepts have been their high cost and historical poor performance of fixed-bed gasifiers, particularly on caking coals. The Gasification Product Improvement Facility (GPIF) project is being developed to solve these problems through the development of a novel coal gasification invention which incorporates pyrolysis (carbonization) with gasification (fixed-bed). It employs a pyrolyzer (carbonizer) to avoid sticky coal agglomeration caused in the conventional process of gradually heating coal through the 400 F to 900 F range. In so doing, the coal is rapidly heated sufficiently such that the coal tar exists in gaseous form rather than as a liquid. Gaseous tars are then thermally cracked prior to the completion of the gasification process. During the subsequent endothermic gasification reactions, volatilized alkali can become chemically bound to aluminosilicates in (or added to) the ash. To reduce NH{sub 3} and HCN from fuel born nitrogen, steam injection is minimized, and residual nitrogen compounds are partially chemically reduced in the cracking stage in the upper gasifier region. Assuming testing confirms successful deployment of all these integrated processes, future IGCC applications will be much simplified, require significantly less mechanical components, and will likely achieve the $1,000/kWe commercialized system cost goal of the GPIF project. This report describes the process and its operation, design of the plant and equipment, site requirements, and the cost and schedule. 23 refs., 45 figs., 23 tabs.

  14. Improving product quality and productivity using better guidelines for concept design

    SciTech Connect

    Hinckley, C.M.; Barkan, P.

    1995-08-01

    The remarkable effectiveness of Japanese practices has led to a growing interest in the US in the development and application of rules and methodologies which attempt to capture design experience. US companies have found unexpected benefits and pitfalls in the application of these rules and methods. In this article, the authors critically examine one of the most widely accepted rules of Design for Manufacturability (DFM): minimize the number of parts. An examination of 240 assemblies and subassemblies has shown that rigid adherence to this rule can lead to unnecessarily complex parts and assembly. Quantitative insights derived from this study have led to a better design goal: minimize and simplify assembly operations. This new rule, which should not be rigidly interpreted, tends to reduce part count, while having the benefit of assuring improved assembly. Another significant advantage of the new design rule is that it results in lower product defect rates as demonstrated by correlations observed for a wide range of products from two different manufacturers. This research links quality to the product concept, enabling a new approach to improving quality at the earliest stages of design.

  15. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

    SciTech Connect

    Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.; Kwang, Jimmy

    2009-09-01

    White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.

  16. Integrating diverse calibration products to improve seismic location

    SciTech Connect

    Schultz, C; Myers, S; Swenson, J; Flanagan, M; Pasyanos, M; Bhattacharyya, J; Dodge, D

    2000-07-17

    The monitoring of nuclear explosions on a global basis requires accurate event locations. As an example, under the Comprehensive Test Ban Treaty, the size of an on-site inspection search area is 1,000 square kilometers or approximately 17 km accuracy assuming a circular area. This level of accuracy is a significant challenge for small events that are recorded using a sparse regional network. In such cases, the travel-time of seismic energy is strongly affected by crustal and upper mantle heterogeneity and large biases can result. This can lead to large systematic errors in location and, more importantly, to invalid error bounds associated with location estimates. Corrections can be developed and integrated to correct for these biases. These path corrections take the form of both three-dimensional model corrections along with three-dimensional empirically based travel time corrections. LLNL is currently working to integrate a diverse set of three-dimensional velocity model and empirical based travel-time products into one consistent and validated calibration set. To perform this task, we have developed a hybrid approach that uses three-dimensional model corrections for a region and then uses reference events when available to improve the path correction. This Bayesian kriging approach uses the best apriori three-dimensional velocity model that is produced for a local region and uses this as a baseline correction. When multiple models are produced for a local region, uncertainties in the models are compared against each other using ground truth data and an optimal model is chosen. We .are in the process of combining three-dimensional models on a region-by-region basis and integrating the uncertainties to form a global correction set. The Bayesian kriging prediction combines this a priori model and its statistics with the empirical calibrations to give an optimal aposteriori calibration estimate. In regions where there is limited or no coverage by reference events the

  17. Production of rice protein by alkaline extraction improves its digestibility.

    PubMed

    Kumagai, Takehisa; Kawamura, Hiroyuki; Fuse, Takao; Watanabe, Toshiyuki; Saito, Yuhi; Masumura, Takehiro; Watanabe, Reiko; Kadowaki, Motoni

    2006-12-01

    Rice seed endosperm has two types of protein bodies (PB). Type I protein body (PB-I) accumulates prolamin and is hard to digest, while type II protein body (PB-II) mainly consists of glutelin, an easily digestible protein. A simple method to process rice protein and improve its digestibility was tested from the viewpoint of its application to food manufacturing. Rice protein prepared by alkaline extraction followed by neutralization sedimentation (AE-RP) was compared with that prepared by starch degradation by alpha-amylase (SD-RP). The crude protein content of AE-RP and SD-RP was 84.7% and 78.2%, respectively. There were no major differences in protein composition among AE-RP, SD-RP and rice flour by SDS-PAGE, except 16 kDa polypeptide. With respect to amino acids, all the groups showed quite similar compositions, although cysteine and methionine were lower in AE-RP. In an in vitro digestion study with pepsin and pancreatin, both the SDS-PAGE analysis of protein pattern and the crude protein content of undigested residue clearly demonstrated that AE-RP has a higher digestibility than SD-RP. To find the cause of the difference in digestibility, the structural property of protein bodies by two production methods was compared using electron microscopy. PB-II of AE-RP was transformed into small, amorphous granules, while that of SD-RP was still kept partial protein body structures. PB-I of AE-RP kept its protein body structure, but produced double layers. From the finding that glutelin-gold was detected by immunochemistry not only in small, amorphous granules but also in PB-I, mainly the cortex layer, in AE-RP, it became clear that PB-I was swollen and fragile as a result of alkali treatment. These results strongly indicate that the improvement in digestibility of AE-RP is a result of the structural change of PB-I and -II caused by alkaline extraction.

  18. Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

    PubMed Central

    Lee, Hee-Jung; Cho, Hansam; Kim, Mi-Gyeong; Heo, Yoon-Ki; Cho, Yeondong; Gwon, Yong-Dae; Park, Ki Hoon; Jin, Hyerim; Kim, Jinyoung; Oh, Yu-Kyoung; Kim, Young Bong

    2015-01-01

    Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×108 copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×109 copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×1010 copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×109 copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses. PMID:25789464

  19. In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter

    PubMed Central

    Luz-Madrigal, Agustín; Clapp, Carmen; Aranda, Jorge; Vaca, Luis

    2007-01-01

    Background Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed in vitro in different mammalian cell lines and in vivo in adult rat retinal vasculature. Results BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, n = 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. In vivo transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, n = 10) in blood vessels, confirming the endothelial phenotype of the transduced cells. Conclusion Taken together, our results indicate that the restricted expression in endothelial cells

  20. Plaque assay of Heliothis zea baculovirus employing a mixed agarose overlay.

    PubMed

    Yamada, K; Maramorosch, K

    1981-01-01

    The nuclear polyhedrosis virus of Heliothis zea has been titrated in Heliothis zea cells by the plaque method, using 1 percent mixed agarose containing a mixture of Seakem and Ultra pure agarose. Visible plaques, formed 8 days postinfection, ranged in diameter from 0.5 to 2 mm. Dose-response experiments indicated that a single particle initiated the formation of a plaque. The titration of Heliothis zea baculovirus by the newly described plaque method provides an accurate technique for the determination of virus concentration.

  1. Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).

    PubMed

    Harder, T C; Osterhaus, A D

    1997-01-20

    A glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids. Phylogenetic analysis gave evidence for a close evolutionary relationship between PhHV-1 and members of the Varicellovirus genus of the alpha-Herpesvirinae and canid herpesvirus in particular. In PhHV-1-infected Crandell feline kidney cells gB is expressed as a 113-kDa glycosylated molecule which is proteolytically cleaved into at least two fragments of 67 and 53-59 kDa apparently forming disulfide-linked heterodimers of 140 kDa. Cell surface expression of PhHV-1 gB was confirmed by FACS analysis. Thus, synthesis and processing of the gB protein of PhHV-1 follows a pattern also observed in other Varicelloviruses. Since the gB protein of herpesviruses, expressed in the baculovirus system, has been shown to be a suitable target for vaccine design, we used this system for expression of PhHV-1 gB. Recombinant (rec) baculovirus-expressed gB was identified as a 105-kDa glycosylated molecule. Proteolytic cleavage into fragments of 62 and 52 kDa was markedly delayed compared to wild-type (wt) gB. Wt and rec gB harbored endoglycosidase H (precursor)- as well as N-glycosidase F-sensitive N-glycans (proteolytic fragments). Baculovirus-expressed gB appeared to be antigenically authentic, since it was recognized in radioimmunoprecipitation and immune peroxidase monolayer assays by PhHV-1-neutralizing seal sera and by gB-specific neutralizing murine monoclonal antibodies. Furthermore, PhHV-1-neutralizing antibodies were induced in mice following immunization with baculovirus-expressed gB, indicating its suitability for incorporation in a candidate vaccine for seals.

  2. Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV.

    PubMed Central

    Mackow, E R; Werner-Eckert, R; Fay, M E; Tao, H; Chen, G

    1993-01-01

    A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization. Images PMID:8386274

  3. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    PubMed

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV.

  4. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    PubMed

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  5. Altering Reservoir Wettability to Improve Production from Single Wells

    SciTech Connect

    W. W. Weiss

    2006-09-30

    Many carbonate reservoirs are naturally fractured and typically produce less than 10% original oil in place during primary recovery. Spontaneous imbibition has proven an important mechanism for oil recovery from fractured reservoirs, which are usually weak waterflood candidates. In some situations, chemical stimulation can promote imbibition of water to alter the reservoir wettability toward water-wetness such that oil is produced at an economic rate from the rock matrix into fractures. In this project, cores and fluids from five reservoirs were used in laboratory tests: the San Andres formation (Fuhrman Masho and Eagle Creek fields) in the Permian Basin of Texas and New Mexico; and the Interlake, Stony Mountain, and Red River formations from the Cedar Creek Anticline in Montana and South Dakota. Solutions of nonionic, anionic, and amphoteric surfactants with formation water were used to promote waterwetness. Some Fuhrman Masho cores soaked in surfactant solution had improved oil recovery up to 38%. Most Eagle Creek cores did not respond to any of the tested surfactants. Some Cedar Creek anticline cores had good response to two anionic surfactants (CD 128 and A246L). The results indicate that cores with higher permeability responded better to the surfactants. The increased recovery is mainly ascribed to increased water-wetness. It is suspected that rock mineralogy is also an important factor. The laboratory work generated three field tests of the surfactant soak process in the West Fuhrman Masho San Andres Unit. The flawlessly designed tests included mechanical well clean out, installation of new pumps, and daily well tests before and after the treatments. Treatments were designed using artificial intelligence (AI) correlations developed from 23 previous surfactant soak treatments. The treatments were conducted during the last quarter of 2006. One of the wells produced a marginal volume of incremental oil through October. It is interesting to note that the field

  6. The Role of Capital in Improving Productivity and Creating Jobs.

    ERIC Educational Resources Information Center

    Carnoy, Martin

    Causes of the significant decrease in productivity growth and dramatic increase in unemployment in the United States since the mid-1960's are examined in order to test the underlying assumption of current economic policies that increasing capital savings and investments will create fuller and more productive employment. Data on trends in…

  7. Cambridge journals blog: Improving feed efficiency in dairy production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Because the cost of feeding animals is one of the greatest expenses in dairy production (40-60% of production costs), research focused on ways to identify and select for animals that are the most efficient at converting feed into milk has greatly expanded during the last decade. The animal Article o...

  8. Improving Knowledge for Green Textile Products: Life Cycle Analysis

    ERIC Educational Resources Information Center

    Nam, Jinhee

    2012-01-01

    Textile products are used heavily every day. The apparel industry is one of the largest industrial polluters, causing damage to both human health and the environment. Despite increasing consumer concern about environmental issues and a growing trend toward supporting sustainable production, consumers are often unable to evaluate accurately which…

  9. Opportunities for improving milk production efficiency in dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing feed costs and the desire to improve environmental stewardship have stimulated renewed interest in improving feed efficiency of livestock, including that of U.S. dairy herds. For instance, USDA cost projections for corn and soybean meal suggest a 20% increase over 2010 pricing for a 16% p...

  10. Opportunities for improving milk production efficiency in dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing feed costs and the desire to improve environmental stewardship have stimulated interest in improving feed efficiency of livestock, including that of U.S. dairy herds. For instance, USDA cost projections for corn and soybean meal suggest a 20% increase over 2010 pricing for a 16% protein ...

  11. Strategies to improve water productivity in a water-stressed future

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the fiscal years 2011 through 2014, ARS scientists and engineers made substantial progress in addressing research problems related to improving water productivity and creating new knowledge, products and outcomes to improve American agricultural production, efficiency of resource use, safety and ...

  12. Summary of strategies for planning Productivity Improvement and Quality Enhancement (PIQE)

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Summary of NASA Strategies for Productivity Improvement and Quality Enhancement respond to NASA's eighth top goal: Establish NASA as a leader in the development and application of advanced technology and management practices which contribute to significant increases in both Agency and national productivity. The Strategies provide the framework for development of the agency-wide Productivity Improvement and Quality Enhancement (PIQE) Plans.

  13. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    SciTech Connect

    Hiraki, Toshiki; Shibayama, Naoya; Yoon, Young-Ho; Yun, Kyung-Mook; Hamamoto, Toshiro; Tame, Jeremy R. H.; Park, Sam-Yong

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  14. How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

    PubMed

    Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

    2010-01-20

    Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

  15. Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells.

