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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. PMID:26458221

  2. 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

  3. 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

  4. RAPID EFFICIENT PRODUCTION OF BACULOVIRUS EXPRESSION VECTORS

    EPA Science Inventory

    Recombinant baculoviruses have been utilized to produce foreign proteins and have the potential to be safe, efficacious insecticides. solation of recombinant virus is usually by plaque phenotype. ypical recombination rates are less than 1%, thus requiring time consuming inspectio...

  5. 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

  6. 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

  7. Quality control and analytical methods for baculovirus-based products.

    PubMed

    Roldão, António; Vicente, Tiago; Peixoto, Cristina; Carrondo, Manuel J T; Alves, Paula M

    2011-07-01

    Recombinant baculoviruses (rBac) are used for many different applications, ranging from bio-insecticides to the production of heterologous proteins, high-throughput screening of gene functions, drug delivery, in vitro assembly studies, design of antiviral drugs, bio-weapons, building blocks for electronics, biosensors and chemistry, and recently as a delivery system in gene therapy. Independent of the application, the quality, quantity and purity of rBac-based products are pre-requisites demanded by regulatory authorities for product licensing. To guarantee maximization utility, it is necessary to delineate optimized production schemes either using trial-and-error experimental setups ("brute force" approach) or rational design of experiments by aid of in silico mathematical models (Systems Biology approach). For that, one must define all of the main steps in the overall process, identify the main bioengineering issues affecting each individual step and implement, if required, accurate analytical methods for product characterization. In this review, current challenges for quality control (QC) technologies for up- and down-stream processing of rBac-based products are addressed. In addition, a collection of QC methods for monitoring/control of the production of rBac derived products are presented as well as innovative technologies for faster process optimization and more detailed product characterization. PMID:21784235

  8. 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.

  9. 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...

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

    PubMed Central

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

    2014-01-01

    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. PMID:24362443

  11. 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.

  12. 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. PMID:26923062

  13. Baculovirus as a vaccine vector

    PubMed Central

    Lu, Hsin-Yu; Chen, Yi-Hsuan; Liu, Hung-Jen

    2012-01-01

    Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has been utilized as RNA interference mediators, gene delivery vectors, and vaccine vectors for a wide variety of applications. This article focuses on the utilization of baculoviruses as vaccine vectors to prepare antigen or subunit vaccines. PMID:22705893

  14. Production of small antibacterial peptides using silkworm-baculovirus protein expression system.

    PubMed

    Fukushima, Mai; Iiyama, Kazuhiro; Yamashita, Jun; Furue, Masutaka; Tsuji, Gaku; Imanishi, Shigeo; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2013-01-01

    The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm-baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus. PMID:23742088

  15. 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

  16. 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

  17. 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.

  18. 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. PMID:26666439

  19. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system.

    PubMed

    Teng, Chao-Yi; van Oers, Monique M; Wu, Tzong-Yuan

    2013-10-01

    The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or β-synuclein (β-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, β-syn + CALR, or β-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three "helper" genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three "helper" genes, up to 10 dpi was observed. Utilization of this "triple-supporters" containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products. PMID:23900798

  20. A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system.

    PubMed

    Schlaeppi, Jean-Marc; Henke, Mario; Mahnke, Marion; Hartmann, Steffen; Schmitz, Rita; Pouliquen, Yann; Kerins, Brendan; Weber, Eric; Kolbinger, Frank; Kocher, Hans P

    2006-12-01

    The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%. PMID:16904904

  1. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

    PubMed

    Liu, Lan-Jun; Suzuki, Takako; Tsunemitsu, Hiroshi; Kataoka, Michiyo; Ngata, Noriyo; Takeda, Naokazu; Wakita, Takaji; Miyamura, Tatsuo; Li, Tian-Cheng

    2008-01-01

    The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity. PMID:18998045

  2. Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

    PubMed

    Johnson, T M; Pease, E A; Li, J K; Tien, M

    1992-08-01

    Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification. PMID:1632652

  3. 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

  4. 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

  5. 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

  6. 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

  7. CHARACTERIZATION OF SHRIMP BACULOVIRUS

    EPA Science Inventory

    The research undertaken involved the partial characterization of a baculovirus of the pink shrimp, Penaeus duorarum. The significance of the study is related to the fact that the shrimp baculovirus is morphologically similar to insect vaculoviruses which were considered unique to...

  8. Improvement in the UV resistance of baculoviruses by displaying nano-zinc oxide-binding peptides on the surfaces of their occlusion bodies.

    PubMed

    Li, Jin; Zhou, Yin; Lei, Chengfeng; Fang, Wei; Sun, Xiulian

    2015-08-01

    The sensitivity of baculoviruses to UV radiation severely limits their large-scale application as biological insecticides. The polyhedron envelope of a baculovirus, which is composed of carbohydrate and polyhedron envelope protein (PEP), is a significant structure for the stability and persistence of occlusion bodies (OBs) under environmental conditions. The results of this study revealed that the rough pitted surface phenotype of a pep-null Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could not be rescued by any of its homologues, such as Helicoverpa armigera nucleopolyhedrovirus pep or Cydia pomonella granulovirus putative peps. In contrast, the N-terminal and middle flexible region (NM region, 1-167 aa) of AcMNPV PEP were able to form an intact OB envelope. Furthermore, this region was capable of carrying eGFP to the surfaces of the OBs. To improve the UV resistance of AcMNPV OBs, two peptides capable of specifically binding to nano-ZnO were separately fused to the NM region of PEP. Under laboratory conditions, infectivity of the recombinant viruses binding to nano-ZnO particles was about ninefold higher than that without the nano-ZnO particles after UV-B irradiation. Pot experiments revealed that the half-life of the recombinant baculovirus binding nano-ZnO particles was 3.3 ± 0.15 days, which was significantly longer than that of the control virus (0.49 ± 0.06 days). These results therefore represent a new approach for the protection the baculoviral insecticides against UV irradiation in the field. PMID:25895092

  9. Baculoviruses: Molecular Biology of Mosquito Baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses found in mosquitoes have been assigned to the Nucleopolyhedroviruses and are of growing interest as they may represent a separate branch within the Baculoviridae that existed prior to the split of lepidopteran nucleopolyhedroviruses and granuloviruses. They may also be ancestral to th...

  10. Chapter 4: Baculoviruses and other occluded insect viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are among the most thoroughly studied insect pathogens. Members of Baculoviridae possess a large, circular double-stranded DNA genome contained within the enveloped nucleoprotein core of a rod-shaped virion. Baculovirus replication is distinguished by the production of two different ...

  11. 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.

  12. 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

  13. 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.

  14. Production and purification of VP2 protein of porcine parvovirus expressed in an insect-baculovirus cell system

    PubMed Central

    2010-01-01

    The porcine parvovirus (PPV) VP2 protein was expressed in an insect-baculovirus cell system and was purified using Ni-NTA affinity column chromatography. The recombinant 6-His-tagged VP2 protein with molecular mass (Mr) of about 64 kDa was detected by anti-his antibody and anti-PPV serum. Electron microscopy showed that the purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The expressed VP2 was antigenically similar to the native capsid protein according to HA and a Western blotting assay performed with polyclonal antibodies collected from an outbreak of PPV in one farm. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of PPV or in the vaccination against PPV in the future. PMID:21143963

  15. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  16. Incorporation of partial polyhedrin homology sequences (PPHS) enhances the production of cloned foreign genes in a baculovirus expression system.

    PubMed

    Gong, Zhaohui; Jin, Yongfeng; Zhang, Yaozhou

    2006-03-01

    Baculovirus expression vector systems (BEVSs) have been used extensively for high-level expression of cloned foreign genes. In many instances, the levels of recombinant protein(s) produced in insect cells and larvae are insufficient for experimental purposes. Thus new techniques and methods are needed to increase significantly the protein expression levels in BEVS. In the present paper, we describe the incorporation of a 15 bp element derived from the 5'-end partial sequence of the polyhedrin gene, which contains the non-coding sequence ATAAAT and the coding sequence ATGCCGAAT, into the 5'-end of the CTB (cholera toxin B subunit)-INS (insulin) fusion gene. With the addition of the PPHS (partial polyhedrin homology sequences), two extra amino acids (Pro-Asn) were added to the N-terminus of the mCTB-INS (modified CTB-INS) fusion protein. This new fusion protein was expressed in both insect cells and larvae using BEVSs. We found that the addition of PPHS enhanced 4-fold the expression of CTB-INS in both insect cells and larvae. Further analysis revealed that the additional two amino acids in mCTB-INS did not significantly affect binding affinity for G(M1) ganglioside. Therefore the PPHS can be used as a constitutive element immediately downstream of the polyhedrin promoter to induce significant increases in the expression levels of cloned foreign genes. PMID:16313236

  17. 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

  18. 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

  19. Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

    PubMed

    George, Steve; Jauhar, Altamash M; Mackenzie, Jennifer; Kieβlich, Sascha; Aucoin, Marc G

    2015-09-01

    The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system. PMID:25850946

  20. 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

  1. Productivity Improvement and Professionalism.

    ERIC Educational Resources Information Center

    Young, Benjamin I., Jr.

    1980-01-01

    The author presents a developmental model designed to help productivity improvement and professional development become an integral part of the natural everyday work experience. Examples and a summarized model are included. (CT)

  2. 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

  3. HSP70 induction during baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are arthropod-specific double-stranded DNA viruses that have been employed as bio-insecticides against crop pests and to produce heterologous proteins in baculovirus expression systems. Although a consensus has emerged on the dominant molecular events driving baculovirus replication i...

  4. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  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. Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

    PubMed

    Kost, T A; Ignar, D M; Clay, W C; Andrews, J; Leray, J D; Overton, L; Hoffman, C R; Kilpatrick, K E; Ellis, B; Emerson, D L

    1997-04-29

    Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA. PMID:9185859

  8. CO-OCCLUSION AND PERSISTENCE OF A BACULOVIRUS MUTANT LACKING THE POLYHEDRIN GENE

    EPA Science Inventory

    A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. oinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Aucographa californica nuclear polyhedrosis...

  9. 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

  10. 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

  11. 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

  12. 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. PMID:25724418

  13. Book review: Baculovirus Molecular Biology, Second Edition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of cell culture and molecular biology methodologies to the study of baculoviruses has resulted in an explosion of information on this group of insect pathogens. The quantity of the corresponding literature on baculoviruses has reached a level difficult for any one researcher to mast...