    PubMed

    Lo, Wen-Hsin; Chen, Chi-Yuan; Yeh, Chia-Ni; Lin, Chin-Yu; Hu, Yu-Chen

    2011-01-01

    Baculovirus is a promising gene delivery vector and can be titrated by constitutive EGFP expression in HeLa cells, which, however, might interfere with target transgene expression and impart cytotoxicity. Here we constructed Bac-ME accommodating egfp under the inducible metallothionein promoter and Bac-MECB harboring an additional BMP-2 gene. Bac-ME effectively transduced HeLa cells with minimal leaky expression, but expressed EGFP robustly upon induction with ZnSO(4), hence allowing for virus titration by transducing HeLa cells with serially diluted virus, subsequent ZnSO(4) induction and flow cytometry analysis of EGFP-positive cells. The titration protocol enabled the generation of discernable titration curves, determination of transducing titers, and discrimination of the transducing abilities of different virus batches. After titration, cell transduction with pre-determined Bac-ME dose revealed consistent transduction efficiency dependence on the dose, regardless of virus batch and cell type. Bac-MECB was similarly titrated by inducible EGFP expression and used to transduce de-differentiated articular chondrocytes without EGFP induction. BMP-2 expression was proportional to the Bac-MECB dose and promoted cartilage-specific matrix synthesis, implicating the potential of Bac-MECB in restoring chondrocyte differentiation. These data confirmed that regulatable EGFP expression enabled rapid, reliable baculovirus titration without interference with subsequent applications.

  16. Bioactive baculovirus nanohybrids for stent based rapid vascular re-endothelialization

    PubMed Central

    Paul, Arghya; Elias, Cynthia B.; Shum-Tim, Dominique; Prakash, Satya

    2013-01-01

    Present study, for the first time, reports the development of a nanohybridized baculovirus based stent that can locally promote vascular re-endothelialization by efficient delivery of pro-angiogenic vascular endothelial growth factor (Vegf) genes. In vitro data demonstrated rapid expression of functionally active Vegf by the bioactive stent-transduced vascular cells. In vivo site-specific transgene expression was observed at the stented regions of balloon-denuded canine femoral artery, which eventually lead to significant endothelial recovery at the injured sites. A significant reduction in neointima formation (2.23 ± 0.56 mm2 vs 2.78 ± 0.49 mm2 and 3.11 ± 0.23 mm2, p < 0.05; n = 8) and percent stenosis was observed in treated stent group compared to negative control and bare metal stent groups. These findings collectively implicate the potential of this newly developed baculovirus based biotherapeutic stent to ameliorate damaged vascular biology and attenuate re-narrowing of stented artery by inhibiting neointima formation. PMID:23917680

  17. Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp.

    PubMed

    Nadala, E C; Tapay, L M; Loh, P C

    1998-07-30

    A non-occluded baculovirus-like agent recently isolated by this laboratory from moribund Penaeus japonicus shrimps obtained from China and named Chinese baculovirus (CBV) was purified and some of its properties characterized. Under the electron microscope, negatively stained virus particles were rod-shaped, enveloped, and measured 322 to 378 nm in length and 130 to 159 nm in diameter. The nucleoprotein core exhibited a unique striated structure and measured 316 to 350 nm in length and 65 to 66 nm in diameter. The striations appear to be the result of the stacking of ring-like structures. These rings consisted of 2 rows of 12 to 14 globular subunits. Each globular subunit measured approximately 10 nm in diameter. SDS-PAGE gels of purified virus preparations showed, among several, 4 prominent protein bands with approximate molecular weights of 19, 23.5, 27.5 and 75 kDa. The structural viral proteins were identified by western blot analysis using polyclonal hyperimmune serum made against purified CBV. The 19, 27.5, and 75 kDa structural proteins were determined to be non-glycosylated components associated with the viral envelope. The 23.5 kDa protein, also non-glycosylated, was identified with the capsid structure. Viral genomic DNA digested with Hind III restriction endonuclease revealed at least 29 different fragments with a conservatively estimated total size of at least 183 kb.

  18. Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

    PubMed

    Roth, D A; Rehemtulla, A; Kaufman, R J; Walsh, C T; Furie, B; Furie, B C

    1993-09-15

    A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase. PMID:8378308

  19. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

    PubMed

    Jin, J; Dong, W; Guarino, L A

    1998-12-01

    The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity. PMID:9811739

  20. Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy

    PubMed Central

    Ang, Wei Xia; Zhao, Ying; Kwang, Timothy; Wu, Chunxiao; Chen, Can; Toh, Han Chong; Mahendran, Ratha; Esuvaranathan, Kesavan; Wang, Shu

    2016-01-01

    Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene therapy triggered significantly increased bladder infiltrations of inflammatory monocytes, CD4+, CD8+ and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC. PMID:27273619

  1. Improving efficiency of sow productivity: nutrition and health

    PubMed Central

    2013-01-01

    This reviews research focused to understand the nutrient requirement and balance to meet the needs of fetal growth, mammary growth, and milk production. This summary will handle how feeding strategies can be adjusted according to the nutrient needs for a sow to enhance productivity and health. Most research data used in this summary are based on the studies conducted by the authors between 1996 and 2013. Nutrient requirements of sows are affected by stage of gestation and parity of sows. Dietary antioxidant concentrations need to be re-evaluated for its sufficiency in sow diets especially to prevent excessive oxidative stress during late gestation and lactation. When feeding sows, consideration of phase feeding of gestating sows and parity feeding of lactating sows could enhances production longevity and health of sows. Use of selected nutrients and additives seems to help productivity and health of sows. PMID:23885840

  2. Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are microbial agents that affects mosquito and lepidoptera larvae. They are characterized by rod-shaped virions containing circular double-stranded DNA and are the most studied insect viruses, due to their role as biological pesticides. The aim of this study was to assess the occurrenc...

  3. Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to assess the occurrence of baculovirus infections in mosquitoes and characterize them by using molecular tools. Fortnightly collections were made of mosquito larvae in the city of Caraguatatuba. Six larvae of Culex quinquefasciatus were isolated that had white cysts (nodul...

  4. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

    PubMed

    Ge, Jingping; An, Qi; Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  5. Renewable microbial lipid production from Oleaginous Yeast: some surfactants greatly improved lipid production of Rhodosporidium toruloides.

    PubMed

    Xu, Jingyang; Du, Wei; Zhao, Xuebing; Liu, Dehua

    2016-07-01

    Microbial oil is drawing increasing interest worldwide as an alternative non-food oil feedstock for biodiesel industry. Nowadays researchers have been increasingly focused on the improvement of microbial oil production process. Oleaginous yeast Rhodosporidium toruloides (R. toruloides) is considered an important candidate due to its excellent capabilities of lipid accumulation, broad adaptabilities to various carbon substrates, and the potential of co-production of some pigments. In present work, the individual effects of non-ionic, cationic, and anionic surfactant on cell growth and lipid accumulation of R. toruloides were investigated for the first time. Interesting results were noticed when some anionic surfactants were supplemented. The most significant effect was observed with addition of 0.2 % (w/v) sodium lignosulfonate, that biomass concentration, lipid concentration, and lipid yield was increased by 25.1, 44.9, and 15.7 %, respectively. The fatty acid compositions of R. toruloides lipids remained unchanged, which is similar to that of vegetable oils, and is considered potential feedstock for biodiesel preparation. PMID:27263002

  6. Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations.

    PubMed

    Xu, Sen; Hoshan, Linda; Chen, Hao

    2016-11-01

    In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10(6) cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.

  7. Improved predictions for J/psi and Upsilon production

    NASA Astrophysics Data System (ADS)

    Artoisenet, P.

    I discuss J/ψ and ϒ production at the Tevatron. Working in the framework of NRQCD, I review the current theoretical status. Motivated by the polarization puzzle at the Tevatron, I present the brand-new computation of higher-order αs corrections to the color-singlet production and discuss the impact of these corrections on the differential cross sections. I finally comment on the relative importance of the various transitions that feed quarkonium hadroproduction.

  8. Performance improvement: an active life cycle product management

    NASA Astrophysics Data System (ADS)

    Cucchiella, Federica; Gastaldi, Massimo; Lenny Koh, S. C.

    2010-03-01

    The management of the supply chain has gained importance in many manufacturing firms. Operational flexibility can be considered a crucial weapon to increase competitiveness in a turbulent marketplace. It reflects the ability of a firm to properly and rapidly respond to a variable and dynamic environment. For the firm operating in a fashion sector, the management of the supply chain is even more complex because the product life cycle is shorter than that of the firm operating in a non-fashion sector. The increase of firm flexibility level can be reached through the application of the real option theory inside the firm network. In fact, real option may increase the project value by allowing managers to more efficiently direct the production. The real option application usually analysed in literature does not take into account that the demands of products are well-defined by the product life cycle. Working on a fashion sector, the life cycle pattern is even more relevant because of an expected demand that grows according to a constant rate that does not capture the demand dynamics of the underlying fashion goods. Thus, the primary research objective of this article is to develop a model useful for the management of investments in a supply chain operating in a fashion sector where the system complexity is increased by the low level of unpredictability and stability that is proper of the mood phenomenon. Moreover, unlike the traditional model, a real option framework is presented here that considers fashion product characterised by uncertain stages of the production cycle.

  9. Improving Employee Productivity: Extensions to Behavioral Performance Management Programs.

    ERIC Educational Resources Information Center

    Clark, Ruth Colvin

    1984-01-01

    Summarizes limitations of current behavioral based employee management programs which rely heavily on reward and punishment techniques to maximize productivity; describes the new cognitive approaches which assume employees think about the meaning of rewards and punishments and act according to beliefs and expectations; and presents examples of…

  10. ADAPTmap: International coordinated data resource for improving goat production effiency

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Goats provide vital food and economic security, particularly in developing countries. We created a database that is a nexus for all performance, type, geographic information system (GIS), production environment, and genome information on goats. This resource provides a platform for meta-analysis tha...

  11. Improved approximations for fermion pair production in inhomogeneous electric fields

    SciTech Connect

    Kim, Sang Pyo; Page, Don N.

    2007-02-15

    Reformulating the instantons in a complex plane for tunneling or transmitting states, we calculate the pair-production rate of charged fermions in a spatially localized electric field, illustrated by the Sauter electric field E{sub 0}sech{sup 2}(z/L), and in a temporally localized electric field such as E{sub 0}sech{sup 2}(t/T). The integration of the quadratic part of WKB instanton actions over the frequency and transverse momentum leads to the pair-production rate obtained by the worldline-instanton method, including the prefactor, of Phys. Rev. D 72, 105004 (2005) and 73, 065028 (2006). It is further shown that the WKB instanton action plus the next-to-leading-order contribution in spinor QED equals the WKB instanton action in scalar QED, thus justifying why the WKB instanton in scalar QED can work for the pair production of fermions. Finally we obtain the pair-production rate in a spatially localized electric field together with a constant magnetic field in the same direction.

  12. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1

    PubMed Central

    Pereira Jr, Nei; Freire, Denise M.G.

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L−1–10.9 g L−1). These results offer promising pathways for the optimization of processes for the production of rhamnolipids. PMID:27257553

  13. Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

    PubMed Central

    Santala, Suvi; Karp, Matti; Santala, Ville

    2014-01-01

    In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications. PMID:25470793

  14. Improving Information Products for System 2 Decision Support

    ERIC Educational Resources Information Center

    Gibson, Neal

    2010-01-01

    The creation, maintenance, and management of Information Product (IP) systems that are used by organizations for complex decisions represent a unique set of challenges. These challenges are compounded when the purpose of such a systems is also for knowledge creation and dissemination. Information quality research to date has focused mainly upon…

  15. Midcontinent operators embrace vlaue in DOE improved production program

    SciTech Connect

    Lyle, D.

    1996-05-01

    In less than four years, the DOE`s fluvial-dominated deltaic reservoir program is paying off in a big way for Midcontinent operators. Many of the projects are getting to the point where they are showing real production impacts.

  16. Health sector employment growth calls for improvements in labor productivity.

    PubMed

    Hofmarcher, Maria M; Festl, Eva; Bishop-Tarver, Leslie

    2016-08-01

    While rising costs of healthcare have put increased fiscal pressure on public finance, job growth in the health sector has had a stabilizing force on overall employment levels - not least in times of economic crises. In 2014 EU-15 countries employed 21 million people in the health and social care sector. Between 2000 and 2014 the share of employed persons in this sector rose from 9.5% to 12.5% of the total labor force in EU-15 countries. Over time labor input growth has shifted towards residential care activities and social work while labor in human health activities including hospitals and ambulatory care still comprises the major share. About half of the human health labor force works in hospital. Variation of health and social care employment is large even in countries with generally comparable institutional structures. While standard measures of productivity in health and social care are not yet comparable across countries, we argue that labor productivity of a growing health work force needs more attention. The long-term stability of the health system will require care delivery models that better utilize a growing health work force in concert with smart investments in digital infrastructure to support this transition. In light of this, more research is needed to explain variations in health and social care labor endowments, to identify effective policy measures of labor productivity enhancement including enhanced efforts to develop comparable productivity indicators in these areas.

  17. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1.

    PubMed

    Soares Dos Santos, Alexandre; Pereira, Nei; Freire, Denise M G

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L(-1)-10.9 g L(-1)). These results offer promising pathways for the optimization of processes for the production of rhamnolipids.

  18. Health sector employment growth calls for improvements in labor productivity.

    PubMed

    Hofmarcher, Maria M; Festl, Eva; Bishop-Tarver, Leslie

    2016-08-01

    While rising costs of healthcare have put increased fiscal pressure on public finance, job growth in the health sector has had a stabilizing force on overall employment levels - not least in times of economic crises. In 2014 EU-15 countries employed 21 million people in the health and social care sector. Between 2000 and 2014 the share of employed persons in this sector rose from 9.5% to 12.5% of the total labor force in EU-15 countries. Over time labor input growth has shifted towards residential care activities and social work while labor in human health activities including hospitals and ambulatory care still comprises the major share. About half of the human health labor force works in hospital. Variation of health and social care employment is large even in countries with generally comparable institutional structures. While standard measures of productivity in health and social care are not yet comparable across countries, we argue that labor productivity of a growing health work force needs more attention. The long-term stability of the health system will require care delivery models that better utilize a growing health work force in concert with smart investments in digital infrastructure to support this transition. In light of this, more research is needed to explain variations in health and social care labor endowments, to identify effective policy measures of labor productivity enhancement including enhanced efforts to develop comparable productivity indicators in these areas. PMID:27370916

  19. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1.

    PubMed

    Soares Dos Santos, Alexandre; Pereira, Nei; Freire, Denise M G

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L(-1)-10.9 g L(-1)). These results offer promising pathways for the optimization of processes for the production of rhamnolipids. PMID:27257553

  20. Rationally engineered synthetic coculture for improved biomass and product formation.

    PubMed

    Santala, Suvi; Karp, Matti; Santala, Ville

    2014-01-01

    In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications. PMID:25470793

  1. Improving Language Production Using Subtitled Similar Task Videos

    ERIC Educational Resources Information Center

    Arslanyilmaz, Abdurrahman; Pedersen, Susan

    2010-01-01

    This study examines the effects of subtitled similar task videos on language production by nonnative speakers (NNSs) in an online task-based language learning (TBLL) environment. Ten NNS-NNS dyads collaboratively completed four communicative tasks, using an online TBLL environment specifically designed for this study and a chat tool in…

  2. Industry Immersion for Reading and Mathematics Improvement. Valley Products Company.

    ERIC Educational Resources Information Center

    Jones, Paul L.; And Others

    An intensive industry immersion program of reading and mathematics was conducted in Spring 1989 for employees at Valley Products, Inc., in a cooperative venture by the Literacy Foundation, Memphis, the Memphis City Schools Adult Education Program, and the company. Employee participants were assessed with the San Diego Quick Assessment Test to…

  3. Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

    PubMed Central

    Hernández-Crespo, Pedro; Sait, Steven M.; Hails, Rosemary S.; Cory, Jenny S.

    2001-01-01

    Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed. PMID:11229903

  4. Genetic improvement of plants for enhanced bio-ethanol production.

    PubMed

    Saha, Sanghamitra; Ramachandran, Srinivasan

    2013-04-01

    The present world energy situation urgently requires exploring and developing alternate, sustainable sources for fuel. Biofuels have proven to be an effective energy source but more needs to be produced to meet energy goals. Whereas first generation biofuels derived from mainly corn and sugarcane continue to be used and produced, the contentious debate between "feedstock versus foodstock" continues. The need for sources that can be grown under different environmental conditions has led to exploring newer sources. Lignocellulosic biomass is an attractive source for production of biofuel, but pretreatment costs to remove lignin are high and the process is time consuming. Genetically modified plants that have increased sugar or starch content, modified lignin content, or produce cellulose degrading enzymes are some options that are being explored and tested. This review focuses on current research on increasing production of biofuels by genetic engineering of plants to have desirable characteristics. Recent patents that have been filed in this area are also discussed.

  5. Opportunities to improve oil productivity in unstructured deltaic reservoirs

    SciTech Connect

    Not Available

    1991-01-01

    This report contains presentations presented at a technical symposium on oil production. Chapter 1 contains summaries of the presentations given at the Department of Energy (DOE)-sponsored symposium and key points of the discussions that followed. Chapter 2 characterizes the light oil resource from fluvial-dominated deltaic reservoirs in the Tertiary Oil Recovery Information System (TORIS). An analysis of enhanced oil recovery (EOR) and advanced secondary recovery (ASR) potential for fluvial-dominated deltaic reservoirs based on recovery performance and economic modeling as well as the potential resource loss due to well abandonments is presented. Chapter 3 provides a summary of the general reservoir characteristics and properties within deltaic deposits. It is not exhaustive treatise, rather it is intended to provide some basic information about geologic, reservoir, and production characteristics of deltaic reservoirs, and the resulting recovery problems.

  6. Heat Exchange System Improvement Saves Energy and Improves Production at a Winery

    SciTech Connect

    2001-08-01

    In 2000, Fetzer Vineyards implemented a project to improve its process heating cycle at its Hopland Winery in Hopland, California. In an effort to reduce expenditures on natural gas, Fetzer reviewed their wine process heating cycle and discovered that they could reduce their natural gas purchases and improve efficiency by installing a heat exchanger.

  7. The Advancement of Geothermal Energy Production through Improved Exploration Methods

    NASA Astrophysics Data System (ADS)

    Thorsteinsson, H.; Klein, K.

    2010-12-01

    Through the American Recovery and Reinvestment Act of 2009, the U.S. Department of Energy’s Geothermal Technologies Program invested $98 million in the geothermal exploration industry, and continues to encourage further research, development and demonstration in this field. The continued development of innovative exploration technologies is essential for wide adoption of geothermal resources. In 2008, the United States Geological Survey estimated that there are approximately 30,000 MW of undiscovered hydrothermal resources in the western United States alone. Improvements in exploration technologies are necessary to discover and define these hidden resources and to reduce up-front risk and cost through more accurate and efficient exploration. Currently, the surface and subsurface are characterized through combinations of ground-based and airborne geophysical surveys, geochemical surveys, satellite imaging and drilling. However, to increase geothermal exploration well success rates, development of improved and new exploration techniques is required.

  8. Induced Mutations for Improving Production on Bread and Durum Wheat

    SciTech Connect

    Stamo, Ilirjana; Ylli, Ariana; Dodbiba, Andon

    2007-04-23

    Wheat is a very important crop and has been bred for food and its improvement is continuous from cross-breeding. Radiation and chemically induced mutations have provided variability in selection for novel varieties. Four bread and one durum wheat cultivars were exposed to gamma rays, Cs 137 with doses 10, 15 and 20 krad (2000 seeds of each dose and cultivars). We have isolated mutant plants with height reduced and on cv Progress spike without chaff.

  9. Composite material and method for production of improved composite material

    NASA Technical Reports Server (NTRS)

    Farley, Gary L. (Inventor)

    1996-01-01

    A laminated composite material with improved interlaminar strength and damage tolerance having short rods distributed evenly throughout the composite material perpendicular to the laminae. Each rod is shorter than the thickness of the finished laminate, but several times as long as the thickness of each lamina. The laminate is made by inserting short rods in layers of prepreg material, and then stacking and curing prepreg material with rods inserted therethrough.

  10. Improved method for determination of light filth in chocolate products.

    PubMed

    Shostak, M T

    1978-07-01

    The recovery of rodent hairs from chocolate has been significantly improved by the introduction of an additional defatting step, substitution of 40% isopropanol for water, and substitution of mineral oil-heptane (85+15) for heptane in the trapping-off step. These changes have no adverse effect on insect fragment recovery. An average recovery of 95% was obtained for rodent hairs, coefficient of variation 7.9%. Insect fragment recoveries were 100%. PMID:681273

  11. Improving rapeseed production practices in the southeastern United States

    SciTech Connect

    Thomas, D.L.; Breve, M.A.; Raymer, P.L.; Minton, N.A.; Sumner, D.R. . Georgia Coastal Plain Experiment Station)

    1990-04-01

    Oilseed rape or rapeseed is a crop which offers a potential for double-cropping in the southeastern United States. This final project report describes the results from a three year study aimed at evaluating the effect of different planting and harvesting practices on establishment and yield of three rape cultivars, and the double cropping potential of rapeseed in the southeastern United States. The project was conducted on two yield sites in Tifton, Georgia during 1986--87, 1987--88 and 1988--89. The general objective of this research is to improve the seed and biomass yield of winter rapeseed in the southeastern United States by developing appropriate agronomic practices for the region. The primary constraint is to grow rapeseed within the allowable period for double cropping with an economically desirable crop, such as peanut or soybean. Planting and harvesting are the most critical steps in this process. Therefore, the specific objectives of this research were: evaluate and improve the emergence of rapeseed by developing planting techniques that enhance the soil, water and seed regimes for winter rapeseed in the southeast, and evaluate and improve the yields of harvested rapeseed by developing techniques for determining the optimum timing of harvest and efficient methods for harvesting winter rapeseed in the southeast. 6 refs., 12 figs., 9 tabs.

  12. New Eye Cleansing Product Improves Makeup-Related Ocular Problems.

    PubMed

    Okura, Masako; Kawashima, Motoko; Katagiri, Mikiyuki; Shirasawa, Takuji; Tsubota, Kazuo

    2015-01-01

    Purpose. This study evaluated the effects of using a newly developed eye cleansing formulation (Eye Shampoo) to cleanse the eyelids for 4 weeks in a parallel-group comparative study in women with chronic eye discomfort caused by heavy use of eye makeup and poor eye hygiene habits. Methods. Twenty women participants who met the inclusion criteria were randomly allocated to 2 groups comprising 10 participants each. The participants were asked to use either artificial tears alone or artificial tears in conjunction with Eye Shampoo for 4 weeks. The participants answered the questionnaire again and were reexamined, and changes in symptoms within each group and variations of symptoms between the two groups were statistically analyzed. Results. In the group using only artificial tears, improvements in subjective symptoms but not in ophthalmologic examination results were found. In the group using Eye Shampoo together with artificial tears, significant improvements were observed in the subjective symptoms, meibomian orifice obstruction, meibum secretion, tear breakup time, and superficial punctate keratopathy. Conclusion. In patients with chronic eye discomfort thought to be caused by heavy eye makeup, maintaining eyelid hygiene using Eye Shampoo caused a marked improvement in meibomian gland blockage and dry eye symptoms.

  13. New Eye Cleansing Product Improves Makeup-Related Ocular Problems

    PubMed Central

    Okura, Masako; Kawashima, Motoko; Katagiri, Mikiyuki; Shirasawa, Takuji; Tsubota, Kazuo

    2015-01-01

    Purpose. This study evaluated the effects of using a newly developed eye cleansing formulation (Eye Shampoo) to cleanse the eyelids for 4 weeks in a parallel-group comparative study in women with chronic eye discomfort caused by heavy use of eye makeup and poor eye hygiene habits. Methods. Twenty women participants who met the inclusion criteria were randomly allocated to 2 groups comprising 10 participants each. The participants were asked to use either artificial tears alone or artificial tears in conjunction with Eye Shampoo for 4 weeks. The participants answered the questionnaire again and were reexamined, and changes in symptoms within each group and variations of symptoms between the two groups were statistically analyzed. Results. In the group using only artificial tears, improvements in subjective symptoms but not in ophthalmologic examination results were found. In the group using Eye Shampoo together with artificial tears, significant improvements were observed in the subjective symptoms, meibomian orifice obstruction, meibum secretion, tear breakup time, and superficial punctate keratopathy. Conclusion. In patients with chronic eye discomfort thought to be caused by heavy eye makeup, maintaining eyelid hygiene using Eye Shampoo caused a marked improvement in meibomian gland blockage and dry eye symptoms. PMID:26347812

  14. New Eye Cleansing Product Improves Makeup-Related Ocular Problems.

    PubMed

    Okura, Masako; Kawashima, Motoko; Katagiri, Mikiyuki; Shirasawa, Takuji; Tsubota, Kazuo

    2015-01-01

    Purpose. This study evaluated the effects of using a newly developed eye cleansing formulation (Eye Shampoo) to cleanse the eyelids for 4 weeks in a parallel-group comparative study in women with chronic eye discomfort caused by heavy use of eye makeup and poor eye hygiene habits. Methods. Twenty women participants who met the inclusion criteria were randomly allocated to 2 groups comprising 10 participants each. The participants were asked to use either artificial tears alone or artificial tears in conjunction with Eye Shampoo for 4 weeks. The participants answered the questionnaire again and were reexamined, and changes in symptoms within each group and variations of symptoms between the two groups were statistically analyzed. Results. In the group using only artificial tears, improvements in subjective symptoms but not in ophthalmologic examination results were found. In the group using Eye Shampoo together with artificial tears, significant improvements were observed in the subjective symptoms, meibomian orifice obstruction, meibum secretion, tear breakup time, and superficial punctate keratopathy. Conclusion. In patients with chronic eye discomfort thought to be caused by heavy eye makeup, maintaining eyelid hygiene using Eye Shampoo caused a marked improvement in meibomian gland blockage and dry eye symptoms. PMID:26347812

  15. Improved method of HIPOT testing of advanced ignition system product

    SciTech Connect

    Baker, P.C.

    1992-04-01

    A new method of high potential (HIPOT) testing of advanced ignition system (AIS) product was developed. The new incorporated using a silver-filled RTV silicone as the electrodes of the HIPOT tester instead of the preformed, semi-rigid aluminum electrodes of the current tester. Initial results indicate that the developed method was more sensitive to the testing requirements of the HIPOT test. A patent for the combination of the material used and the method of testing developed was attempted but was withdrawn following a patent search by the US Patent Office.

  16. Productivity improvement by frontier horizontal drilling in Italy

    SciTech Connect

    Schenato, A.

    1995-12-31

    Italian domestic activity on horizontal wells has been specially addressed to carbonate reservoir and specifically targeted to re-entry in existing wells. The speech will focus on the specific experience matured in frontier applications in Italy, from 1989 with the short radius drain holes in Sicily, throughout world record deep water short radius in the southern part of Adriatic sea and depth world record medium radius in a HP/HT reservoir in the Po Valley. Production results will be reported as well as the achieved technological aspects.

  17. Proctor & Gamble: Compressed Air System Upgrade Saves Energy & Improves Production at a Paper Mill

    SciTech Connect

    2004-05-01

    In 2002, Procter & Gamble applied a system-level strategy to optimize a compressed air system at its paper products mill in Mehoopany, Pennsylvania. The project improved production, improved system performance, and saved 7.6 million kWh per year and $309,000 per year in maintenance costs.

  18. Ohio Aluminum Industries: Compressed air system improvement project saves energy and improves product quality

    SciTech Connect

    None, None

    2003-11-01

    In 2001, Ohio Aluminum Industries implemented the first phase of a compressed air system improvement project at its Cleveland, Ohio, plant. By completing this phase, the plant stabilized the system's pressure and improved its performance. Furthermore, it yielded annual energy savings of 716,000 kilowatt-hours and $73,200. The total cost for the project's first phase was $83,500, making the simple payback slightly more than 1 year.

  19. Improved alcohol production employing SSF with thermotolerant yeast

    SciTech Connect

    Tsao, G.T.; Cao, N.; Gong, C.S.