  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. PMID:26743500

  15. 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

  16. 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

  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. [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. PMID:23233923

  19. Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

    PubMed

    Mahiou, J; Abastado, J P; Cabanie, L; Godeau, F

    1998-03-01

    We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart. PMID:9480929

  20. Improving haul truck productivity

    SciTech Connect

    Fiscor, S.

    2007-06-15

    The paper reviews developments in payload management and cycle times. These were discussed at a roundtable held at the Haulage and Loading 2007 conference held in May in Phoenix, AZ, USA. Several original equipment manufacturers (OEMs) explaind what their companies were doing to improve cycle times for trucks, shovels and excavators used in surface coal mining. Quotations are given from Dion Domaschenz of Liebherr and Steve Plott of Cat Global Mining. 4 figs.

  1. RADIOIMMUNOASSAY ANALYSIS OF BACULOVIRUS GRANULINS AND POLYHEDRINS

    EPA Science Inventory

    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 Spodotera frugiperda. Antisera were...

  2. Visualization of Intracellular Pathways of Engineered Baculovirus in Mammalian Cells

    PubMed Central

    Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

    2014-01-01

    Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy. PMID:24457070

  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. Improving Productivity via QWL Centers.

    ERIC Educational Resources Information Center

    Bentley, Marion T.; Hansen, Gary B.

    1980-01-01

    Gives a brief history of productivity improvement legislation in the United States and of the development and demise of the National Center for Productivity and Quality of Working Life (QWL). Describes existing productivity and QWL centers, including their locations, scope, services, and activities, and urges greater support at the federal level.…

  5. Peptide-mediated interference with baculovirus transduction.

    PubMed

    Mäkelä, Anna R; Närvänen, Ale; Oker-Blom, Christian

    2008-03-20

    Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells. PMID:18294718

  6. [Research Advances in Baculovirus Occlusion-derived Virions].

    PubMed

    Zhu, Shimao; Li, Hui

    2016-01-01

    Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses. PMID:27295890

  7. Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of the 42 baculovirus genomes that have been sequenced to date, some are from closely related viruses that share overall nucleotide sequence identities in excess of 95%. Comparative analysis of genomic sequences from closely related viruses can provide insight into how baculoviruses evolve and adap...

  8. 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

  9. 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

  10. GENETICALLY ENGINEERED BACULOVIRUSES AS AGENTS FOR PEST CONTROL

    EPA Science Inventory

    Baculoviruses constitute one of the largest and most diverse groups of insect pathogenic viruses. Following World War II, research on baculovirus intensified with the realization that these relatively virulent insect-specific viruses were widespread in nature among economically i...

  11. Use of Bacterial Artificial Chromosomes in Baculovirus Research and Recombinant Protein Expression: Current Trends and Future Perspectives

    PubMed Central

    Roy, Polly; Noad, Rob

    2012-01-01

    The baculovirus expression system is one of the most successful and widely used eukaryotic protein expression methods. This short review will summarise the role of bacterial artificial chromosomes (BACS) as an enabling technology for the modification of the virus genome. For many years baculovirus genomes have been maintained in E. coli as bacterial artificial chromosomes, and foreign genes have been inserted using a transposition-based system. However, with recent advances in molecular biology techniques, particularly targeting reverse engineering of the baculovirus genome by recombineering, new frontiers in protein expression are being addressed. In particular, BACs have facilitated the propagation of disabled virus genomes that allow high throughput protein expression. Furthermore, improvement in the selection of recombinant viral genomes inserted into BACS has enabled the expression of multiprotein complexes by iterative recombineering of the baculovirus genome. PMID:23762754

  12. 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.

  13. 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.

  14. 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

  15. Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) and a wild-type isolate (Sf3) of the same baculovirus. Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioas...

  16. Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus

    PubMed Central

    2012-01-01

    Background West Nile Virus (WNV) is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells. Results In the present study, recombinant baculoviruses expressing WNV premembrane (prM) and envelope (E) proteins under the cytomegalovirus (CMV) promoter with or without vesicular stomatitis virus glycoprotein (VSV/G) were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse) induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ) in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6. Conclusions These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus. PMID:22799608

  17. 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

  18. Functional characterization of hesp018, a baculovirus-encoded serpin gene.

    PubMed

    Ardisson-Araujo, Daniel M P; Rohrmann, George F; Ribeiro, Bergmann M; Clem, Rollie J

    2015-05-01

    The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects. PMID:25573886

  19. Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

    PubMed Central

    Erdmann, Dirk; Zimmermann, Catherine; Fontana, Patrizia; Hau, Jean-Christophe; De Pover, Alain; Chène, Patrick

    2010-01-01

    Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for

  20. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    PubMed

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  1. 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

  2. Productivity improvement for longwall development

    SciTech Connect

    Whipkey, K.

    2005-08-01

    Industry survey reveals coal operators thoughts about the use of different techniques to keep development ahead of longwall production. Factors considered that can optimise productivity include mine design (the number of entries, size of pillars etc.), work schedules, preventative maintenance programs and good management. The article was adapted from a presentation to Longwall USA 2005, in June 2005 (Pittsburgh, PA, USA). 3 figs.

  3. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    PubMed

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  4. Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design.

    PubMed

    Lin, Shih-Yeh; Yeh, Chia-Tsui; Li, Wan-Hua; Yu, Cheng-Ping; Lin, Wen-Chin; Yang, Jyh-Yuan; Wu, Hsueh-Ling; Hu, Yu-Chen

    2015-10-01

    Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease in the Asia-Pacific region. To produce the virus-like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co-express EV71 P1 polypeptide and 3CD protease using the Bac-to-Bac(®) vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v-cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF-P1-C3CD, a recombinant baculovirus constructed using the flashBAC GOLD(TM) system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five(TM) cells with BacF-P1-C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross-protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High Five(TM) culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. PMID:25997678

  5. Hormone activation of baculovirus expressed progesterone receptors.

    PubMed

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  6. 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.

  7. 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

  8. 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. PMID:25993013

  9. [Advances in baculovirus per os infection and per os infectivity factor--A review].

    PubMed

    Li, Hui; Guo, Caiping; Zhu, Shimao

    2015-04-01

    Baculoviruses are a family of arthropod-specific viruses that mainly affect insects of the orders Lepidoptera, Hymenoptera, and Diptera. In nature, baculoviruses establish infection in their hosts orally and a battery of proteins designated as per os infectivity factors play pivotal roles in baculovirus per os infection. This review summarizes the basic characteristics of baculovirus and discusses the main events that baculovirus establishes per os infection, including the evolutionary advantages for baculovirus to initiate infection through the oral route, the binding and fusion of baculovirus virions with insect midgut microvilli and the functional roles of baculovirus per os infectivity factors. These achievements and advances should promise to shed light on the understanding and utilization of baculovirus for bio-control and exogenous gene expression in the future. PMID:26211313

  10. Ancient Coevolution of Baculoviruses and Their Insect Hosts

    PubMed Central

    Herniou, Elisabeth A.; Olszewski, Julie A.; O'Reilly, David R.; Cory, Jenny S.

    2004-01-01

    If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts. PMID:15016845

  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. Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells

    PubMed Central

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul

    2012-01-01

    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

  13. 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

  14. Control of programmed cell death by the baculovirus genes p35 and iap.

    PubMed Central

    Clem, R J; Miller, L K

    1994-01-01

    The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells. Images PMID:8035800

  15. The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

    PubMed Central

    Prikhod’ko, Elena A.; Miller, Lois K.

    1999-01-01

    While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection. PMID:10400766

  16. 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)

  17. Interchange. Program Improvement Products Identified through Networking.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. National Center for Research in Vocational Education.

    This catalog lists exemplary field-based program improvement products identified by the Dissemination and Utilization Products and Services Program (D&U) at the National Center for Research in Vocational Education. It is designed to increase awareness of these products among vocational educators and to provide information about them that…

  18. 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.

  19. DEVELOPMENT OF GENETICALLY ENHANCED BACULOVIRUS PESTICIDES

    EPA Science Inventory

    The assessment of potential environmental impacts of genetically improved viral pesticides will include an evaluation of the properties of the foreign gene product(s) as well as the biological properties of altered virus itself. It is anticipated that in the near future several t...

  20. 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. PMID:17141223

  1. 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. PMID:24401775

  2. Improving product introduction through effective design reviews.

    PubMed

    Pelnik, Tammy M

    2003-01-01

    The design review process is a part of the manufacturer's due diligence in developing a safe and effective product. Design review provides early and on-going independent feedback to developers. By adopting a proactive review process, design improvements can be pursued at an optimum time in the product development effort, i.e., when it will cost less to implement changes and when these changes may have the greatest impact. Effective implementation of the design review requirement will lead to better medical products and improved product introduction results. PMID:12677754

  3. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    PubMed Central

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  4. 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…

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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 ...

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    PubMed Central

    2011-01-01

    Background Foot-and-mouth disease (FMD) is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV) under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE), and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA) and indirect sandwich-ELISA (IS-ELISA) showed both recombinant baculoviruses (with or without T-cell epitopes) were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ). Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P < 0.01) and higher neutralizing antibody titer (P < 0.05). Although the inactivated vaccine produced the highest titer of neutralizing antibodies, a lower IFN-γ expression was observed compared to the two recombinant pseudotype baculoviruses. Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection

  16. Enhanced effect of fluorescent whitening agent on peroral infection for recombinant baculovirus in the host Bombyx mori L.

    PubMed

    Wang, Bing; Shang, Jinyan; Liu, Xunli; Cui, Weizheng; Wu, Xiaofeng; Zhao, Na

    2007-01-01

    The low efficiency of the oral infectivity of recombinant polyhedrin-negative baculovirus is a major bottleneck in the application of the baculovirus expression system in the silkworm (Bombyx mori L). In this study, the effects of a fluorescent whitening agent on improving the oral infection for the recombinant Bombyx mori nuclear polyhedrosis virus in silkworm larva and their possible mechanism were investigated. The results showed that the peroral infection can be remarkably enhanced by adding VBL into the larval artificial diet. The maximum infection rate reached as high as 90% with the concentration of VBL (1%), which was then considered as optimal. The total protease activity and pH value of the larval intestinal juice were found to be lower when compared to the control, indicating an abnormal physiological change of the larval digestive system by VBL, which, in turn, resulted in improved peroral infection of recombinant virus. PMID:17160363

  17. [Expression and characterization of rabies virus nucleoprotein in baculovirus].