    1996-12-31

    Simultaneous saccharification and fermentation (SSF) involves the enzymatic hydrolysis of cellulose and the yeast fermentation of sugars to ethanol simultaneously in the same reactor. For the effective SSF process to produce ethanol from lignocellulose, it is required to remove the physical and chemical barrier around cellulose fibers and make cellulose more accessible to cellulose. Furthermore, it is preferred to have the compatible fermentation and saccharification conditions (e.g., temperature and pH). The process for pretreatment of lignocellulosic biomass involves the steeping in ammonia solution to remove lignin followed by dilute acid (1%, w/w) hydrolysis of hemicellulose fraction. The ammonia steeping removes over 70% of lignin and consequently facilitates the removal of hemicellulose by dilute acid. Dilute acid hydrolysis of hemicellulose yielding hydrolysate with sugar concentration of up to 8%. This fraction was used as substrate for ethanol production with xylose fermenting yeast strain. After lignin and hemicellulose were removed, the cellulose fraction was used as substrate in the SSF process for ethanol production. High yield of ethanol of over 60 g/L was produced by the thermotolerant yeast within 80 hours of SSF with a low enzyme loading of 8 IFPU/g cellulose.

  20. Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii.

    PubMed

    Yao, Shuo; Mikkelsen, Marie Just

    2010-09-01

    Thermoanaerobacter mathranii can produce ethanol from lignocellulosic biomass at high temperatures, but its biotechnological exploitation will require metabolic engineering to increase its ethanol yield. With a cofactor-dependent ethanol production pathway in T. mathranii, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was previously deleted from T. mathranii to eliminate an NADH oxidation pathway. To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD(+)-dependent glycerol dehydrogenase was expressed in T. mathranii. One of the resulting recombinant strains, T. mathranii BG1G1 (Deltaldh, P(xyl)GldA), showed increased ethanol yield in the presence of glycerol using xylose as a substrate. With an inactivated lactate pathway and expressed glycerol dehydrogenase activity, the metabolism of the cells was shifted toward the production of ethanol over acetate, hence restoring the redox balance. It was also shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reduced substrate glycerol resulted in a higher ethanol yield.

  1. Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

    PubMed

    Grant, Rebecca J; Kelley, Karen L; Maruniak, James E; Garcia-Maruniak, Alejandra; Barrett, Tom; Manire, Charles A; Romero, Carlos H

    2010-07-14

    The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals. PMID:20005643

  2. Transcriptome analyses of insect cells to facilitate baculovirus-insect expression.

    PubMed

    Yu, Kai; Yu, Yang; Tang, Xiaoyan; Chen, Huimin; Xiao, Junyu; Su, Xiao-Dong

    2016-05-01

    The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.

  3. Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

    PubMed

    Fleming-Davies, Arietta E; Dwyer, Greg

    2015-12-01

    A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen. PMID:26655986

  4. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells.

    PubMed

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

    2006-05-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type. PMID:16545777

  5. Immunogenicity of a Trivalent Human Papillomavirus L1 DNA-Encapsidated, Non-Replicable Baculovirus Nanovaccine

    PubMed Central

    Heo, Yoon-Ki; Cho, Yeondong; Gwon, Yong-Dae; Kim, Mi-Gyeong; Park, Ki Hoon; Oh, Yu-Kyoung; Kim, Young Bong

    2014-01-01

    Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58. PMID:24759938

  6. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  7. The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells.

    PubMed

    Chen, Yun-Ru; Zhong, Silin; Fei, Zhangjun; Hashimoto, Yoshifumi; Xiang, Jenny Z; Zhang, Shiying; Blissard, Gary W

    2013-06-01

    Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5' capped transcription start sites and 3' polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3' ends showed that 92 (77%) of the 3' PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

  8. Improvement of ectoine productivity by using sugar transporter-overexpressing Halomonas elongata.

    PubMed

    Tanimura, Kosuke; Matsumoto, Takuya; Nakayama, Hideki; Tanaka, Tsutomu; Kondo, Akihiko

    2016-07-01

    We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar.

  9. Improving Jet Reactor Configuration for Production of Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Povitsky, Alex

    2000-01-01

    The jet mixing reactor has been proposed for the industrial production of fullerene carbon nanotubes. Here we study the flowfield of this reactor using the SIMPLER algorithm. Hot peripheral jets are used to enhance heating of the central jet by mixing with the ambiance of reactor. Numerous configurations of peripheral jets with various number of jets, distance between nozzles, angles between the central jet and a peripheral jets, and twisted configuration of nozzles are considered. Unlike the previous studies of jet mixing, the optimal configuration of peripheral jets produces strong non-uniformity of the central jet in a cross-section. The geometrical shape of reactor is designed to obtain a uniform temperature of a catalyst.

  10. Productivity improvement in xanthan gum fermentation using multiple substrate optimization.

    PubMed

    Chaitali, Mandal; Kapadi, Mangesh; Suraishkumar, G K; Gudi, R D

    2003-01-01

    A novel and more comprehensive formulation of the optimal control problem that reflects the operational requirements of a typical industrial fermentation has been proposed in this work. This formulation has been applied to a fed-batch bioreactor with three control variables, i.e., feed rates of carbon source, nitrogen source, and an oxygen source, to result in a 148.7% increase in product formation. Xanthan gum production using Xanthomonas campestris has been used as the model system for this optimization study, and the liquid-phase oxygen supply strategy has been used to supply oxygen to the fermentation. The formulated optimization problem has several constraints associated with it due to the nature of the system. A robust stochastic technique, differential evolution, has been used to solve this challenging optimization problem. The infinite dimensional optimization problem has been approximated to a finite dimensional one by control vector parametrization. The state constraints that are path constraints have been addressed by using penalty functions and by integrating them over the total duration to ensure a feasible solution. End point constraints on final working volume of the reactor and on the final residual concentrations of carbon and nitrogen sources have been included in the problem formulation. Further, the toxicity of the oxygen source, H(2)O(2), has been addressed by imposing a constraint on its maximum usable concentration. In addition, the initial volume of the bioreactor contents and feed concentrations have been handled as decision variables, which has enabled a well-grounded choice for their values from the optimization procedure; adhoc values are normally used in the industry. All results obtained by simulation have been validated experimentally with good agreements between experimental and simulated values.

  11. Pathway engineering to improve ethanol production by thermophilic bacteria

    SciTech Connect

    Lynd, L.R.

    1998-12-31

    Continuation of a research project jointly funded by the NSF and DOE is proposed. The primary project goal is to develop and characterize strains of C. thermocellum and C. thermosaccharolyticum having ethanol selectivity similar to more convenient ethanol-producing organisms. An additional goal is to document the maximum concentration of ethanol that can be produced by thermophiles. These goals build on results from the previous project, including development of most of the genetic tools required for pathway engineering in the target organisms. As well, we demonstrated that the tolerance of C. thermosaccharolyticum to added ethanol is sufficiently high to allow practical utilization should similar tolerance to produced ethanol be demonstrated, and that inhibition by neutralizing agents may explain the limited concentrations of ethanol produced in studies to date. Task 1 involves optimization of electrotransformation, using either modified conditions or alternative plasmids to improve upon the low but reproducible transformation, frequencies we have obtained thus far.

  12. [Application of systems biology and synthetic biology in strain improvement for biofuel production].

    PubMed

    Zhao, Xinqing; Bai, Fengwu; Li, Yin

    2010-07-01

    Biofuels are renewable and environmentally friendly, but high production cost makes them economically not competitive, and the development of robust strains is thus one of the prerequisites. In this article, strain improvement studies based on the information from systems biology studies are reviewed, with a focus on their applications on stress tolerance improvement. Furthermore, the contribution of systems biology, synthetic biology and metabolic engineering in strain development for biofuel production is discussed, with an expectation for developing more robust strains for biofuel production.

  13. Potential Roles of Vocational Education in Improving the Productivity of the Workforce.

    ERIC Educational Resources Information Center

    Illinois Univ., Urbana. Coll. of Education.

    This monograph is a report on the impact vocational education can have on improving worker productivity. Specifically, the monograph presents a discussion of the relationship between vocational education and productivity and identifies potential areas of impact. The first chapter discusses the concept of productivity and its relationship to…

  14. Weyerhaeuser: Compressed Air System Improvement Saves Energy and Improves Production at a Sawmill

    SciTech Connect

    2004-11-01

    In 2000, Weyerhaeuser Company, a U.S. Department of Energy Allied Partner in the Industrial Technologies Program, increased the efficiency of the compressed air system at its sawmill facility in Coburg, Oregon. This improved the system's performance and will save about 1.3 million kWh annually. Total project costs were $55,000; because annual energy cost savings were also $55,000, the simple payback period was only 1 year. Subsequent improvements at six other company plants and mills are yielding 6.8 million kWh in energy savings and reducing annual energy costs by $250,000.

  15. Reducing phosphorus runoff and improving poultry production with alum.

    PubMed

    Moore, P A; Daniel, T C; Edwards, D R

    1999-05-01

    This is a review paper on the effects of aluminum sulfate (alum) on ammonia volatilization and P runoff from poultry litter. Initially, laboratory studies were conducted that showed P solubility could be reduced in poultry litter with Al, Ca, and Fe amendments, indicating that these amendments may reduce P runoff. These results were confirmed in small plot studies in which alum applications to litter were shown to decrease P concentrations in runoff by as much as 87%, while improving tall fescue yields. Leaf tissue analyses indicated that the yield improvements were due to increased N availability, which we hypothesized was due to reduced NH3 volatilization. This result was confirmed in laboratory studies that showed that alum was one of the most effective (and cost-effective) compounds for reducing NH3 volatilization. Field trials conducted at commercial broiler farms in conjunction with the Environmental Protection Agency showed that alum additions to poultry litter lowered litter pH, particularly during the first 3 to 4 wk of each growout, which resulted in less NH3 volatilization and lower atmospheric NH3. Ammonia volatilization rates were reduced by 97% for the first 4 wk of the growout. Broilers grown on alum-treated litter were heavier than the controls (1.73 vs 1.66 kg) and had lower mortality (3.9 vs 4.2%) and better feed efficiency (1.98 vs 2.04). Electricity and propane use were lower for alum-treated houses. As a result of these economic benefits to the integrator and grower, the benefit:cost ratio of alum addition was 1.96. Phosphorus concentrations in runoff from small watersheds were 75% lower from alum-treated litter than normal litter over a 3-yr period. Long-term small plot studies on alum use have shown that alum-treated litter results in lower soil test P levels than normal litter and does not increase Al availability in soils or uptake by plants.

  16. High-density plasma deposition manufacturing productivity improvement

    NASA Astrophysics Data System (ADS)

    Olmer, Leonard J.; Hudson, Chris P.

    1999-09-01

    High Density Plasma (HDP) deposition provides a means to deposit high quality dielectrics meeting submicron gap fill requirements. But, compared to traditional PECVD processing, HDP is relatively expensive due to the higher capital cost of the equipment. In order to keep processing costs low, it became necessary to maximize the wafer throughput of HDP processing without degrading the film properties. The approach taken was to optimize the post deposition microwave in-situ clean efficiency. A regression model, based on actual data, indicated that number of wafers processed before a chamber clean was the dominant factor. Furthermore, a design change in the ceramic hardware, surrounding the electrostatic chuck, provided thermal isolation resulting in an enhanced clean rate of the chamber process kit. An infra-red detector located in the chamber exhaust line provided a means to endpoint the clean and in-film particle data confirmed the infra-red results. The combination of increased chamber clean frequency, optimized clean time and improved process.

  17. Improving ethanol production by membrane technology: The continuous saccharification reactor

    SciTech Connect

    Cheryan, M.; Escobar, J.

    1993-12-31

    The saccharification of liquefied starch is typically done in a batch mode, taking 30--72 hours and requiring large quantities of enzyme (since each dose of enzyme is used only once). The process can be improved considerably by using a membrane reactor in which the reaction vessel is connected in a semi-closed loop configuration to a membrane module of the appropriate chemical nature and physical configuration. The continuous membrane reactor (CMR) concept was first evaluated with a dead-end cell, and later scaled-up to a cross-flow recycle configuration using hollow fibers or spiral wound modules. The CMR results in a dramatic reduction in reaction time to 5--10 hours, and reduces overall enzyme usage by 50--70%. In addition, the dextrose stream is crystal clear with little or no suspended particles, protein or fat, thus potentially reducing downstream costs. The CMR has been scaled up to a pilot-scale system of 1,500 liters with a membrane capacity of 30--65 cm{sup 2}, which is presently undergoing on-site trials at a large ethanol plant. The economics of this operation appear to be quite attractive.

  18. Quality improvement methodologies increase autologous blood product administration.

    PubMed

    Hodge, Ashley B; Preston, Thomas J; Fitch, Jill A; Harrison, Sheilah K; Hersey, Diane K; Nicol, Kathleen K; Naguib, Aymen N; McConnell, Patrick I; Galantowicz, Mark

    2014-03-01

    Whole blood from the heart-lung (bypass) machine may be processed through a cell salvaging device (i.e., cell saver [CS]) and subsequently administered to the patient during cardiac surgery. It was determined at our institution that CS volume was being discarded. A multidisciplinary team consisting of anesthesiologists, perfusionists, intensive care physicians, quality improvement (QI) professionals, and bedside nurses met to determine the challenges surrounding autologous blood delivery in its entirety. A review of cardiac surgery patients' charts (n = 21) was conducted for analysis of CS waste. After identification of practices that were leading to CS waste, interventions were designed and implemented. Fishbone diagram, key driver diagram, Plan-Do-Study-Act (PDSA) cycles, and data collection forms were used throughout this QI process to track and guide progress regarding CS waste. Of patients under 6 kg (n = 5), 80% had wasted CS blood before interventions, whereas those patients larger than 36 kg (n = 8) had 25% wasted CS before interventions. Seventy-five percent of patients under 6 kg who had wasted CS blood received packed red blood cell transfusions in the cardiothoracic intensive care unit within 24 hours of their operation. After data collection and didactic education sessions (PDSA Cycle I), CS blood volume waste was reduced to 5% in all patients. Identification and analysis of the root cause followed by implementation of education, training, and management of change (PDSA Cycle II) resulted in successful use of 100% of all CS blood volume. PMID:24783313

  19. Quality improvement methodologies increase autologous blood product administration.

    PubMed

    Hodge, Ashley B; Preston, Thomas J; Fitch, Jill A; Harrison, Sheilah K; Hersey, Diane K; Nicol, Kathleen K; Naguib, Aymen N; McConnell, Patrick I; Galantowicz, Mark

    2014-03-01

    Whole blood from the heart-lung (bypass) machine may be processed through a cell salvaging device (i.e., cell saver [CS]) and subsequently administered to the patient during cardiac surgery. It was determined at our institution that CS volume was being discarded. A multidisciplinary team consisting of anesthesiologists, perfusionists, intensive care physicians, quality improvement (QI) professionals, and bedside nurses met to determine the challenges surrounding autologous blood delivery in its entirety. A review of cardiac surgery patients' charts (n = 21) was conducted for analysis of CS waste. After identification of practices that were leading to CS waste, interventions were designed and implemented. Fishbone diagram, key driver diagram, Plan-Do-Study-Act (PDSA) cycles, and data collection forms were used throughout this QI process to track and guide progress regarding CS waste. Of patients under 6 kg (n = 5), 80% had wasted CS blood before interventions, whereas those patients larger than 36 kg (n = 8) had 25% wasted CS before interventions. Seventy-five percent of patients under 6 kg who had wasted CS blood received packed red blood cell transfusions in the cardiothoracic intensive care unit within 24 hours of their operation. After data collection and didactic education sessions (PDSA Cycle I), CS blood volume waste was reduced to 5% in all patients. Identification and analysis of the root cause followed by implementation of education, training, and management of change (PDSA Cycle II) resulted in successful use of 100% of all CS blood volume.

  20. Quality Improvement Methodologies Increase Autologous Blood Product Administration

    PubMed Central

    Hodge, Ashley B.; Preston, Thomas J.; Fitch, Jill A.; Harrison, Sheilah K.; Hersey, Diane K.; Nicol, Kathleen K.; Naguib, Aymen N.; McConnell, Patrick I.; Galantowicz, Mark

    2014-01-01

    Abstract: Whole blood from the heart–lung (bypass) machine may be processed through a cell salvaging device (i.e., cell saver [CS]) and subsequently administered to the patient during cardiac surgery. It was determined at our institution that CS volume was being discarded. A multidisciplinary team consisting of anesthesiologists, perfusionists, intensive care physicians, quality improvement (QI) professionals, and bedside nurses met to determine the challenges surrounding autologous blood delivery in its entirety. A review of cardiac surgery patients’ charts (n = 21) was conducted for analysis of CS waste. After identification of practices that were leading to CS waste, interventions were designed and implemented. Fishbone diagram, key driver diagram, Plan–Do–Study–Act (PDSA) cycles, and data collection forms were used throughout this QI process to track and guide progress regarding CS waste. Of patients under 6 kg (n = 5), 80% had wasted CS blood before interventions, whereas those patients larger than 36 kg (n = 8) had 25% wasted CS before interventions. Seventy-five percent of patients under 6 kg who had wasted CS blood received packed red blood cell transfusions in the cardiothoracic intensive care unit within 24 hours of their operation. After data collection and didactic education sessions (PDSA Cycle I), CS blood volume waste was reduced to 5% in all patients. Identification and analysis of the root cause followed by implementation of education, training, and management of change (PDSA Cycle II) resulted in successful use of 100% of all CS blood volume. PMID:24783313

  1. Improving Epithermal Transmission Measurements by Optimizing Neutron Production and Detection

    NASA Astrophysics Data System (ADS)

    Piela, Sean Garrigan

    Methods of improving the neutron count rate in the energy range 3 to 100-300 [keV] at the 100 [m] detector station for the Gaerttner LINAC Center were studied. One part of this undertaking was the explore alterations to the main photonuclear target used for this energy range. Detailed simulations using Monte Carlo N-Particle Transport Code (MCNP, version 5) were carried out and the results used to synthesize an optimized target design. Simulation predicted a gain in neutron intensity of 1.63 at 4.49 [keV], above the current target capability. Experiment found a gain of 1.42 at 4.89 [keV] over the current target. An addition of 1% boric acid to the water in the moderating ring of the current target is predicted to result in a factor of 111 +/- 12 increase in the neutron to photon ratio above baseline for 3.51 [keV] neutrons at 100 [m]; this value is for photons above 700 [keV]. The gain in neutron intensity for the optimized target comes with a gain in photon intensity. A 1% boric acid addition to this design has same neutron to photon ratio as with the boric acid addition to the current target (for 3.51 [keV] neutrons). In an alternate approach to enhancing the neutron counts, a prototype plastic scintillator was studied as a possible replacement for the extant lithium glass scintillators, with experiments finding major increases to the neutron counts in the energy range of interest. The plastic scintillator prototype was found to produce gains of 1.86, 3.61, 1.76 and 3.16 at 70.1, 127, 219, and 307 [keV], respectively, over a lithium glass detector. However, at 24.5 [keV] the plastic prototype count rate was only 36% of that measured with the lithium glass detector. The prototype detector geometry and its associated electronics need to be altered in order to actualize the true potential of the plastic scintillator detector.

  2. Improved Digitization of Lunar Mare Ridges with LROC Derived Products

    NASA Astrophysics Data System (ADS)

    Crowell, J. M.; Robinson, M. S.; Watters, T. R.; Bowman-Cisneros, E.; Enns, A. C.; Lawrence, S.

    2011-12-01

    Lunar wrinkle ridges (mare ridges) are positive-relief structures formed from compressional stress in basin-filling flood basalt deposits [1]. Previous workers have measured wrinkle ridge orientations and lengths to investigate their spatial distribution and infer basin-localized stress fields [2,3]. Although these plots include the most prominent mare ridges and their general trends, they may not have fully captured all of the ridges, particularly the smaller-scale ridges. Using Lunar Reconnaissance Orbiter Wide Angle Camera (WAC) global mosaics and derived topography (100m pixel scale) [4], we systematically remapped wrinkle ridges in Mare Serenitatis. By comparing two WAC mosaics with different lighting geometry, and shaded relief maps made from a WAC digital elevation model (DEM) [5], we observed that some ridge segments and some smaller ridges are not visible in previous structure maps [2,3]. In the past, mapping efforts were limited by a fixed Sun direction [6,7]. For systematic mapping we created three shaded relief maps from the WAC DEM with solar azimuth angles of 0°, 45°, and 90°, and a fourth map was created by combining the three shaded reliefs into one, using a simple averaging scheme. Along with the original WAC mosaic and the WAC DEM, these four datasets were imported into ArcGIS, and the mare ridges of Imbrium, Serenitatis, and Tranquillitatis were digitized from each of the six maps. Since the mare ridges are often divided into many ridge segments [8], each major component was digitized separately, as opposed to the ridge as a whole. This strategy enhanced our ability to analyze the lengths, orientations, and abundances of these ridges. After the initial mapping was completed, the six products were viewed together to identify and resolve discrepancies in order to produce a final wrinkle ridge map. Comparing this new mare ridge map with past lunar tectonic maps, we found that many mare ridges were not recorded in the previous works. It was noted

  3. Importance of Indigenous Breeds of Chicken for Rural Economy and Their Improvements for Higher Production Performance

    PubMed Central

    Padhi, Mahendra Kumar

    2016-01-01

    Indigenous/native breeds of chickens are playing an important role in rural economies in most of the developing and underdeveloped countries. They play a major role for the rural poor and marginalised section of the people with respect to their subsidiary income and also provide them with nutritious chicken egg and meat for their own consumption. Performance of native fowl can be improved by change in husbandry, feeding, and better health cover. However, genetic improvement may be made either through selection and crossbreeding or by utilisation of both selection and crossbreeding. Improvement through selection may be time consuming but the improvement will be permanent. Through crossbreeding improvement may be faster but research has to aim for the production of native-type birds with higher production potential. In the present review efforts have been made to present the importance of native fowl to rural economy and their improvement for higher production performance. PMID:27144053

  4. Expression of the human interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector.

    PubMed

    Raivio, E; Oetken, C; Oker-Blom, C; Engberg, C; Akerman, K; Lindqvist, C

    1995-04-01

    The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis. PMID:7899821

  5. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    SciTech Connect

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  6. Isolation and Characterization of a Baculovirus Associated with the Insect Parasitoid Wasp, Cotesia marginiventris, or Its Host, Trichoplusia ni

    PubMed Central

    Grasela, James J.; McIntosh, Arthur H.; Shelby, Kent S.; Long, Steve

    2008-01-01

    A multiple nucleopolyhedrovirus (MNPV) was isolated from Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae that had been stung by the parasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). The wild type virus was plaque purified by infecting a Heliothis subflexa (BCIRL- HsAM1) cell line and isolating several clones. The mean estimated genomic size of this virus based on PstI, BstEII, StyI, HindIII restriction profiles was estimated to be 106 ± 2.5 kbp (mean±SE). A clone designated as TnMNPV/CmBCL9 was used in bioassays against several lepidopteran pests and in comparative studies with the baculoviruses AcMNPV, AgMNPV, AfMNPV, PxMNPV and HzSNPV of Autographa califomica, Anticarsia gemmatalis, Anagrapha falcifera, Plutella xylostella, and Helicoverpa zea, respectively. Infectivity studies showed that TnMNPV/CmBCL9 was highly infectious for Heliothis subflexa and T. ni, with an LC50 value 0.07 occlusion bodies/mm2 in both species and also infectious for H. zea and Heliothis virescens with LC50 values of 0.22 and 0.27 occlusion bodies/mm2, respectively. Restriction endonuclease analysis of the isolate and selected baculoviruses revealed profiles that were very similar to AfMNPV but different from the restriction endonuclease profiles of the other baculoviruses. Hybridization studies suggest that the TnMNPV/CmBCL9 was closely related to AfMNPV and AcMNPV-HPP. Further support for this comes from a phylogenetic analysis employing a split-graphs network, comparing the polh, egt, and p10 genes from TnMNPV/CmBCL9 with those from other baculoviruses and suggests that this virus is closely related to the AcMNPV variants, AfMNPV and RoMNPV of Rachiplusia ou. PMID:20334593

  7. DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses.

    PubMed

    Nielsen, Cydney B; Cooper, Dawn; Short, Steven M; Myers, Judith H; Suttle, Curtis A

    2002-11-01

    Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests. Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided. DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses. We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity. We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses. McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada. Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity). Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity). Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses.

  8. Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

    PubMed

    Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P; Small, J Victor

    2014-01-01

    Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion. PMID:24453943

  9. Generation of a p10-based baculovirus expression vector in yeast with infectivity for insect larvae and insect cells.

    PubMed

    Heldens, J G; Kester, H A; Zuidema, D; Vlak, J M

    1997-10-01

    A new, versatile baculovirus vector was developed for the generation of recombinants in the yeast Saccharomyces cerevisiae and for the expression of foreign proteins in both insect larvae and in insect cells. This vector is based on Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) and exploits the 10-kDa protein promoter (p10) for the expression of the foreign gene. The p10 locus was used for the insertion of a yeast-selectable marker system (ARS-URA-URA3) and of a gene for screening and titration of recombinants in insect cells (beta-galactosidase). The polyhedron-positive phenotype of this vector is maintained allowing its use in insect larvae, by feeding polyhedra, and in insect cells, by infecting with budded virus. The generation of this baculovirus vector requires a single recombination step in yeast prior to infection of insect cells, but has the advantage over the vector designed previously (Patel et al., A new method for the isolation of recombinant baculovirus, Nucleic Acids Research 20 (1992) 97-104) that these vectors can also be used in insects.

  10. Improving microbial biogasoline production in Escherichia coli using tolerance engineering

    SciTech Connect

    Foo, Jee Loon; Jensen, Heather M.; Dahl, Robert H.; George, Kevin; Keasling, Jay D.; Lee, Taek Soon; Leong, Susanna; Mukhopadhyay, Aindrila

    2014-11-04

    Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production.

  11. Improvement of inter-field CDU by using on-product focus control

    NASA Astrophysics Data System (ADS)

    Park, Kyeong Dong; Park, Tony; Hwang, Jong Hyun; Choi, Jin Phil; Kang, Young Seog

    2014-04-01

    This paper introduces to improve inter-field CDU with on-product focus control by diffraction based focus (DBF) method. For DBF target selection, a robust focus metrology for focus control was obtained, and the selected DBF target was integrated on each seven spot of a product reticle. For on-product focus control, previously on-product focus monitoring was performed, and the monitored lots showed a stable focus fingerprint. Based on the result, Z and Z/ Rx/Ry corrections per field on wafers were applied. Focus uniformity of controlled wafers was improved up to 29% in comparison with non-corrected ones. To demonstrate the improvement of inter-field CDU, Full CDs on wafers were measured by SEM. As a result, inter-field CDU for controlled wafers was improved by 16% (3σ) compared with noncontrolled wafers.

  12. NGS for the Masses: Empowering Biologists to Improve Bioinformatics Productivity ( 7th Annual SFAF Meeting, 2012)

    ScienceCinema

    Qaadri, Kashef [Biomatters

    2016-07-12

    Kashef Qaadri on "NGS for the Masses: Empowering biologists to improve bioinformatic productivity" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  13. NGS for the Masses: Empowering Biologists to Improve Bioinformatics Productivity ( 7th Annual SFAF Meeting, 2012)

    SciTech Connect

    Qaadri, Kashef

    2012-06-01

    Kashef Qaadri on "NGS for the Masses: Empowering biologists to improve bioinformatic productivity" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  14. Strategies to improve water productivity in a water-stressed future

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As water shortages become more commonplace, strategies to increase crop water productivity are increasingly sought by producers. Common strategies include increasing irrigation application efficiency, changing irrigation application methods, improving irrigation scheduling, using sensing systems to ...