    PubMed

    Cao, Lei; Tang, Qing; Tao, Xiao-yan; Huang, Ying; Du, Jia-liang; Zhang, Shou-feng; Hu, Rong-liang

    2010-09-01

    To construct a recombinant expression plasmid Bacmid-N containing the N gene of Rabies Virus, the N gene of RV CVS-11 strain was cloned into the baculovirus shuttle vector (Bacmid). Recombinant Baculovirus AcMNPV-N (P1 Viral stock) was obtained by transfecting the Bacmid-N into the insect cell line of Sf9. The expressed nucleoprotein was identified and analysised by ELISA, FA, SDS-PAGE and Western blot assays. The results showed that the NP protein was expressed intracellularly and had a good antigenicity, which would be potentially used for further study on the diagnostic reagent of rabies virus detection. PMID:21043133

  18. Improving Educational Productivity and School Finance.

    ERIC Educational Resources Information Center

    Odden, Allan; Clune, William

    1995-01-01

    Presents strategies for improving student academic achievement and U.S. school productivity. Among the strategies are the following: making student achievement a priority goal for schools, enhancing and toughening the curriculum, managing all educational resources at the school and classroom levels, providing a variety of system incentives towards…

  19. How Online Faculty Improve Student Learning Productivity

    ERIC Educational Resources Information Center

    Meyer, Katrina A.; McNeal, Larry

    2011-01-01

    Ten experienced online faculty were interviewed to elicit examples of how they improved student learning productivity in their online courses. The ten faculty represented nine different states, 13 different fields or disciplines, and all were tenured or tenure-track at master's or doctoral level higher education institutions. Based on a thematic…

  20. Blast optimization for improved dragline productivity

    SciTech Connect

    Humphreys, M.; Baldwin, G.

    1994-12-31

    A project aimed at blast optimization for large open pit coal mines is utilizing blast monitoring and analysis techniques, advanced dragline monitoring equipment, and blast simulation software, to assess the major controlling factors affecting both blast performance and subsequent dragline productivity. This has involved collaborative work between the explosives supplier, mine operator, monitoring equipment manufacturer, and a mining research organization. The results from trial blasts and subsequently monitored dragline production have yielded promising results and continuing studies are being conducted as part of a blast optimization program. It should be stressed that the optimization of blasting practices for improved dragline productivity is a site specific task, achieved through controlled and closely monitored procedures. The benefits achieved at one location can not be simply transferred to another minesite unless similar improvement strategies are first implemented.

  1. 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

  2. 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.

  3. 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.

  4. The 3Rs of Productivity Improvement: Responsibility, Recognition, Reward.

    ERIC Educational Resources Information Center

    Training, 1979

    1979-01-01

    Describes the American Production Center and a productivity improvement system in which people become part of the productivity solution when given responsibility, recognition, and reward for productivity improvement. (LRA)

  5. Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus.

    PubMed

    Braconi, Carla Torres; Ardisson-Araújo, Daniel Mendes Pereira; Paes Leme, Adriana Franco; Oliveira, Juliana Velasco de Castro; Pauletti, Bianca Alves; Garcia-Maruniak, Alejandra; Ribeiro, Bergmann Morais; Maruniak, James E; Zanotto, Paolo Marinho de Andrade

    2014-04-01

    Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery. PMID:24443474

  6. Hybrid of baculovirus and galactosylated PEI for efficient gene carrier.

    PubMed

    Kim, You-Kyoung; Choi, Jae Young; Jiang, Hu-Lin; Arote, Rohidas; Jere, Dhananjay; Cho, Myung-Haing; Je, Yeon Ho; Cho, Chong-Su

    2009-04-25

    Baculovirus, containing an appropriate eukaryotic promoter, is considered an attractive approach for an efficient and safe gene delivery vehicle. However, the drawbacks of baculovirus, such as the lack of specificity and the inactivation of baculovirus by the complement system in human serum, negatively affect efficient gene delivery. Therefore, a hybrid system utilizing the positive aspects of both viral and non-viral vector systems would be useful to overcome the obstacles of either system alone. In this study, we constructed a hybrid system composed of baculovirus (B) and galactosylated polyethylenimine (GP)/DNA complexes through electrostatic interaction. The resulting GP/B hybrid had suitable physicochemical properties and low cytotoxicity for use in gene therapy. Furthermore, the GP/B significantly enhanced transduction efficiency and showed good cell-specificity compared to either viral or non-viral vector systems. These results suggest that the GP/B hybrid system can be used in gene therapy to enhance transduction efficiency and hepatocyte specificity. PMID:19272627

  7. 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...

  8. Improved Production Of Wrought Articles From Powders

    NASA Technical Reports Server (NTRS)

    Thomas, James R.; Singleton, Ogle R.

    1994-01-01

    Improved technique for consolidation of powders into dense articles developed. Peripheral bands used in consolidation, forging, and rolling operations. Facilitates consolidation of dispersion-hardened aluminous powders and composite mixtures for processing to such useful wrought articles as plates and sheets. Potential use in production of plates and sheets and perhaps other objects from "hard" powders, particularly from powders, objects made from which have propensity to crack when mechanically worked to other forms.

  9. Toward Improving Streamflow Forecasts Using SNODAS Products

    NASA Astrophysics Data System (ADS)

    Barth, C.; Boyle, D. P.; Lamorey, G. W.; Bassett, S. D.

    2007-12-01

    As part of the Water 2025 initiative, researchers at the Desert Research Institute in collaboration with the U.S. Bureau of Reclamation are developing and improving water decision support system (DSS) tools to make seasonal streamflow forecasts for management and operations of water resources in the mountainous western United States. Streamflow forecasts in these areas may have errors that are directly related to uncertainties resulting from the lack of direct high resolution snow water equivalent (SWE) measurements. The purpose of this study is to investigate the possibility of improving the accuracy of streamflow forecasts through the use of Snow Data Assimilation System (SNODAS) products, which are high-resolution daily estimates of snow cover and associated hydrologic variables such as SWE and snowmelt runoff that are available for the coterminous United States. To evaluate the benefit of incorporating the SNODAS product into streamflow forecasts, a variety of Ensemble Streamflow Predictions (ESP) are generated using the Precipitation-Runoff Modeling System (PRMS). A series of manual and automatic calibrations of PRMS to different combinations of measured (streamflow) and estimated (SNODAS SWE) hydrologic variables is performed for several watersheds at various scales of spatial resolution. This study, which is embedded in the constant effort to improve streamflow forecasts and hence water operations DSS, shows the potential of using a product such as SNODAS SWE estimates to decrease parameter uncertainty related to snow variables and enhance forecast skills early in the forecast season.

  10. 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

  11. Generating a host range-expanded recombinant baculovirus.

    PubMed

    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

  12. Actions for productivity improvement in crew training

    NASA Technical Reports Server (NTRS)

    Miller, G. E.

    1985-01-01

    Improvement of the productivity of astronaut crew instructors in the Space Shuttle program and beyond is proposed. It is suggested that instructor certification plans should be established to shorten the time required for trainers to develop their skills and improve their ability to convey those skills. Members of the training cadre should be thoroughly cross trained in their task. This provides better understanding of the overall task and greater flexibility in instructor utilization. Improved facility access will give instructors the benefit of practical application experience. Former crews should be integrated into the training of upcoming crews to bridge some of the gap between simulated conditions and the real world. The information contained in lengthy and complex training manuals can be presented more clearly and efficiently as computer lessons. The illustration, animation and interactive capabilities of the computer combine an effective means of explanation.

  13. 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.

  14. 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...

  15. 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.

  16. Display of VP1 on the Surface of Baculovirus and Its Immunogenicity against Heterologous Human Enterovirus 71 Strains in Mice

    PubMed Central

    Kiener, Tanja K.; Chow, Vincent T. K.; Kwang, Jimmy

    2011-01-01

    Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization. Methodology/Principal Finding In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 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 VP1 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 VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains. Conclusion Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns. PMID:21747954

  17. 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

  18. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.

    PubMed

    Urakawa, T; Ferguson, M; Minor, P D; Cooper, J; Sullivan, M; Almond, J W; Bishop, D H

    1989-06-01

    A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses. PMID:2543785

  19. Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

    PubMed Central

    Iatrou, K; Meidinger, R G; Goldsmith, M R

    1989-01-01

    Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required. Images PMID:2556701

  20. Improvement of wool production through genetic engineering.

    PubMed

    Rogers, G E

    1990-01-01

    Wool is a natural fibre popular in the manufacture of outer clothing and is a vital source of income for several countries. Fundamental knowledge of how wool grows has been gained from research on the biology of sheep, and the prospects ahead are to take advantage of the development and application of recombinant DNA technology to improve the efficiency of production of wool and its quality. This review surveys the possibilities for producing transgenic sheep, modifying rumen bacteria and forage crops, thus increasing the nutritional benefit sheep gain from pasture. PMID:1366571

  1. 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. PMID:26944455

  2. 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.

  3. WFC3: Improved WFC3 Calibration Products

    NASA Astrophysics Data System (ADS)

    Gunning, Heather C.; Sosey, M. L.; Anderson, J.; Lee, J. C.; Pirzkal, N.; MacKenty, J. W.; Kozhurina-Platais, V.; Deustua, S. E.; Hammer, D.; Dahlen, T.; Sabbi, E.; Mack, J.; Baggett, S. M.; WFC3 Team

    2014-01-01

    The Wide Field Camera 3 (WFC3) is a fourth-generation UV/visible and IR imaging instrument on the Hubble Space Telescope (HST). Installed in May 2009, during HST servicing mission 4, both channels have been performing very well on-orbit. To provide optimum calibrated data, the WFC3 team routinely updates and refines the calibration software and associated files, designated as calibration products. We present some of the recently improved calibration products that will be of interest to current and future users of WFC3, including information on the chip-dependent zeropoints and flat fields, post-flash calibrations, and detector-to-image distortion corrections. The latter results in four new extensions (two per chip and dimension), in all UVIS FLTs retrieved from MAST after September 10, 2013. The D2IMFILE contains astrometric corrections for shifts of the raw X and Y positions induced by the lithographic-mask pattern. We discuss the migration of CALWF3 from the STSDAS package to HSTCAL, a package independent of IRAF; as a consequence, the IRAF/STSDAS version of CALWF3 is no longer being updated. Finally, we summarize recent improvements to aXe, a PyRAF/IRAF software package that enables automated extraction of spectra from WFC3 slitless spectral (grism) images. Updated versions of aXe are made available as part of the STSDAS testing environment (SSBX).