  15. Improved product energy intensity benchmarking metrics for thermally concentrated food products.

    PubMed

    Walker, Michael E; Arnold, Craig S; Lettieri, David J; Hutchins, Margot J; Masanet, Eric

    2014-10-21

    Product energy intensity (PEI) metrics allow industry and policymakers to quantify manufacturing energy requirements on a product-output basis. However, complexities can arise for benchmarking of thermally concentrated products, particularly in the food processing industry, due to differences in outlet composition, feed material composition, and processing technology. This study analyzes tomato paste as a typical, high-volume concentrated product using a thermodynamics-based model. Results show that PEI for tomato pastes and purees varies from 1200 to 9700 kJ/kg over the range of 8%-40% outlet solids concentration for a 3-effect evaporator, and 980-7000 kJ/kg for a 5-effect evaporator. Further, the PEI for producing paste at 31% outlet solids concentration in a 3-effect evaporator varies from 13,000 kJ/kg at 3% feed solids concentration to 5900 kJ/kg at 6%; for a 5-effect evaporator, the variation is from 9200 kJ/kg at 3%, to 4300 kJ/kg at 6%. Methods to compare the PEI of different product concentrations on a standard basis are evaluated. This paper also presents methods to develop PEI benchmark values for multiple plants. These results focus on the case of a tomato paste processing facility, but can be extended to other products and industries that utilize thermal concentration.

  16. Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

    PubMed Central

    2013-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections. Methods Immunoblotting analysis of recombinant baculovirus and immunofluorescent staining of baculovirus-infected cells were followed using anti-ORF2 monoclonal antibodies. The BALB/c mice were immunized intramuscularly with this baculovirus. The titers of antibodies were mensurated with a Cap-protein-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The IFN-γ response in splenocytes harvested from immunized mice was measured by ELISA. Student's t-test was used to compare immune responses of different groups. Results In this study, we successfully constructed a dual-expression-system-based recombinant baculovirus BV-GD-ORF2, which can display the PCV2 capsid (Cap) protein and VSV-G protein on the viral envelope and also expressing Cap protein on transduced mammalian cells, thereby functioning as both a subunit and a DNA vaccine. After infection, the Cap protein was expressed and displayed on the viral surface, as demonstrated with an indirect fluorescence assay and immunoblotting. The vaccination of mice with recombinant baculovirus BV-GD-ORF2 successfully induced robust Cap-protein-specific humoral and cellular immune responses. Conclusions Our findings collectively demonstrate that the recombinant baculovirus BV-GD-ORF2 is a potential vaccine against PCV2 infections. PMID:24161107

  17. Applying Value Stream Mapping Technique for Production Improvement in a Manufacturing Company: A Case Study

    NASA Astrophysics Data System (ADS)

    Jeyaraj, K. L.; Muralidharan, C.; Mahalingam, R.; Deshmukh, S. G.

    2013-01-01

    The purpose of this paper is to explain how value stream mapping (VSM) is helpful in lean implementation and to develop the road map to tackle improvement areas to bridge the gap between the existing state and the proposed state of a manufacturing firm. Through this case study, the existing stage of manufacturing is mapped with the help of VSM process symbols and the biggest improvement areas like excessive TAKT time, production, and lead time are identified. Some modifications in current state map are suggested and with these modifications future state map is prepared. Further TAKT time is calculated to set the pace of production processes. This paper compares the current state and future state of a manufacturing firm and witnessed 20 % reduction in TAKT time, 22.5 % reduction in processing time, 4.8 % reduction in lead time, 20 % improvement in production, 9 % improvement in machine utilization, 7 % improvement in man power utilization, objective improvement in workers skill level, and no change in the product and semi finished product inventory level. The findings are limited due to the focused nature of the case study. This case study shows that VSM is a powerful tool for lean implementation and allows the industry to understand and continuously improve towards lean manufacturing.

  18. Improving Student Concern for Safety in a Production Technology Lab through the Use of Teambuilding.

    ERIC Educational Resources Information Center

    Lacina, Dale Robert

    The effectiveness of team building as a strategy for improving students' concern for safety in a production technology laboratory was examined in a study involving a group of grade 9 and 10 production technology students from an urban, lower-middle-class community in western Illinois. Students' safety test scores, teacher checklists, and…

  19. Remediation/Restoration of Degraded Soil to Improve Productivity In The Central Great Plains Region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The quality and productivity of some farmlands in the central Great Plains Region (CGPR) have been lost through wind and water erosion induced by tillage and poor soil management. Productivity of degraded/eroded soils can be restored using organic amendments such as manure and improved crop and soil...

  20. The Role of Breeding and Genetics in Animal Production Improvement in the Developing Countries

    PubMed Central

    Rendel, Jan

    1974-01-01

    Availability of animal protein for human consumption is very low in the developing countries mainly because of low productivity of existing livestock; ways and means to improve productivity through breeding are discussed and some basic issues requiring further research pointed out. PMID:17248670

  1. [Problems of improving working conditions for workers engaged in the production of nitrogen mineral fertilizers].

    PubMed

    Rumiantsev, G I; Kozlova, T A; Atiakina, I K; Pavlova, A P

    1989-01-01

    The study results show that significant improvement of labour conditions can be achieved due to the introduction of a new distribution pattern of technological equipment, further improvement of equipment supply of the technological process, further mechanization of production operations, rational arrangement and disposition of control desks for technological processes, utilization of possible means of protection against noise, improvement of measuring machines, rational organization of the workplace for packing of stock-produced products and application of the means of small-scale mechanization, rational distribution of the sections of packing, filling and loading.

  2. Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange.

    PubMed

    Fernandes, Fabiana; Dias, Mafalda M; Vidigal, João; Sousa, Marcos F Q; Patrone, Marco; Teixeira, Ana P; Alves, Paula M

    2014-02-10

    A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles. PMID:24333128

  3. Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst.

    PubMed

    Wang, Dahui; Ju, Xiaomin; Zhang, Gaochuan; Wang, Donghua; Wei, Gongyuan

    2016-10-01

    The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

  4. Uranium Mill Tailings Remedial Action Project: Cost Reduction and Productivity Improvement Program Project Plan

    SciTech Connect

    Not Available

    1991-11-01

    The purpose of the Cost Reduction/Productivity Improvement Program Plan is to formalize and improve upon existing efforts to control costs which have been underway since project inception. This program plan has been coordinated with the Department of Energy (DOE) Office of Environmental Management (EM) and the DOE Field Office, Albuquerque (AL). It incorporates prior Uranium Mill Tallings Remedial Action (UMTRA) Project Office guidance issued on the subject. The opportunities for reducing cosh and improving productivity are endless. The CR/PIP has these primary objectives: Improve productivity and quality; heighten the general cost consciousness of project participants, at all levels of their organizations; identify and implement specific innovative employee ideas that extend beyond what is required through existing processes and procedures; emphasize efforts that create additional value for the money spent by maintaining the project Total Estimated Cost (TEC) at the lowest possible level.

  5. Uranium Mill Tailings Remedial Action Project: Cost Reduction and Productivity Improvement Program Project Plan. Revised

    SciTech Connect

    Not Available

    1991-11-01

    The purpose of the Cost Reduction/Productivity Improvement Program Plan is to formalize and improve upon existing efforts to control costs which have been underway since project inception. This program plan has been coordinated with the Department of Energy (DOE) Office of Environmental Management (EM) and the DOE Field Office, Albuquerque (AL). It incorporates prior Uranium Mill Tallings Remedial Action (UMTRA) Project Office guidance issued on the subject. The opportunities for reducing cosh and improving productivity are endless. The CR/PIP has these primary objectives: Improve productivity and quality; heighten the general cost consciousness of project participants, at all levels of their organizations; identify and implement specific innovative employee ideas that extend beyond what is required through existing processes and procedures; emphasize efforts that create additional value for the money spent by maintaining the project Total Estimated Cost (TEC) at the lowest possible level.

  6. Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad-spectrum resistance to disease

    PubMed Central

    Lincoln, James E.; Richael, Craig; Overduin, Bert; Smith, Kathy; Bostock, Richard; Gilchrist, David G.

    2002-01-01

    The sphinganine analog mycotoxin, AAL-toxin, induces a death process in plant and animal cells that shows apoptotic morphology. In nature, the AAL-toxin is the primary determinant of the Alternaria stem canker disease of tomato, thus linking apoptosis to this disease caused by Alternaria alternata f. sp. lycopersici. The product of the baculovirus p35 gene is a specific inhibitor of a class of cysteine proteases termed caspases, and naturally functions in infected insects. Transgenic tomato plants bearing the p35 gene were protected against AAL-toxin-induced death and pathogen infection. Resistance to the toxin and pathogen co-segregated with the expression of the p35 gene through the T3 generation, as did resistance to A. alternata, Colletotrichum coccodes, and Pseudomonas syringae pv. tomato. The p35 gene, stably transformed into tomato roots by Agrobacterium rhizogenes, protected roots against a 30-fold greater concentration of AAL-toxin than control roots tolerated. Transgenic expression of a p35 binding site mutant (DQMD to DRIL), inactive against animal caspases-3, did not protect against AAL-toxin. These results indicate that plants possess a protease with substrate-site specificity that is functionally equivalent to certain animal caspases. A biological conclusion is that diverse plant pathogens co-opt apoptosis during infection, and that transgenic modification of pathways regulating programmed cell death in plants is a potential strategy for engineering broad-spectrum disease resistance in plants. PMID:12403830

  7. Self-Assembly and Release of Peste des Petits Ruminants Virus-Like Particles in an Insect Cell-Baculovirus System and Their Immunogenicity in Mice and Goats

    PubMed Central

    Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

    2014-01-01

    Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR. PMID:25117931

  8. Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus.

    PubMed

    Michalsky, Ronald; Pfromm, Peter H; Czermak, Peter; Sorensen, Christopher M; Passarelli, A Lorena

    2008-11-01

    Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used virus. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (AcMNPV), vHSGFP, was incubated at 15-65 degrees C. A 2-log decrease in virus infectivity occurred after virus incubation above 45 degrees C. The activation energy of virus deactivation was circa 108 kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150+/-19 to 249+/-13 nm at 55 degrees C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4 degrees C or exposed to shear stress from stirring (100 rpm, 1.02x10(-5) psi) and pumping (50-250 ml/min, 1.45x10(-5) to 7.25x10(-5) psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.

  9. Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction.

    PubMed

    Pfohl, Jeffrey L; Worley, Jennings F; Condreay, J Patrick; An, Gang; Apolito, Christopher J; Kost, Tom A; Truax, James F

    2002-01-01

    A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.

  10. Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

    PubMed

    Huang, Yong; Zou, Quan; Shen, Xing Jia; Yu, Xue Li; Wang, Zhan Bin; Cheng, Xiang Chao

    2012-01-01

    MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells. PMID:22937569

  11. Method to express and purify nm23-H2 protein from baculovirus-infected cells.

    PubMed

    Garzia, L; André, A; Amoresano, A; D'Angelo, A; Martusciello, R; Cirulli, C; Tsurumi, T; Marino, G; Zollo, M

    2003-08-01

    High-throughput protein expression and purification are major bottlenecks in the postgenomic and proteomic era. We show here an automated method to express and purify nm23-H2, a nucleoside diphosphate kinase (NDPK), in a 96-well format, by the use of a robotic workstation, from insect Spodoptera frugiperda (Sf9) baculovirus-infected cells using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads. The automated method is coupled to mass spectrometry for a validation and quality-control analysis. To verify the bona fide of the recombinant protein, several tests have been produced, including NDPK assay, Western blotting, and in vitro phosphorylation experiments, thus confirming the value of the protocol developed. The method has been validated for the expression of several proteins, thus confirming the value of this automated protocol. The research presented here is a useful method both for industrial and academic environments to produce in a high-throughput mode recombinant eukaryotic proteins to be assayed for a specific function in a systematic manner.

  12. Baculovirus Induced Transcripts in Hemocytes from the Larvae of Heliothis virescens

    PubMed Central

    Breitenbach, Jonathan E.; Shelby, Kent S.; Popham, Holly J.R.

    2011-01-01

    Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae. PMID:22163334

  13. RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein.

    PubMed

    Gross, C H; Shuman, S

    1998-12-01

    Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases. PMID:9811740

  14. Population genomics supports baculoviruses as vectors of horizontal transfer of insect transposons

    PubMed Central

    Gilbert, Clément; Chateigner, Aurélien; Ernenwein, Lise; Barbe, Valérie; Bézier, Annie; Herniou, Elisabeth A.; Cordaux, Richard

    2014-01-01

    Horizontal transfer (HT) of DNA is an important factor shaping eukaryote evolution. Although several hundreds of eukaryote-to-eukaryote HTs of transposable elements (TEs) have been reported, the vectors underlying these transfers remain elusive. Here, we show that multiple copies of two TEs from the cabbage looper (Trichoplusia ni) transposed in vivo into genomes of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) during caterpillar infection. We further demonstrate that both TEs underwent recent HT between several sympatric moth species (T. ni, Manduca sexta, Helicoverpa spp.) showing different degrees of susceptibility to AcMNPV. Based on two independent population genomics data sets (reaching a total coverage >330,000X), we report a frequency of one moth TE in ~8,500 AcMNPV genomes. Together, our results provide strong support for the role of viruses as vectors of TE HT between animals, and they call for a systematic evaluation of the frequency and impact of virus-mediated HT on the evolution of host genomes. PMID:24556639

  15. Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

    PubMed Central

    Seong, Baik Lin; Nguyen, Huan Huu; Chang, Jun

    2016-01-01

    The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic. PMID:27023684

  16. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function.

    PubMed

    Biernat, Magdalena A; Eker, André P M; van Oers, Monique M; Vlak, Just M; van der Horst, Gijsbertus T J; Chaves, Inês

    2012-02-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses. PMID:22306969

  17. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    SciTech Connect

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-03-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.

  18. [Horizontal transmission routes of baculovirus infection in gypsy moth (Lymantria dispar L.)].

    PubMed

    Kolosov, A V; Kosogova, T A; Bulychev, L E; Sergeev, A N

    2010-01-01

    The paper considers horizontal transmission routes of baculovirus infection in the gypsy moth (Lymantria dispar L.). The original method for modeling natural processes in controllable conditions allowed one to estimate the influence of factors on the occurrence of epizooties. The authors investigated 3 possible models of virus transmission from infected to uninfected gypsy moths: 1) infected and test caterpillars were kept and fed together (a complex route); 2) those which were in the immediate vicinity, but deprived of eating together (an aerial route); 3) test caterpillars were fed on the leaves on which infected caterpillars had eaten (an oral route). The investigations have shown that the complex and oral routes out of the considered models may be considered to be effective infection transmission routes for the horizontal spread of epizooties. Furthermore, the availability of sufficient amount of infected caterpillars in the population leads to a reduction in the resistance of healthy insects to other diseases. Thus, by taking into account the capacity of larvae for passive migration, the purpose of insecticidal treatment is to set up a few infection foci that will be a source for the spread of epizootias and contribute to an overall viability reduction of a pest population.

  19. The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.

    PubMed

    Kuroda, K; Geyer, H; Geyer, R; Doerfler, W; Klenk, H D

    1990-02-01

    The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains. PMID:2407026

  20. Improving ethanol production from alfalfa stems via ambient-temperature acid pretreatment and washing.

    PubMed

    Zhou, Shengfei; Weimer, Paul J; Hatfield, Ronald D; Runge, Troy M; Digman, Matthew

    2014-10-01

    The concept of co-production of liquid fuel (ethanol) along with animal feed on farm was proposed, and the strategy of using ambient-temperature acid pretreatment, ensiling and washing to improve ethanol production from alfalfa stems was investigated. Alfalfa stems were separated and pretreated with sulfuric acid at ambient-temperature after harvest, and following ensiling, after which the ensiled stems were subjected to simultaneous saccharification and fermentation (SSF) for ethanol production. Ethanol yield was improved by ambient-temperature sulfuric acid pretreatment before ensiling, and by washing before SSF. It was theorized that the acid pretreatment at ambient temperature partially degraded hemicellulose, and altered cell wall structure, resulted in improved cellulose accessibility, whereas washing removed soluble ash in substrates which could inhibit the SSF. The pH of stored alfalfa stems can be used to predict the ethanol yield, with a correlation coefficient of +0.83 for washed alfalfa stems.

  1. Catalytic partial oxidation coupled with membrane purification to improve resource and energy efficiency in syngas production.

    PubMed

    Iaquaniello, G; Salladini, A; Palo, E; Centi, G

    2015-02-01

    Catalytic partial oxidation coupled with membrane purification is a new process scheme to improve resource and energy efficiency in a well-established and large scale-process like syngas production. Experimentation in a semi industrial-scale unit (20 Nm(3)  h(-1) production) shows that a novel syngas production scheme based on a pre-reforming stage followed by a membrane for hydrogen separation, a catalytic partial oxidation step, and a further step of syngas purification by membrane allows the oxygen-to-carbon ratio to be decreased while maintaining levels of feed conversion. For a total feed conversion of 40 %, for example, the integrated novel architecture reduces oxygen consumption by over 50 %, with thus a corresponding improvement in resource efficiency and an improved energy efficiency and economics, these factors largely depending on the air separation stage used to produce pure oxygen.

  2. Catalytic partial oxidation coupled with membrane purification to improve resource and energy efficiency in syngas production.

    PubMed

    Iaquaniello, G; Salladini, A; Palo, E; Centi, G

    2015-02-01

    Catalytic partial oxidation coupled with membrane purification is a new process scheme to improve resource and energy efficiency in a well-established and large scale-process like syngas production. Experimentation in a semi industrial-scale unit (20 Nm(3)  h(-1) production) shows that a novel syngas production scheme based on a pre-reforming stage followed by a membrane for hydrogen separation, a catalytic partial oxidation step, and a further step of syngas purification by membrane allows the oxygen-to-carbon ratio to be decreased while maintaining levels of feed conversion. For a total feed conversion of 40 %, for example, the integrated novel architecture reduces oxygen consumption by over 50 %, with thus a corresponding improvement in resource efficiency and an improved energy efficiency and economics, these factors largely depending on the air separation stage used to produce pure oxygen. PMID:25571881

  3. Activating transhydrogenase and NAD kinase in combination for improving isobutanol production.

    PubMed

    Shi, Aiqin; Zhu, Xinna; Lu, Jiao; Zhang, Xueli; Ma, Yanhe

    2013-03-01

    Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic

  4. Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse.

    PubMed

    Li, Qiuling; Zhai, Qiongxiang; Geng, Jia; Zheng, Hui; Chen, Fei; Kong, Jie; Zhang, Cheng

    2012-08-01

    In this study, recombinant baculovirus carrying the microdystrophin and β-catenin genes was used to infect adipose-derived stem cells from a dystrophin-utrophin double knock-out mouse. Results showed that, after baculovirus transgene infection, microdystrophin and β-catenin genes were effectively expressed in adipose-derived stem cells from the dystrophin-utrophin double knock-out mouse. Furthermore, this transgenic expression promoted adipose-derived stem cell differentiation into muscle cells, but inhibited adipogenic differentiation. In addition, protein expression related to the microdystrophin and Wnt/β-catenin signaling pathway was upregulated. Our experimental findings indicate that baculovirus can successfully deliver the microdystrophin and β-catenin genes into adipose-derived stem cells, and the microdystrophin and Wnt/β-catenin signaling pathway plays an important role in myogenesis of adipose-derived stem cells in the dystrophin-utrophin double knock-out mouse.

  5. Hyperactivity and tree-top disease induced by the baculovirus AcMNPV in Spodoptera exigua larvae are governed by independent mechanisms

    NASA Astrophysics Data System (ADS)

    van Houte, Stineke; Ros, Vera I. D.; van Oers, Monique M.

    2014-04-01

    Although many parasites are known to manipulate the behavior of their hosts, the mechanisms underlying such manipulations are largely unknown. Baculoviruses manipulate the behavior of caterpillar hosts by inducing hyperactivity and by inducing climbing behavior leading to death at elevated positions (tree-top disease or Wipfelkrankheit). Whether hyperactivity and tree-top disease are independent manipulative strategies of the virus is unclear. Recently, we demonstrated the involvement of the protein tyrosine phosphatase ( ptp) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity in Spodoptera exigua larvae. Here we show that AcMNPV ptp is not required for tree-top disease, indicating that in S. exigua baculovirus-induced hyperactivity and tree-top disease are independently induced behaviors that are governed by distinct mechanisms.

  6. Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

    PubMed

    Martínez, Juan Andrés; Bolívar, Francisco; Escalante, Adelfo

    2015-01-01

    Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the

  7. Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production

    PubMed Central

    Martínez, Juan Andrés; Bolívar, Francisco; Escalante, Adelfo

    2015-01-01

    Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several “intuitive” metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for

  8. Improving Production of Treated and Untreated Verbs in Aphasia: A Meta-Analysis

    PubMed Central

    de Aguiar, Vânia; Bastiaanse, Roelien; Miceli, Gabriele

    2016-01-01

    Background: Demographic and clinical predictors of aphasia recovery have been identified in the literature. However, little attention has been devoted to identifying and distinguishing predictors of improvement for different outcomes, e.g., production of treated vs. untreated materials. These outcomes may rely on different mechanisms, and therefore be predicted by different variables. Furthermore, treatment features are not typically accounted for when studying predictors of aphasia recovery. This is partly due to the small numbers of cases reported in studies, but also to limitations of data analysis techniques usually employed. Method: We reviewed the literature on predictors of aphasia recovery, and conducted a meta-analysis of single-case studies designed to assess the efficacy of treatments for verb production. The contribution of demographic, clinical, and treatment-related variables was assessed by means of Random Forests (a machine-learning technique used in classification and regression). Two outcomes were investigated: production of treated (for 142 patients) and untreated verbs (for 166 patients). Results: Improved production of treated verbs was predicted by a three-way interaction of pre-treatment scores on tests for verb comprehension and word repetition, and the frequency of treatment sessions. Improvement in production of untreated verbs was predicted by an interaction including the use of morphological cues, presence of grammatical impairment, pre-treatment scores on a test for noun comprehension, and frequency of treatment sessions. Conclusion: Improvement in the production of treated verbs occurs frequently. It may depend on restoring access to and/or knowledge of lexeme representations, and requires relative sparing of semantic knowledge (as measured by verb comprehension) and phonological output abilities (including working memory, as measured by word repetition). Improvement in the production of untreated verbs has not been reported very often

  9. Improving farming practices reduces the carbon footprint of spring wheat production.

    PubMed

    Gan, Yantai; Liang, Chang; Chai, Qiang; Lemke, Reynald L; Campbell, Con A; Zentner, Robert P

    2014-01-01

    Wheat is one of the world's most favoured food sources, reaching millions of people on a daily basis. However, its production has climatic consequences. Fuel, inorganic fertilizers and pesticides used in wheat production emit greenhouse gases that can contribute negatively to climate change. It is unknown whether adopting alternative farming practices will increase crop yield while reducing carbon emissions. Here we quantify the carbon footprint of alternative wheat production systems suited to semiarid environments. We find that integrating improved farming practices (that is, fertilizing crops based on soil tests, reducing summerfallow frequencies and rotating cereals with grain legumes) lowers wheat carbon footprint effectively, averaging -256 kg CO2 eq ha(-1) per year. For each kg of wheat grain produced, a net 0.027-0.377 kg CO2 eq is sequestered into the soil. With the suite of improved farming practices, wheat takes up more CO2 from the atmosphere than is actually emitted during its production. PMID:25405548

  10. Improving farming practices reduces the carbon footprint of spring wheat production.

    PubMed

    Gan, Yantai; Liang, Chang; Chai, Qiang; Lemke, Reynald L; Campbell, Con A; Zentner, Robert P

    2014-11-18

    Wheat is one of the world's most favoured food sources, reaching millions of people on a daily basis. However, its production has climatic consequences. Fuel, inorganic fertilizers and pesticides used in wheat production emit greenhouse gases that can contribute negatively to climate change. It is unknown whether adopting alternative farming practices will increase crop yield while reducing carbon emissions. Here we quantify the carbon footprint of alternative wheat production systems suited to semiarid environments. We find that integrating improved farming practices (that is, fertilizing crops based on soil tests, reducing summerfallow frequencies and rotating cereals with grain legumes) lowers wheat carbon footprint effectively, averaging -256 kg CO2 eq ha(-1) per year. For each kg of wheat grain produced, a net 0.027-0.377 kg CO2 eq is sequestered into the soil. With the suite of improved farming practices, wheat takes up more CO2 from the atmosphere than is actually emitted during its production.

  11. Improved methane production from brown algae under high salinity by fed-batch acclimation.

    PubMed

    Miura, Toyokazu; Kita, Akihisa; Okamura, Yoshiko; Aki, Tsunehiro; Matsumura, Yukihiko; Tajima, Takahisa; Kato, Junichi; Nakashimada, Yutaka

    2015-01-01

    Here, a methanogenic microbial community was developed from marine sediments to have improved methane productivity from brown algae under high salinity. Fed-batch cultivation was conducted by adding dry seaweed at 1wt% total solid (TS) based on the liquid weight of the NaCl-containing sediment per round of cultivation. The methane production rate and level of salinity increased 8-fold and 1.6-fold, respectively, at the 10th round of cultivation. Moreover, the rate of methane production remained high, even at the 10th round of cultivation, with accumulation of salts derived from 10wt% TS of seaweed. The salinity of the 10th-round culture was equivalent to 5% NaCl. The improved methane production was attributed to enhanced acetoclastic methanogenesis because acetate became rapidly converted to methane during cultivation. The family Fusobacteriaceae and the genus Methanosaeta, the acetoclastic methanogen, predominated in bacteria and archaea, respectively, after the cultivation.

  12. Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells

    PubMed Central

    Hansen, Henning Gram; Nilsson, Claes Nymand; Lund, Anne Mathilde; Kol, Stefan; Grav, Lise Marie; Lundqvist, Magnus; Rockberg, Johan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

    2015-01-01

    Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells. PMID:26657798

  13. Viral infection. II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells.

    PubMed

    Saha, D; Wu, S; Bose, A; Chatterjee, N; Chakraborty, A; Chatterjee, M; Gupta, N K

    1997-06-15

    The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia viral infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of hemin to the growth medium. Maximum enhancement was observed at 5 microM hemin. Data suggest that hemin prevents the activation of latent p67-DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus-infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells). In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells. Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin-deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells. We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG. PMID:9186500

  14. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

    PubMed Central

    2013-01-01

    Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

  15. Mid-term financial impact of animal welfare improvements in Dutch broiler production.

    PubMed

    Gocsik, E; Lansink, A G J M Oude; Saatkamp, H W

    2013-12-01

    This study used a stochastic bioeconomic simulation model to simulate the business and financial risk of different broiler production systems over a 5-yr period. Simulation analysis was conducted using the @Risk add-in in MS Excel. To compare the impact of different production systems on economic feasibility, 2 cases were considered. The first case focused on the economic feasibility of a completely new system, whereas the second examined economic feasibilities when a farm switches from a conventional to an animal welfare-improving production system. A sensitivity analysis was conducted to assess the key drivers of economic feasibility and to reveal systematic differences across production systems. The study shows that economic feasibility of systems with improved animal welfare predominantly depends on the price that farmers receive. Moreover, the study demonstrates the importance of the level and variation of the price premium for improved welfare, particularly in the first 5 yr after conversion. The economic feasibility of the production system increases with the level of welfare improvements for a sufficiently high price level for broiler meat and low volatility in producer prices. If this is not the case, however, risk attitudes of farmers become important as well as the use of potential risk management instruments.

  16. UMTRA Project-Level Cost Reduction/Productivity Improvement Program manual

    SciTech Connect

    1995-06-01

    Mission of the Uranium Mill Tailings Remedial Action (UMTRA) Project Cost Reduction/Productivity Improvement Program (CR/PIP) is to contribute to the UMTRA Project`s environmental restoration mission by providing the means to achieve and recognize continuous improvements and cost savings. This manual includes program definition, description of UMTRA project organizational responsibilities and interfaces with existing project functions, guidance to contractors, and definition of project-level functions.