  4. Molten carbonate fuel cell product design improvement

    SciTech Connect

    P. Voyentzie; T. Leo; A. Kush; L. Christner; G. Carlson; C. Yuh

    1998-12-20

    Drawing on the manufacture, field test, and post-test experience of the sixteen Santa Clara Demonstration Project (SCDP) stacks, ERC is finalizing the next generation commercial entry product design. The second generation cells are 50% larger in area, 40% lighter on equal geometric area basis, and 30% thinner than the earlier design. These improvements have resulted in doubling of the full-height stack power. A low-cost and high-strength matrix has also been developed for improving product ruggedness. The low-cost advanced cell design incorporating these improvements has been refined through six short stack tests. Power production per cell of two times the SCDP maximum power operation, over ten thermal cycles, and overall operating flexibility with respect to load and thermal changes have been demonstrated in these short stack tests. An internally insulated stack enclosure has been designed and fabricated to eliminate the need for an inert gas environment during operation. ERC has acquired the capability for testing 400kW full-height direct fuel ceil (DFC) stack and balance-of-plant equipment. With the readiness of the power plant test facility, the cell package design, and the stack module, full-height stack testing has begun. The first full- height stack incorporating the post-SCDP second generation design was completed. The stack reached a power level of 253 kW, setting a world record for the highest power production from the advanced fuel cell system. Excellent performance uniformity at this power level affirmed manufacturing reproducibility of the components at the factory. This unoptimized small size test has achieved pipeline natural gas to DC electricity conversion efficiency of 47% (based on lower heating value - LHV) including the parasitic power consumed by the BOP equipment; that should translate to more than 50% efficiency in commercial operation, before employing cogeneration. The power plant system also operated smoothly. With the success of this

  5. [Estimate the safety of baculovirus insecticides: the history and the status Quo].

    PubMed

    Xiao, H Z; Qi, Y P; Yang, F H

    2001-05-01

    This article reviews the evaluation of security of baculovirus used as a pesticide. Since 1970s, the scholars have done a lot of experiments with kinds of baculovirus to test the security of a large number kinds of living things even our human beings. Almost all experiments proved that baculovirus is secure, but some experiments came to different conclusions. These gave rise to great debate twice when they were published, but these conclusions have been proved to be wrong with later test by other scientists or the author himself. Since 90s baculovirus have been used a great deal as the vector to express the foreign gene. Some of them reported the expression in mammalian cells, which brought the suspicious of the baculovirus safety. This article made an analysis and a conclusion about them. Also, this article laid emphasis on the security of recombination baculovirus with the results of the security experiment of self-made recombination baculovirus pesticide. In the last analysis, this article draws a conclusion that the baculovirus and recombinant baculovirus insecticides are secure. PMID:11517591

  6. Use of Whole Genome Sequence Data To Infer Baculovirus Phylogeny

    PubMed Central

    Herniou, Elisabeth A.; Luque, Teresa; Chen, Xinwen; Vlak, Just M.; Winstanley, Doreen; Cory, Jennifer S.; O'Reilly, David R.

    2001-01-01

    Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses. PMID:11483757

  7. 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.

  8. Molten Carbonate Fuel Cell Product Design Improvement

    SciTech Connect

    1996-03-01

    This annual report provides results of Energy Research Corporation`s technical approach to performing the program `Molten Carbonate Fuel Cell (MCFC) Product Design Improvement` covered under the DOE-ERC Cooperative Agreement DE-FC21-95MC31184. This work is supported by DOE/METC and DOD/DARPA as well as ERC Team funds. The objective of the DOE-sponsored program is to advance the direct carbonate fuel cell technology to a level suitable for commercial entry for civilian applications. The overall objective of the DOD/DARPA initiative is to adapt the civilian 2 MW-Class fuel cell power plant for dual fuel DOD applications. This program is designed to advance the carbonate fuel cell technology from the power plant demonstration status to the commercial entry early production unit design stage. The specific objectives which will allow attainment of these overall program goals are: (1) Provide environmental information to support DOE evaluation with respect to the National Environmental Policy Act (NEPA), (2) Define market-responsive power plant requirements and specifications, (3) Establish design for multifuel, low-cost, modular, market-responsive power plant, (4) Resolve power plant manufacturing issues and define the design for the commercial manufacturing facility, (5) Acquire capabilities to support developmental testing of 0370 stacks and BOP equipment as required to prepare for commercial design, and (6) Resolve stack and BOP equipment technology issues and design, build, and field test a modular commercial prototype power plant to demonstrate readiness of the power plant for commercial entry.

  9. 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.

  10. 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

  11. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    PubMed Central

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  12. Improving Infrared Astronomical Satellite Data Products

    NASA Astrophysics Data System (ADS)

    Melis, Carl

    The InfraRed Astronomical Satellite (IRAS) revolutionized astrophysics when it surveyed the sky at mid- and far-infrared wavelengths for the first time. Since then other missions have built upon the legacy of IRAS, but none have surpassed its far-infrared dataset. It is unlikely that a new all-sky far-infrared survey will fly any time in the near future thus making IRAS data a valuable commodity as the most sensitive and complete all-sky survey at far-infrared wavelengths that will likely remain so for at least another decade. Given its importance, it is prudent to make any attempt to extend the IRAS dataset to its limits. Using advanced image processing techniques we believe it is possible to improve IRAS data both in sensitivity and resolution by an order of magnitude or more. We will use revised IRAS data products to explore dusty circumstellar material, especially its temporal evolution, with an emphasis on protoplanetary and debris disks. Before embarking on a full-scale re-analysis of all IRAS survey data, we first propose a pilot survey in which we will validate our methodology. The proposal goals are well- aligned with the scope of the Astrophysics Data Analysis program and directly inform the NASA Science Mission Directorate to understand our cosmic origins, especially to determine how planets form around young stars, and will likely have broader impacts in all areas of astrophysics much like the original IRAS survey did.

  13. 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...

  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. Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.

    PubMed

    Pajot-Augy, E; Bozon, V; Remy, J J; Couture, L; Salesse, R

    1999-03-01

    The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors. PMID:10102991

  17. 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

  18. 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

  19. 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.

  20. 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

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

    PubMed

    Rebolledo, Dulce; Lasa, Rodrigo; Guevara, Roger; Murillo, Rosa; Williams, Trevor

    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. Productivity Sharing Programs: Can They Contribute to Productivity Improvement?

    ERIC Educational Resources Information Center

    General Accounting Office, Washington, DC.

    Productivity sharing plans were studied to determine how they operate, what benefits result, and whether long-term increases in productivity can be realized through the program. Thirty-six firms were interviewed that had productivity sharing plans. Nine firms that had either rejected adoption of a productivity sharing plan or were still…

  3. 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.

  4. Productivity improvement through cycle time analysis

    NASA Astrophysics Data System (ADS)

    Bonal, Javier; Rios, Luis; Ortega, Carlos; Aparicio, Santiago; Fernandez, Manuel; Rosendo, Maria; Sanchez, Alejandro; Malvar, Sergio

    1996-09-01

    A cycle time (CT) reduction methodology has been developed in the Lucent Technology facility (former AT&T) in Madrid, Spain. It is based on a comparison of the contribution of each process step in each technology with a target generated by a cycle time model. These targeted cycle times are obtained using capacity data of the machines processing those steps, queuing theory and theory of constrains (TOC) principles (buffers to protect bottleneck and low cycle time/inventory everywhere else). Overall efficiency equipment (OEE) like analysis is done in the machine groups with major differences between their target cycle time and real values. Comparisons between the current value of the parameters that command their capacity (process times, availability, idles, reworks, etc.) and the engineering standards are done to detect the cause of exceeding their contribution to the cycle time. Several friendly and graphical tools have been developed to track and analyze those capacity parameters. Specially important have showed to be two tools: ASAP (analysis of scheduling, arrivals and performance) and performer which analyzes interrelation problems among machines procedures and direct labor. The performer is designed for a detailed and daily analysis of an isolate machine. The extensive use of this tool by the whole labor force has demonstrated impressive results in the elimination of multiple small inefficiencies with a direct positive implications on OEE. As for ASAP, it shows the lot in process/queue for different machines at the same time. ASAP is a powerful tool to analyze the product flow management and the assigned capacity for interdependent operations like the cleaning and the oxidation/diffusion. Additional tools have been developed to track, analyze and improve the process times and the availability.

  5. 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. PMID:25800821

  6. Baculovirus vectors for antiangiogenesis-based cancer gene therapy.

    PubMed

    Luo, W-Y; Shih, Y-S; Lo, W-H; Chen, H-R; Wang, S-C; Wang, C-H; Chien, C-H; Chiang, C-S; Chuang, Y-J; Hu, Y-C

    2011-09-01

    Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors. PMID:21701531

  7. Modularity and evolutionary constraints in a baculovirus gene regulatory network

    PubMed Central

    2013-01-01

    Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates

  8. Can humic products become mainstream amendments for improving crop production?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Humic products have been used in cropland production for several decades but only by small numbers of farmers. Appreciable proportions of field studies demonstrate efficacy of the products for numerous crops, justifying their further evaluation. Their adoption by mainstream farmers could be accelera...

  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. Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

    Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection. PMID:9311875

  12. Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.

    2012-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals. PMID:23035220

  13. 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

  14. 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.

  15. 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.

  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. PMID:23484123

  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. Overexpression of PNGase at from baculovirus-infected insect cells.

    PubMed

    Ftouhi Paquin, N; Tarentino, A L; Plummer, T H

    1998-11-01

    Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme. PMID:9790895

  20. Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells.

    PubMed

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

    2012-03-01

    Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus-insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell-baculovirus systems for bioprocess optimization. PMID:22166686

  1. A Model to Study the Phenotypic Changes of Insect Cell Transfection by Copepod Super Green Fluorescent Protein (cop-GFP) in Baculovirus Expression System

    PubMed Central

    Shokrollahi, Narjes; Shahbazzadeh, Delavar; Pooshang-Bagheri, Kamran; Habibi-Anbouhi, Mahdi; Jahanian-Najafabadi, Ali; Behdani, Mahdi

    2016-01-01

    Background: Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells. Methods: The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein (copGFP). In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells. Results: Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis. Conclusion: The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection. PMID:26518237

  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. Guide to Productivity Improvement Projects. Third Edition.

    ERIC Educational Resources Information Center

    International City Management Association, Washington, DC.

    This edition features over 400 projects that have effected cost savings and/or improved services in a broad range of functional areas of city management, a jurisdictional index as well as a topical index, in-depth reports on selected projects, and project description forms that readers can use to share their successful projects with other readers.…

  4. Improving cow herd production through early weaning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early weaning, in spring calving production systems, has intrigued many producers to consider this alternative management practice especially during extended droughts and as a tool to promote stayability within a cow herd for young developing cows. The first objective of this study was to evaluate ...

  5. Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses.

    PubMed

    Grasela, James J; McIntosh, Arthur H; Ringbauer, Joseph; Goodman, Cynthia L; Carpenter, James E; Popham, Holly J R

    2012-05-01

    The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) in Australia, Hawaii, South Africa, and various Caribbean islands, has posed great concern as to the possible threat to native, endangered species of cactus in the southeastern USA as well as with the potential to cause a major infestation of commercial and agricultural cactus crops in Mexico. A number of control measures have been investigated with varying degrees of success including, field exploration for cactus moth-specific parasitoids, insecticides, fungal, bacterial, and nematode agents. Current tactics used by the USA-Mexico binational program to eradicate cactus moth from Mexico and mitigate its westward movement in the USA include host plant removal, the manual removal and destruction of egg sticks and infected cacti stems, and the Sterile Insect Technique. One other approach not taken until now is the development of a cactus moth cell line as a tool to facilitate the investigation of baculoviruses as an alternative biocontrol method for the cactus moth. Consequently, we established C. cactorum cell lines derived from adult ovarian tissue designated as BCIRL-Cc-AM and BCIRL-Cc-JG. The mean cell population doubling time was 204.3 and 112 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with weekly medium change, while the doubling time was 176.6 and 192.6 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with a daily change of medium. In addition, the daily versus weekly change in medium was reflected in the percentage viability with both cell lines showing higher levels with a daily medium change. Of the three baculoviruses tested, only the recombinant AcMNPV-hsp70Red and GmMNPV at a multiplicity of infection (MOI) of 1.0 were able to demonstrate significant production of extracellular virus (ECV) in each of the cell lines, whereas both cell lines were

  6. 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

  7. Some new ideas for improving production operations

    SciTech Connect

    Wright, T.R. Jr.

    1983-04-01

    Recent innovations in equipment and technology for producing oil and gas are surveyed. A new concept called the fluid rod production system uses production fluid as a power transfer medium to transmit fluid power from a skid mounted surface unit to a down hole stroker that activates a standard API type rod pump. This system is used in low-volume wells. An automatic chemical feed system, an unusual pump jack, an electric downhole steam generator, and methods for producing gas hydrates are surveyed. Finally, a pressure activated tubing plug, which eliminates the problem of debris settling on top of a wireline retrievable plug, is discussed. The key aspect of this tool is the floating cylinder, as specified.

  8. 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).

  9. 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.

  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. 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.

  12. 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

  13. 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.

  14. Improved maintenance productivity through software application

    SciTech Connect

    Rohrig, S.D.

    1987-01-01

    Some twelve years ago, MRC - Mound was one of the first Department of Energy (DOE) weapons complex sites to define and implement an engineering performance standards system to aid in the management of site maintenance operations. Over succeeding years, the scope of the system was broadened to include preventive maintenance and equipment management. Portions of the system were eventually automated through applications programming utilizing Mound's mainframe computer. The Automated Maintenance Management Operations, AMMO, software package will support all the principal functions of the maintenance mission at Mound. In the subsequent pages, a few unique features of the AMMO System will be highlighted. Also to be discussed, will be some of the productivity philosophies that influenced the AMMO design, as well as, the mechanisms within the system that will monitor the success of the system in achieving the desired results. Piror to this discussion, however, an overview of Mounds's present maintenance work order execution must be briefly discussed. The AMMO System software was designed around this mode of operation while incorporating some innovative new philosophies.

  15. 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

  16. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

    PubMed Central

    Luckow, V A; Lee, S C; Barry, G F; Olins, P O

    1993-01-01

    The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells. Images PMID:8392598

  17. The Effect of MicroRNA bantam on Baculovirus AcMNPV Infection in Vitro and in Vivo

    PubMed Central

    Shi, Xiaojie; Ran, Zihan; Li, Sisi; Yin, Juan; Zhong, Jiang

    2016-01-01

    The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction. PMID:27196923

  18. The Effect of MicroRNA bantam on Baculovirus AcMNPV Infection in Vitro and in Vivo.

    PubMed

    Shi, Xiaojie; Ran, Zihan; Li, Sisi; Yin, Juan; Zhong, Jiang

    2016-01-01

    The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction. PMID:27196923

  19. 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

  20. Improvements in large window and optics production

    NASA Astrophysics Data System (ADS)

    Hallock, Bob; Messner, Bill; Hall, Chris; Supranowitz, Chris

    2007-04-01

    are virtually eliminated with the MRF process. This paper presents some recent results of the deterministic finishing typified by the QED product line and more specifically of its large-aperture machines, presently capable of finishing optics up to one meter in size. Examples of large sapphire windows and meter-class aspheric glass optics will be reviewed. Associated metrology concerns will also be discussed.

  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. Improving microbial biogasoline production in Escherichia coli using tolerance engineering

    DOE PAGESBeta

    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

  3. 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

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

    PubMed

    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

  5. A Trainer's Guide to Successful Productivity Improvement Planning.

    ERIC Educational Resources Information Center

    Smith, Judson

    1982-01-01

    Discusses the concept of productivity improvement which must be studied before selecting an approach. Concepts include increasing efficiency without decreasing quality; using employees to redefine the job; and planning for long-term effort. (JOW)

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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…

  11. 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.

  12. 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…

  13. Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system☆

    PubMed Central

    Lu, Huiyuan; Ma, Jun; Liu, Nian; Wang, Shoulin

    2010-01-01

    Objective CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe3+ or hemin alone. Either co-addition of 5-ALA and Fe3+ or addition of 5-ALA or Fe3+ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). Conclusion 5-ALA and Fe3+ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression. PMID:23554636

  14. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    SciTech Connect

    Vanarsdall, Adam L.; Mikhailov, Victor S.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-08-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout

  15. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    PubMed Central

    Vanarsdall, Adam L.; Mikhailov, Victor S.; Rohrmann, George F.

    2009-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using pulsed-field gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 hours post-infection, DBP co-localized with replicated DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp

  16. Untapped opportunities for improving performance and increasing smelter production

    SciTech Connect

    Forberg, H.O.

    1996-10-01

    A review has been made of a large number of aluminum smelters concerning opportunities for improving performance and increasing production. Smelters have been grouped according to current efficiency. Opportunities for improvement of older and newer smelters are discussed. For older smelters to remain competitive and extend economic life, it is important to find new ways to increase production. These efforts have been going on for many years and the most successful producers have been able to stay viable. Opportunities are still available for many older facilities as further incremental increases of production wait to be harvested. Newer smelters are also interested in decreasing cost by increasing production. Even if the operating costs of newer plants are lower than those of older ones, it is important that newer plants make improvements that generate an acceptable rate of return on invested capital.

  17. 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

  18. Improvements to the MODIS Land Products in Collection Version 6

    NASA Astrophysics Data System (ADS)

    Wolfe, R. E.; Devadiga, S.; Masuoka, E. J.; Running, S. W.; Vermote, E.; Giglio, L.; Wan, Z.; Riggs, G. A.; Schaaf, C.; Myneni, R. B.; Friedl, M. A.; Wang, Z.; Sulla-menashe, D. J.; Zhao, M.

    2013-12-01

    The MODIS (Moderate Resolution Imaging Spectroradiometer) Adaptive Processing System (MODAPS), housed at the NASA Goddard Space Flight Center (GSFC), has been processing the earth view data acquired by the MODIS instrument aboard the Terra (EOS AM) and Aqua (EOS PM) satellites to generate suite of land and atmosphere data products using the science algorithms developed by the MODIS Science Team. These data products are used by diverse set of users in research and other applications from both government and non-government agencies around the world. These validated global products are also being used in interactive Earth system models able to predict global change accurately enough to assist policy makers in making sound decisions concerning the protection of our environment. Hence an increased emphasis is being placed on generation of high quality consistent data records from the MODIS data through reprocessing of the records using improved science algorithms. Since the launch of Terra in December 1999, MODIS land data records have been reprocessed four times. The Collection Version 6 (C6) reprocessing of MODIS Land and Atmosphere products is scheduled to start in Fall 2013 and is expected to complete in Spring 2014. This presentation will describe changes made to the C6 science algorithms to correct issues in the C5 products, additional improvements made to the products as deemed necessary by the data users and science teams, and new products introduced in this reprocessing. In addition to the improvements from product specific changes to algorithms, the C6 products will also see significant improvement in the calibration by the MODIS Calibration Science Team (MCST) of the C6 L1B Top of the Atmosphere (TOA) reflectance and radiance product, more accurate geolocation, and an improved Land Water mask. For the a priori land cover input, this reprocessing will use the multi-year land cover product generated with three years of MODIS data as input as opposed to one

  19. Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.