  17. Clavulanic acid production by Streptomyces clavuligerus: biogenesis, regulation and strain improvement.

    PubMed

    Paradkar, Ashish

    2013-07-01

    Clavulanic acid (CA) is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus and has been successfully used in combination with β-lactam antibiotics (for example, Augmentin) to treat infections caused by β-lactamase-producing pathogens. Since the discovery of CA in the late 1970s, significant information has accumulated on its biosynthesis, and regarding molecular mechanisms involved in the regulation of its production. Notably, the genes directing CA biosynthesis are clustered along with the genes responsible for the biosynthesis of the β-lactam antibiotic, cephamycin C, and co-regulated, which makes this organism unique in that the production of an antibiotic and production of a small molecule to protect the antibiotic from its enzymatic degradation are controlled by shared mechanisms. Traditionally, the industrial strain improvement programs have relied significantly on random mutagenesis and selection approach. However, the recent availability of the genome sequence of S. clavuligerus along with the capability to build metabolic models, and ability to engineer the organism by directed approaches, has created exciting opportunities to improve strain productivity more efficiently. This review will include focus mainly on the gene organization of the CA biosynthetic genes, regulatory mechanisms that affect its production, and will include perspectives on improving strain productivity.

  18. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

    PubMed

    Cho, Kwang-Soo; Han, Eun-Heui; Kwak, Sang-Soo; Cho, Ji-Hong; Im, Ju-Seong; Hong, Su-Young; Sohn, Hwang-Bae; Kim, Yun-Hee; Lee, Shin-Woo

    2016-01-01

    Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

  19. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

    PubMed

    Cho, Kwang-Soo; Han, Eun-Heui; Kwak, Sang-Soo; Cho, Ji-Hong; Im, Ju-Seong; Hong, Su-Young; Sohn, Hwang-Bae; Kim, Yun-Hee; Lee, Shin-Woo

    2016-01-01

    Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits. PMID:27212605

  20. Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

    PubMed Central

    Korth, K L; Levings, C S

    1993-01-01

    The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms. Images Fig. 1 Fig. 2 Fig. 5 Fig. 6 PMID:8475086

  1. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    PubMed

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  2. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  3. Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus.

    PubMed

    Yamada, Yoji; Matsuyama, Takahiro; Quan, Guo-Xing; Kanda, Toshio; Tamura, Toshiki; Sahara, Ken; Asano, Shin-ichiro; Bando, Hisanori

    2002-12-01

    An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN. PMID:12457979

  4. Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection.

    PubMed Central

    Laurent, S; Vautherot, J F; Madelaine, M F; Le Gall, G; Rasschaert, D

    1994-01-01

    VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response. Images PMID:8084017

  5. Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay.

    PubMed Central

    He, J; Ching, W M; Yarbough, P; Wang, H; Carl, M

    1995-01-01

    We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus. PMID:8586723

  6. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    PubMed

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  7. Aromatic/heterocyclic amino acids and the simulated sunlight-ultraviolet inactivation of the Heliothis/Helicoverpa baculovirus

    SciTech Connect

    Ignoffo, C.M.; Garcia, C.

    1995-04-01

    Tryptophan, of five aromatic/heterocyclic amino acids (tyrosine, phenylalanine, proline, histidine) provided significant protection of the Heliothis baculovirus (HzSNPV) from inactivation by simulated ultraviolet (SUV). Fifty percent of SUV protection of HzSNPV with tryptophan or tyrosine was obtained at 0.03 mg/ml and 0.5 mg/ml, respectively. Rates as high as 100.0 mg/ml of phenylalanine, histidine, or proline provided <50% protection. The extent of tryptophan protection was correlated with its absorption in the sunlight UV-B spectra. 16 refs., 2 tabs.

  8. Productivity Improvement for the SHX--SEN's Single-Wafer High-Current Ion Implanter

    SciTech Connect

    Ninomiya, Shiro; Ochi, Akihiro; Kimura, Yasuhiko; Yumiyama, Toshio; Kudo, Tetsuya; Kurose, Takeshi; Kariya, Hiroyuki; Tsukihara, Mitsukuni; Ishikawa, Koji; Ueno, Kazuyoshi

    2011-01-07

    Equipment productivity is a critical issue for device fabrication. For ion implantation, productivity is determined both by ion current at the wafer and by utilization efficiency of the ion beam. Such improvements not only result in higher fabrication efficiency but also reduce consumption of both electrical power and process gases. For high-current ion implanters, reduction of implant area is a key factor to increase efficiency. SEN has developed the SAVING system (Scanning Area Variation Implantation with Narrower Geometrical pattern) to address this opportunity. In this paper, three variations of the SAVING system are introduced along with discussion of their effects on fab productivity.

  9. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

    PubMed Central

    Lebacq-Verheyden, A M; Kasprzyk, P G; Raum, M G; Van Wyke Coelingh, K; Lebacq, J A; Battey, J F

    1988-01-01

    The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands. Images PMID:3211139

  10. Lepidopteran Ortholog of Drosophila Breathless Is a Receptor for the Baculovirus Fibroblast Growth Factor

    PubMed Central

    Katsuma, Susumu; Daimon, Takaaki; Mita, Kazuei; Shimada, Toru

    2006-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an ∼110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis. PMID:16699027

  11. Baculovirus p35 increases pancreatic {beta}-cell resistance to apoptosis

    SciTech Connect

    Hollander, Kenneth; Bar-Chen, Michal; Efrat, Shimon . E-mail: sefrat@post.tau.ac.il

    2005-07-01

    {beta}-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in {beta}-cells exposed to cytokines and hIAPP, as well as the effects of p35 on {beta}-cell function. The p35 gene was introduced into {beta}TC-tet cells, a differentiated murine {beta}-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted {beta}-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from {beta}-cell damage caused by hIAPP in type 2 diabetes.

  12. Altered nutrient intake by baculovirus-challenged insects: Self-medication or compensatory feeding?

    PubMed

    Shikano, Ikkei; Cory, Jenny S

    2016-09-01

    Infection by parasites can alter the feeding behaviour of hosts. Some animals seek out substances that can therapeutically clear infections (self-medication), some may seek out resources to recoup resources lost while fighting off infection (compensatory feeding) and others may be manipulated to ingest substances that benefit parasite fitness (parasite manipulation of host). Recent studies have indicated that pathogen-challenged insects can self-medicate by increasing their protein intake relative to carbohydrate, which is thought to act by boosting the insect's immune response. However, increased protein intake could also be due to compensatory feeding or pathogen manipulation of the host, and a rigorous examination of all four of the testable predictions, which is necessary for verifying self-medication behaviour, has not been conducted. The therapeutic behaviour must (1) only be employed by infected individuals and (2) alleviate the potential fitness loss of the infected individual. (3) If an uninfected individual engages in the behaviour, they suffer a decrease in fitness, and lastly, (4) the parasite cannot benefit from the behaviour. In response to baculovirus-challenge (AcMNPV) at 24°C, the cabbage looper, Trichoplusia ni, increased proportional protein intake, by increasing protein intake rather than decreasing carbohydrate intake. Increased protein intake did not benefit virus fitness, but it also did not increase the probability of host survival. Increased proportional protein intake did not occur in response to TnSNPV-challenge at 24°C or in response to AcMNPV-challenge at a higher temperature (32°C), indicating that the virus-induced change in nutrient intake depends on virus identity and temperature. Since virus-challenged T. ni did not show the typical costs associated with infection, the altered nutrient intake is likely to be a compensatory response. Understanding the motivation behind pathogen-induced changes in feeding behaviour could have

  13. Baculovirus-mediated miRNA regulation to suppress hepatocellular carcinoma tumorigenicity and metastasis.

    PubMed

    Chen, Chiu-Ling; Wu, Jaw-Ching; Chen, Guan-Yu; Yuan, Pei-Hsiang; Tseng, Yen-Wen; Li, Kuei-Chang; Hwang, Shiaw-Min; Hu, Yu-Chen

    2015-01-01

    MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expressed in HCC cells. MiR-151 is aberrantly overexpressed in HCC cells and promotes HCC metastasis yet its roles on HCC tumorigenicity are unknown. To combat HCC tumorigenicity/metastasis, we developed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors that expressed (i) miR-122 precursors (pre-miR-122), (ii) miR-151 sponges, or (iii) pre-miR-122 and miR-151 sponges. Transduction of aggressive HCC cells (Mahlavu) with the pre-miR-122-expressing BV tremendously enhanced miR-122 levels for >6 weeks, suppressed the levels of downstream effectors (e.g., ADAM10 and Bcl-w), proliferation, anchorage-independent growth, motility and migration/invasion in vitro. Intratumoral injection of the pre-miR-122-expressing BV attenuated the HCC growth/metastasis. The miR-151 sponges-expressing BV diminished the miR-151 levels for 6 weeks, enhanced RhoGDIA expression, suppressed RhoGTPases, as well as motility and migration/invasion of Mahlavu cells. Intratumoral injection of the miR-151 sponge-expressing BV impeded not only HCC metastasis but also cell proliferation, MMP expression and tumor growth in vivo. The BV co-expressing pre-miR-122 and miR-151 sponges also simultaneously enhanced miR-122 expression and inhibited miR-151, and conferred antitumor/anti-metastasis effects albeit lack of synergism. These data implicate the potentials of the SB-based hybrid BV for persistently modulating miRNA and suppressing HCC tumorigenicity/metastasis. PMID:25023326

  14. Resolution and Content Improvements to MISR Aerosol and Land Surface Products

    NASA Astrophysics Data System (ADS)

    Garay, M. J.; Bull, M. A.; Diner, D. J.; Hansen, E. G.; Kalashnikova, O. V.

    2015-12-01

    Since early 2000, the Multi-angle Imaging SpectroRadiometer (MISR) instrument on NASA's Terra satellite has been providing operational Level 2 (swath-based) aerosol optical depth (AOD) and particle property retrievals at 17.6 km spatial resolution and atmospherically corrected land surface products at 1.1 km resolution. The performance of the aerosol product has been validated against ground-based Aerosol Robotic Network (AERONET) observations, model comparisons, and climatological assessments. This product has played a major role in studies of the impacts of aerosols on climate and air quality. The surface product has found a variety of uses, particularly at regional scales for assessing vegetation and land surface change. A major development effort has led to the release of an update to the operational (Version 22) MISR Level 2 aerosol and land surface retrieval products, which has been in production since December 2007. The new release is designated Version 23. The resolution of the aerosol product has been increased to 4.4 km, allowing more detailed characterization of aerosol spatial variability, especially near local sources and in urban areas. The product content has been simplified and updated to include more robust measures of retrieval uncertainty and other fields to benefit users. The land surface product has also been updated to incorporate the Version 23 aerosol product as input and to improve spatial coverage, particularly over mountainous terrain and snow/ice-covered surfaces. We will describe the major upgrades incorporated in Version 23 and present validation of the aerosol product against both the standard AERONET historical database, as well as high spatial density AERONET-DRAGON deployments. Comparisons will also be shown relative to the Version 22 aerosol and land surface products. Applications enabled by these product updates will be discussed.

  15. Evaluation of FGD-gypsum to improve forage production and reduce phosphorus lossed from Piedmont soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flue gas desulfurization gypsum (FGD-gypsum), a byproduct from coal fired electricity generators, has the potential for beneficial use in agricultural systems as a soil amendment. Similar to mined gypsum it can improve soil chemical and physical properties and increase crop productivity. FGD-gypsum ...

  16. Potential Nationwide Improvements in Productivity and Health from Better Indoor Environments

    SciTech Connect

    Fisk, W.J.; Rosenfeld, A.H.

    1998-05-01

    Theoretical considerations and empirical data suggest that existing technologies and procedures can improve indoor environments in a manner that significantly increases productivity and health. Existing literature contains moderate to strong evidence that characteristics of buildings and indoor environments significantly influence rates of respiratory disease, allergy and asthma symptoms, sick building symptoms, and worker performance. While there is considerable uncertainty in our estimates of the magnitudes of productivity gains that may be obtained by providing better indoor environments, the projected gains are very large. For the U.S., we estimate potential annual savings and productivity gains of $6 to $19 billion from reduced respiratory disease, $1 to $4 billion from reduced allergies and asthma, $10 to $20 billion from reduced sick building syndrome symptoms, and $12 to $125 billion from direct improvements in worker performance that are unrelated to health. In two example calculations, the potential financial benefits of improving indoor environments exceed costs by a factor of 8 and 14. Productivity gains that are quantified and demonstrated could serve as a strong stimulus for energy efficiency measures that simultaneously improve the indoor environment.

  17. Improving Productivity of School Systems Through Educational Technology. Final Report of Symposium.

    ERIC Educational Resources Information Center

    Scanlon, Robert G.; Weinberger, JoAnn

    Full texts of ten commissioned papers are presented, along with discussants' remarks and an analytical review by the editors. The symposium was held to explore the potentials and implications of using advanced communications and technological systems to improve educational productivity. Major foci included: 1) the use of management models to…

  18. Improving feed efficiency in dairy production systems – challenges and possibilities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improving production efficiency has always been a goal of animal agriculture to ensure an abundant food and fiber supply, and to maintain producer profitability. In recent decades, the concept of sustainable agriculture emerged, which includes the additional goals of safeguarding natural resources, ...

  19. Determinants of Adoption of Improved Fish Production Technologies among Fish Farmers in Delta State, Nigeria

    ERIC Educational Resources Information Center

    Ofuoku, A. U.; Olele, N. F.; Emah, G. N.

    2008-01-01

    This study was conducted to isolate the determinants of improved fish production technologies in Delta State, Nigeria. Data were collected from a sample population of 250 fish farmers from ten randomly selected Local Government Areas of Delta State. The data were elicited from respondents with the use of structured interview schedule while…

  20. Compressed Air System Retrofitting Project Improves Productivity at a Foundry (Cast Masters, Bowling Green, OH)

    SciTech Connect

    2002-06-01

    This case study highlights International Truck and Engine Corporation's optimization project on the compressed air system that serves its foundry, Indianapolis Casting Corporation. Due to the project's implementation, the system's efficiency was greatly improved, allowing the foundry to operate with less compressor capacity, which resulted in reduced energy consumption, significant maintenance savings, and more reliable production.