    PubMed Central

    Ritchie, P J; Decout, A; Amey, J; Mann, C J; Read, J; Rosseneu, M; Scott, J; Shoulders, C C

    1999-01-01

    The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex. PMID:10036224

  20. Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

    PubMed

    Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

    2010-11-01

    Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. PMID:20713098

  1. 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. PMID:18713650

  2. Adaptive Immune Responses Elicited by Baculovirus and Impacts on Subsequent Transgene Expression In Vivo

    PubMed Central

    Luo, Wen-Yi; Lin, Shih-Yeh; Lo, Kai-Wei; Lu, Chia-Hsin; Hung, Chang-Lin; Chen, Chi-Yuan; Chang, Chien-Chung

    2013-01-01

    Baculovirus (BV) is a promising gene therapy vector and typically requires readministration because BV mediates transient expression. However, how the prime-boost regimen triggers BV-specific adaptive responses and their impacts on BV readministration, transgene expression, and therapeutic/vaccine efficacy remain unknown. Here we unraveled that BV injection into BALB/c mice induced the production of BV-specific antibodies, including IgG1 and IgG2a, which could neutralize BV by antagonizing the envelope protein gp64 and impede BV-mediated transgene expression. Moreover, humans did not possess preexisting anti-BV antibodies. BV injection also elicited BV-specific Th1 and Th2 responses as well as CD4+ and CD8+ T cell responses. gp64 was a primary immunogen to activate the antibody and CD8+ T cell response, with its peptide at positions 457 to 465 (peptide 457-465) being the major histocompatibility complex (MHC) class I epitope to stimulate CD8+ T cell and cytotoxic responses. Nonetheless, a hybrid Sleeping Beauty-based BV enabled long-term expression for >1 year by a single injection, indicating that the T cell responses did not completely eradicate BV-transduced cells and implicating the potential of this hybrid BV vector for gene therapy. These data unveil that BV injection triggers adaptive immunity and benefit rational design of BV administration schemes for gene therapy and vaccination. PMID:23408634

  3. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    SciTech Connect

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M. )

    1990-03-06

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ({sup 3}H)azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the ({sup 3}H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

  4. Evolutionary analysis of the ubiquitin gene of baculovirus and insect hosts.

    PubMed

    Ma, S S; Zhang, Z; Xia, H C; Chen, L; Yang, Y H; Yao, Q; Chen, K P

    2015-01-01

    Baculovirus is the only virus that has been found to encode the ubiquitin protein. In this study, ubiquitin sequences from 16 insects and 49 viruses were collected and compared. The resulting sequences were aligned with virus genomes. Then MAGE 5.0, k-estimated software, as well as other software programs were used for systemic evolutionary, selection pressure, and evolutionary distance analysis. The results of the pairwise ratio of non-synonymous to synonymous substitution values and evolutionary distances showed that ubiquitin from baculovirus and insect hosts have been under purifying selection during evolution and are thus evolutionarily conserved. Moreover, genes from insect hosts were more conserved than those in baculovirus. Analysis of the non-synonymous to synonymous substitution rates at each site and entropy calculations revealed the evolutionary status of every site in the ubiquitin genes of baculovirus and their hosts. Genome locations and phylogenetic trees indicated that granuloviruses and non-photosynthetic vegetation evolved, and granulovirus evolution was more similar to that of insect hosts. Our results suggest that the ubiquitin gene in baculovirus may have been acquired through horizontal transfer from the host. PMID:26345932

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

    PubMed Central

    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. PMID:26490731

  6. 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

  7. 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

  8. 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

  9. CAD/CAM improves productivity in nonaerospace job shops

    NASA Astrophysics Data System (ADS)

    Koenig, D. T.

    1982-12-01

    Business cost improvements that can result from Computer Aided Design/Computer Aided Manufacturing (CAD/CAM), when properly applied, are discussed. Emphasis is placed on the use of CAD/CAM for machine and process control, design and planning control, and production and measurement control. It is pointed out that the implementation of CAD/CAM should be based on the following priorities: (1) recognize interrelationships between the principal functions of CAD/CAM; (2) establish a Systems Council to determine overall strategy and specify the communications/decision-making system; (3) implement the communications/decision-making system to improve productivity; and (4) implement interactive graphics and other additions to further improve productivity.

  10. 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.

  11. 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

  12. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.

    PubMed

    Zhang, Ziguo; Yang, Jing; Barford, David

    2016-02-15

    The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells. PMID:26454197

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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. PMID:26117660

  2. 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…

  3. Managing soil under vegetable production to improve soil quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over the years, soil quality has eroded as soil organic matter has declined on farms across North Carolina. This study is assessing the effects of tillage practice, winter cover cropping and compost use on changes in soil function and improvement in soil quality under vegetable production. The field...

  4. 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…

  5. Improvement of the production of Trypanosoma vivax antigens.

    PubMed

    Staak, C

    1975-09-01

    An improved technique for the production of Trypanosoma vivax antigen from infected cattle is described. This technique is based upon 1. the application of immunosuppressive drug, 2. the isolation of trypanosomes by centrifugation, and 3. the clearing of the trypanosome suspension from non sedimented blood cells by an anti-serum against bovine blood cells, raised in rabbits. PMID:1189022

  6. 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.

  7. 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.

  8. 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...

  9. Polymer mud improves chalk gas production and profits

    SciTech Connect

    Cornett, J.E.

    1984-01-01

    A new polymer drilling fluid is being used successfully in the Austin Chalk area of Texas to improve wellbore stability, decrease formation damage and significantly increase production rates. This article discusses development of the fluid, laboratory test results, field performance and benefits to operators.

  10. Production does not improve memory for face-name associations.

    PubMed

    Hourihan, Kathleen L; Smith, Alexis R S

    2016-06-01

    Strategies for learning face-name associations are generally difficult and time-consuming. However, research has shown that saying a word aloud improves our memory for that word relative to words from the same set that were read silently. Such production effects have been shown for words, pictures, text material, and even word pairs. Can production improve memory for face-name associations? In Experiment 1, participants studied face-name pairs by reading half of the names aloud and half of the names silently, and were tested with cued recall. In Experiment 2, names were repeated aloud (or silently) for the full trial duration. Neither experiment showed a production effect in cued recall. Bayesian analyses showed positive support for the null effect. One possibility is that participants spontaneously implemented more elaborate encoding strategies that overrode any influence of production. However, a more likely explanation for the null production effect is that only half of each stimulus pair was produced-the name, but not the face. Consistent with this explanation, in Experiment 3 a production effect was not observed in cued recall of word-word pairs in which only the target words were read aloud or silently. Averaged across all 3 experiments, aloud targets were more likely to be recalled than silent targets (though not associated with the correct cue). The production effect in associative memory appears to require both members of a pair to be produced. Surprisingly, production shows little promise as a strategy for improving memory for the names of people we have just met. (PsycINFO Database Record PMID:27244356

  11. The nutrition intervention improved adult human capital and economic productivity.

    PubMed

    Martorell, Reynaldo; Melgar, Paul; Maluccio, John A; Stein, Aryeh D; Rivera, Juan A

    2010-02-01

    This article reviews key findings about the long-term impact of a nutrition intervention carried out by the Institute of Nutrition of Central America and Panama from 1969 to 1977. Results from follow-up studies in 1988-89 and 2002-04 show substantial impact on adult human capital and economic productivity. The 1988-89 study showed that adult body size and work capacity increased for those provided improved nutrition through age 3 y, whereas the 2002-04 follow-up showed that schooling was increased for women and reading comprehension and intelligence increased in both men and women. Participants were 26-42 y of age at the time of the 2002-04 follow-up, facilitating the assessment of economic productivity. Wages of men increased by 46% in those provided with improved nutrition through age 2 y. Findings for cardiovascular disease risk factors were heterogeneous; however, they suggest that improved nutrition in early life is unlikely to increase cardiovascular disease risk later in life and may indeed lower risk. In conclusion, the substantial improvement in adult human capital and economic productivity resulting from the nutrition intervention provides a powerful argument for promoting improvements in nutrition in pregnant women and young children. PMID:20032473

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

    PubMed

    Zhang, Congqiang; Chen, Xixian; Stephanopoulos, Gregory; Too, Heng-Phon

    2016-08-01

    Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc. PMID:26804325

  17. [Improving 3-dehydroshikimate production by metabolically engineered Escherichia coli].

    PubMed

    Yuan, Fei; Chen, Wujiu; Jia, Shiru; Wang, Qinhong

    2014-10-01

    In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS. PMID:25726580

  18. 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. PMID:25448534

  19. 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. PMID:27005492

  20. 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

  1. 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

  2. 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.

  3. 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. PMID:18502077

  4. A Framework for Process Improvement in Software Product Management

    NASA Astrophysics Data System (ADS)

    Bekkers, Willem; van de Weerd, Inge; Spruit, Marco; Brinkkemper, Sjaak

    This paper presents a comprehensive overview of all the important areas within Software Product Management (SPM). The overview has been created and validated in collaboration with many experts from practice and the scientific community. It provides a list of 68 capabilities a product software organization should implement to reach a full grown SPM maturity. The overview consists of the SPM Competence Model that shows the areas of importance to SPM, and the SPM Maturity Matrix that lists all important activities within those areas in a best practice implementation order. SPM organizations can use this matrix to map and improve their SPM practices incrementally.

  5. Productivity improvements through the use of CAD/CAM

    NASA Astrophysics Data System (ADS)

    Wehrman, M. D.

    This paper focuses on Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) productivity improvements that occurred in the Boeing Commercial Airplane Company (BCAC) between 1979 and 1983, with a look at future direction. Since the introduction of numerically controlled machinery in the 1950s, a wide range of engineering and manufacturing applications has evolved. The main portion of this paper includes a summarized and illustrated cross-section of these applications, touching on benefits such as reduced tooling, shortened flow time, increased accuracy, and reduced labor hours. The current CAD/CAM integration activity, directed toward capitalizing on this productivity in the future, is addressed.

  6. The polyhedra of the occluded baculoviruses of marine decapod crustacea: A unique structure, crystal organization, and proposed model

    PubMed

    Bonami; Aubert; Mari; Poulos; Lightner

    1997-11-01

    The baculoviruses of marine penaeid shrimp, PmSNPV and PvSNPV (MBV type and BP type, respectively), have distinctly different occlusion bodies (OBs) from those of the insect baculoviruses. In contrast to insect baculovirus, the penaeid baculovirus OB is unenveloped and formed by large subunits (SuOBs), as observed by electron microscopy after negative staining. The polyhedrin subunits measure 17 to 23 nm in diameter and appear icosahedral, resembling full and empty viral particles. Although these SuOBs look similar in morphometrics to shrimp parvoviruses, their density, polypeptide composition, and UV spectra are more characteristic of proteins than nucleoproteins. Common to the two shrimp baculovirus OBs that were investigated is the aggregation of three icosahedral SuOBs into a triplet. The observed difference in their crystalline structure is directly related to the way in which triplets attach to each other to form the OB. In the BP-type OB, the triplets form alternating parallel rows in all three dimensions. On the other hand, in the MBV-type OB, four triplets form a hollow sphere which we call a "rosette," the building blocks of the MBV-type OB. We assembled models for the penaeid baculovirus OB as an alternative to those hypothesized for insect baculovirus OBs. Copyright 1997 Academic Press. Copyright 1997 Academic Press PMID:9417978

  7. Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene▿

    PubMed Central

    Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, WonKyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

    2010-01-01

    Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph. PMID:20219904

  8. 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

  9. Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions.

    PubMed

    Nguyen, Quan; Palfreyman, Robin W; Chan, Leslie C L; Reid, Steven; Nielsen, Lars K

    2012-01-01

    The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from cultured H. zea cells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection of H. zea cells with H. Armigera single-capsid nucleopolyhedrovirus (HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base for H. zea insect-HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of

  10. Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system.

    PubMed

    Ring, M; Meredith, J; Wiens, C; Macins, A; Brock, H W; Phillips, J E; Theilmann, D A

    1998-01-01

    We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl- transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2-3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270-fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses. PMID:9612936

  11. 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

  12. 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.

  13. Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

  14. 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...

  15. MEMBRANOUS LABYRINTH IN BACULOVIRUS-INFECTED CRUSTRACEAN CELLS: POSSIBLE ROLES IN VIRAL REPRODUCTION

    EPA Science Inventory

    The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. t is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and o...

  16. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  17. Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus.

    PubMed Central

    Johnson, T M; Mann, W R; Dragland, C J; Anderson, R C; Nemecek, G M; Bell, P A

    1995-01-01

    The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively. Images Figure 1 Figure 2 Figure 4 PMID:7626037

  18. Changes in trace metals in hemolymph of baculovirus infected noctuid larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied how biologically relevant trace metals (i.e., micronutrients) in the plasma of larvae of Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of plas...

  19. Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haema...

  20. 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...

  1. 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...

  2. 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...

  3. Iron levels change in larval Heliothis virescens tissues following baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inductively-coupled plasma mass spectrometry (ICP-MS) and 59Fe radiotracers were used to investigate changes in levels of iron (Fe) in the tissues of Heliothis virescens following baculovirus infection. Fe concentrations were determined by ICP-MS in hemolymph collected from 4th instar larvae infect...

  4. CHARACTERIZATION OF THE DNA OF A NONOCCLUDED BACULOVIRUS, HZ-1V

    EPA Science Inventory

    The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsC1-ethidium bromide density gr...

  5. 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

  6. 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

  7. 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

  8. 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

  9. Improvement of halophilic cellulase production from locally isolated fungal strain

    PubMed Central

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

    2014-01-01

    Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett–Burman design and the Face Centered Central Composite Design (FCCCD). Plackett–Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization. PMID:26150755

  10. Improvement of halophilic cellulase production from locally isolated fungal strain.

    PubMed

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

    2015-07-01

    Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett-Burman design and the Face Centered Central Composite Design (FCCCD). Plackett-Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization. PMID:26150755

  11. New improved method for fructooligosaccharides production by Aureobasidium pullulans.

    PubMed

    Dominguez, Ana; Nobre, Clarisse; Rodrigues, Lígia R; Peres, António M; Torres, Duarte; Rocha, Isabel; Lima, Nelson; Teixeira, José

    2012-08-01

    Fructooligosaccharides are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. In this study, an integrated one-stage method for FOS production via sucrose fermentation by Aureobasidium pullulans was developed and optimized using experimental design tools. Optimization of temperature and agitation speed for maximizing the FOS production was performed using response surface methodology. Temperature was found to be the most significant parameter. The optimum fermentation conditions were found to be 32 °C and 385 rpm. Under these conditions, the model predicted a total FOS production yield of 64.7 gFOS/gsucrose. The model was validated at optimal conditions in order to check its adequacy and accuracy and an experimental yield of 64.1 (±0.0) gFOS/gsucrose was obtained. A significant improvement of the total FOS production yields by A. pullulans using a one-stage process was obtained. PMID:24750929

  12. 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. PMID:23881318

  13. 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.

  14. The role of productivity in improving the environmental sustainability of ruminant production systems.

    PubMed

    Capper, Judith L; Bauman, Dale E

    2013-01-01

    The global livestock industry is charged with providing sufficient animal source foods to supply the global population while improving the environmental sustainability of animal production. Improved productivity within dairy and beef systems has demonstrably reduced resource use and greenhouse gas emissions per unit of food over the past century through the dilution of maintenance effect. Further environmental mitigation effects have been gained through the current use of technologies and practices that enhance milk yield or growth in ruminants; however, the social acceptability of continued intensification and use of productivity-enhancing technologies is subject to debate. As the environmental impact of food production continues to be a significant issue for all stakeholders within the field, further research is needed to ensure that comparisons among foods are made based on both environmental impact and nutritive value to truly assess the sustainability of ruminant products. PMID:25387028

  15. 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

  16. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

  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. Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production.

    PubMed

    Zhou, Jiewen; Wu, Kang; Rao, Christopher V

    2016-10-01

    The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc. PMID:27002479

  19. 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...

  20. Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.

    PubMed

    Wu, Qunfeng; Yu, Fulai; Xu, Jinfang; Li, Yang; Chen, Huanchun; Xiao, Shaobo; Fu, Zhen F; Fang, Liurong

    2014-06-25

    Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. PMID:24793501

  1. 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. PMID:26069440

  2. 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

  3. Vector Design for Improved DNA Vaccine Efficacy, Safety and Production

    PubMed Central

    Williams, James A.

    2013-01-01

    DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy. PMID:26344110

  4. 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

  5. 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

  6. In Situ Propellant Production for improved sample return mission performance

    NASA Technical Reports Server (NTRS)

    Stancati, M. L.; Niehoff, J. C.; Wells, W. C.; Feingold, H.; Ash, R. L.

    1980-01-01

    In Situ Propellant Production (ISPP) on the surface of a target body is evaluated as a potential way to relax sample return mass constraints and to improve mission performance. Utilization of an oxygen/methane bipropellant combination for primary outbound and return propulsion has a significant favorable impact upon Earth escape requirements. A small sample can be returned from Mars using a single Shuttle/IUS(Twin) launch. Performance and design data are presented for the Mars mission. For sample returns from selected Galilean satellites, launch requirements are reduced by fifteen to forty percent. An assessment is made of overall utility of ISPP to planetary missions.

  7. 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.

  8. Improved enzymatic production of phenolated acylglycerols through alkyl phenolate intermediates.

    PubMed

    Yang, Zhiyong; Feddern, Vivian; Glasius, Marianne; Guo, Zheng; Xu, Xuebing

    2011-04-01

    A novel approach is reported for the synthesis of dihydrocaffoylated glycerols that consists of two steps: enzymatic synthesis of octyl dihydrocaffeate (as a synthetic intermediate) from octanol and dihydrocaffeic acid, and enzymatic interesterification of triacylglycerols with octyl dihydrocaffeate. Due to the good compatibility of the intermediate with triacylglycerols, an improved volumetric productivity [147 mol h(-1)(kg Novozym 435)(-1)] and high enzyme specific activity [up to 9.6 μmol(-1) min(-1)(g Novozym 435)(-1)] have been obtained. PMID:21120584

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Transient and Stable Expression of the Neurotensin Receptor NTS1: A Comparison of the Baculovirus-Insect Cell and the T-REx-293 Expression Systems

    PubMed Central

    Xiao, Su; White, Jim F.; Betenbaugh, Michael J.; Grisshammer, Reinhard; Shiloach, Joseph

    2013-01-01

    Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [3H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs. PMID:23696845

  16. Natural products discovery needs improved taxonomic and geographic information.

    PubMed

    Leal, Miguel C; Hilário, Ana; Munro, Murray H G; Blunt, John W; Calado, Ricardo

    2016-06-01

    Covering: up to 2016Marine and terrestrial organisms yield a remarkable chemical diversity and are important sources for discovery of new chemical products. In order to maximize the bioprospecting efficiency of natural products (NP), taxonomy, geography and biodiversity are starting to be used to draw conclusions on which taxonomic groups and/or regions may be of interest for future research. However, accurate taxonomic information and sampling location of source organisms have often been overlooked. Although these issues were already reported a few decades ago and improvements have been made, such outstanding problems are still recurrent in recent peer-reviewed literature. Here, we focus on the importance of taxonomic and geographic identification of source material and illustrate how taxonomic and geographic data of source organisms continues to be poorly handled. It is our opinion that this issue needs to be discussed within the NP community with the ultimate goal of improving publication standards and guaranteeing the scientific principle of research reproducibility. Moreover, by doing so, it will be possible to take advantage of information available in the literature to develop cross-disciplinary meta-analyses that may help to advance the state of the art of NP research and future bioprospecting endeavours. PMID:26892141

  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. Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus

    PubMed Central

    Nie, Zuo-Ming; Zhang, Zhi-Fang; Wang, Dan; He, Ping-An; Jiang, Cai-Ying; Song, Li; Chen, Fang; Xu, Jie; Yang, Ling; Yu, Lin-Lin; Chen, Jian; Lv, Zheng-Bing; Lu, Jing-Jing; Wu, Xiang-Fu; Zhang, Yao-Zhou

    2007-01-01

    Background The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome. Results The genome of AnpeNPV, which infects Chinese tussah silkworm (Antheraea pernyi), was sequenced and analyzed. The genome was 126,629 bp in size. The G+C content of the genome, 53.4%, was higher than that of most of the sequenced baculoviruses. 147 open reading frames (ORFs) that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. Of the 147 ORFs, 143 appeared to be homologous to other baculovirus genes, and 4 were unique to AnpeNPV. Furthermore, there are still 29 and 33 conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. In addition, the total number of genes common to all lepidopteran NPVs is sill 74, however the 74 genes are somewhat different from the 74 genes identified before because of some new sequenced NPVs. Only 6 genes were found exclusively in all lepidopteran NPVs and 12 genes were found exclusively in all Group I NPVs. AnpeNPV encodes v-trex(Anpe115, a 3' to 5' repair exonuclease), which was observed only in CfMNPV and CfDEFNPV in Group I NPVs. This gene potentially originated by horizontal gene transfer from an ancestral host. In addition, AnpeNPV encodes two conotoxin-like gene homologues (ctls), ctl1 and ctl2, which were observed only in HycuNPV, OpMNPV and LdMNPV. Unlike other baculoviruses, only 3 typical homologous regions (hrs) were identified containing 2~9 repeats of a 30 bp-long palindromic core. However, 24 perfect or imperfect direct repeats (drs) with a high degree of AT content were found within the intergenic spacer regions that

  19. 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.

  20. 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.

  1. Road Sediment Production and Delivery: Processes, Rates, and Possible Improvements

    NASA Astrophysics Data System (ADS)

    MacDonald, Lee; Ramos-Scharron, Carlos; Coe, Drew; Stafford, Alexander; Welsh, Matthew; Korte, Abby; Libohova, Zamir; Brown, Ethan; James, Cajun

    2013-04-01

    -dominated area. Connectivity was less than 20% in permeable volcanics in California and coarse-textured soils in the Colorado Front Range. Road-stream crossings accounted for most of the connected segments, as in temperate forests drainage rills and sediment plumes rarely extend for more than 50 m. Few studies have quantified the effects of unpaved roads on watershed-scale sediment yields, but in northern California the estimated sediment yield was 1.4 Mg yr-1 per kilometer of road, while in Colorado the long-term, chronic sediment production from roads was estimated to be about 1 Mg km-2 yr-1. On St. John roads were estimated to increase watershed-scale sediment production by 3-9 times, indicating that the road sediments are a critical stressor to near-shore coral reefs. Relatively simple management actions can greatly reduce sediment production and delivery from unpaved roads, but the necessary improvements are constrained by the lack of political will and funding.

  2. 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.

  3. The cellular substrate: a very important requirement for baculovirus in vitro replication.

    PubMed

    Miltenburger, H G; Naser, W L; Harvey, J P; Huber, J; Huger, A M

    1984-01-01

    We established more than 200 primary cell lines of Cydia pomonella (coding moth). 81 of them were selected and screened for replication of two baculoviruses (from two different subgroups): the Choristoneura murinana NPV and the Cydia pomonella GV. Although all these cell lines had been derived from the same insect species, they varied largely in their response to challenge with the NPV. Most of them showed CPE or produced different amounts of polyhedra. Interestingly, we also found a few cell lines that were permissive for GV replication. To our knowledge this is the first time that GV replication in cell lines has been obtained. Our results show that cell line properties are most important for baculovirus in vitro replication. PMID:6516539

  4. 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

  5. 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

  6. 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

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

    PubMed

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

    2016-06-01

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

  8. Enterovirus 71 virus-like particle vaccine: improved production conditions for enhanced yield.

    PubMed

    Chung, Cheng-Yu; Chen, Chi-Yuan; Lin, Shih-Yeh; Chung, Yao-Chi; Chiu, Hsin-Yi; Chi, Wei-Kuang; Lin, Yu-Li; Chiang, Bor-Luen; Chen, Wei-Jheng; Hu, Yu-Chen

    2010-10-01

    To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production. PMID:20797455

  9. An Improved Tropospheric Ozone Residual Product: Limitations and Applications

    NASA Astrophysics Data System (ADS)

    Witte, J. C.; Douglass, A. R.; Olsen, M. A.; Thompson, A. M.

    2012-12-01

    The Tropospheric Ozone Residual (TOR) product is derived from Aura's Ozone (O3) Monitoring Instrument (OMI) and Microwave Limb Sounder (MLS). OMI and MLS can estimate the TOR by subtracting the MLS stratospheric O3 from the OMI total column O3. Our improved TOR is a subset of the 2005-2012 database that takes into account (1) outliers that occur regularly in the mid to high latitudes, and (2) decreased sensitivity in the OMI O3 total column below clouds that contributes to a low-bias in the TOR compared to in-situ ozonesonde observations. To remove outliers, we have devised a filtering criteria based on the expected range of tropospheric O3 values from sondes, in combination with the tropospheric column fraction amount represented by the ratio of the OMI total column O3 to the MLS stratospheric column amount. We take advantage of a new cloud height algorithm - the optical centroid cloud pressure algorithm (OCCP) - that is sensitive to O3 below the nominal cloud-top pressure and can extend significantly below previous estimates of the assigned effective cloud height. Applying the new algorithm significantly improves the agreement with sondes for effective cloud pressures greater than 800-850 hPa, thereby reducing the need to assign a below-cloud O3 climatology. Finally, we assess the viability of applying the improved TOR for pollution studies and monitoring in the northern mid-latitudes. We examine O3 variability in the improved TOR over case study regions of Europe, Southeast Asia, and the US between 2005 and 2012, along with available sonde observations. We record the frequency, dates, and locations of the best and worst agreements with sondes to estimate when and where the TOR can be optimally used, and show cases where the variability of the TOR is representative of O3 variability in the boundary layer. A potential application of the improved TOR is to study inter-annual variability in summertime continental pollution outflow over regions of the US and

  10. Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

    PubMed Central

    Greenfield, C; Patel, G; Clark, S; Jones, N; Waterfield, M D

    1988-01-01

    The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production. Images PMID:2834199

  11. Improvements of SMILES Level 2 products on ISAS/JAXA

    NASA Astrophysics Data System (ADS)

    Mitsuda, Chihiro; Suzuki, Makoto; Iwata, Yoshitaka; Manago, Naohiro; Takahashi, Chikako; Imai, Koji; Shiotani, Masato; Sano, Takuki; Takayanagi, Masahiro; Taniguchi, Hirotomo

    The Superconducting Submillimeter-wave Limb-Emission Sounder (SMILES),which was jointly developed by JAXA and NICT, had been launched and aboard the Japanese Experiment Mod-ule (JEM) of the International Space Station (ISS) in September, 2009. The SMILES carries 4 K-cooled Superconductor-Insulator-Superconductor (SIS) mixers to demonstrate a sensitive instrument for sub-millimeter limb sounding. SMILES system noise temperature (Tsys) is less than 500K (random noise ¡ 1 K). Since ISS has a non-sun-synchronous orbit, SMILES can observe diurnal variations of ClO, BrO, HO2 and mesospheric O3 etc. Standard L2 products, which are defined as O3, HCl, ClO, HNO3, CH3CN, HOCl, HO2, BrO, O3-isotopes on stratospheres, began to be released to RA PIs on January, 2010. The L2 data is currently 4 85 km, with 3 km interval (geometrical altitude) in HDF ver.5 file format similar to EOS-HDF including time, location etc.However, release data (ver. 005-06-0024) is a test version which is retrieved by prelaunch algorithms (Rodgers 1976. SMILES mission plan 2002. Takahashi et al., 2010. Imai et al., 2010.), and has some known issues. The one of the main issue is less product data. SMILES observes 1630 scans per a day. However, release products include only 60 percents of observation data. There are two reasons for this. The first is that antenna main beam is interfered by the ISS solar paddles. The second is that star tracker cameras, which determine SMILES observation points, are interfered by the Sun. In latter case, if we estimate the points by other information, geophysical parameters are retrieved since observed spectrums are useful. In new release data, we use both STT and ISS information and 80 percents of the data are included to products. In this presentation, we will introduce improvements of operational level 2 processing for new release product version.

  12. Improved bounds on the heavy neutrino productions at the LHC

    NASA Astrophysics Data System (ADS)

    Das, Arindam; Okada, Nobuchika

    2016-02-01

    The Majorana neutrino in the type-I seesaw and the pseudo-Dirac neutrinos in the inverse seesaw can have sizable mixings with the light neutrinos in the standard model, through which the heavy neutrinos can be produced at the LHC. In producing the heavy neutrinos, we study a variety of initial states such as quark-quark, quark-gluon and gluon-gluon as well as photon mediated processes. For the Majorana heavy neutrino production, we consider a same-sign dilepton plus dijet as the signal events. Using the recent ATLAS and CMS data at √{s }=8 TeV with 20.3 and 19.7 fb-1 luminosities, respectively, we obtain direct upper bounds on the light-heavy neutrino mixing angles. For the pseudo-Dirac heavy neutrino production we consider the final sate with a trilepton plus missing energy as the signal events. Using the recent anomalous multilepton search by CMS at √{s }=8 TeV with 19.5 fb-1 luminosity, we obtain upper bounds on the mixing angles. Taking the varieties of initial states into account, the previously obtained upper bounds on the mixing angles have been improved. We scale our results at the 8 TeV LHC to obtain a prospective search reach at the 14 TeV LHC with high luminosities.

  13. 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

  14. Kraft mill sludge to improve vegetal production in Chilean Andisol.

    PubMed

    Gallardo, F; Mora, M L; Diez, M C

    2007-01-01

    The effect of kraft mill sludge addition (25 to 75 ton/ha) to soil derived from volcanic ashes (Andisol) on wheat (Triticum aestivum L.cv. Puken) biomass production, and in the nutrient absorption by the plants was evaluated. Respiration activity and seed germination tests were carried out on the soil/sludge mixtures, in order to evaluate possible toxic effects due to the sludge addition to the soil. Soil without sludge was used as a control treatment. The plants were grown in a greenhouse (25 degrees C, 14 h-photoperiod) during 120 days, then the plants were collected and dried at 65 degrees C for 72 h for the determination of biomass production (root and aerial) and analyzed for mineral content (Ca, Mg, K and P). The mixtures of soil/sludge showed no toxicity. Seed germination and respiration activity increased with the increment of the sludge. The accumulated CO2 in the soil without sludge was 41.66 mg CO2/100; this value shows a low microbial activity. The biomass increased with the increment of sludge addition to the soil and five times more biomass was obtained when 75 ton/ha sludge was added to the soil. The nutrient absorption efficiency was also improved with the sludge addition. PMID:17486832

  15. 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. PMID:25728446

  16. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    PubMed

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  17. 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

  18. Gene organization and transcription of TED, a lepidopteran retrotransposon integrated within the baculovirus genome.

    PubMed Central

    Friesen, P D; Nissen, M S

    1990-01-01

    A single copy of the retrotransposon TED, from the moth Trichoplusia ni (a lepidopteran noctuid), was identified within the DNA genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Determination of the complete nucleotide sequence (7,510 base pairs) of the integrated copy indicated that TED belongs to the family of retrotransposons that includes Drosophila melanogaster elements 17.6 and gypsy and thus represents the first nondipteran member of this invertebrate group to be identified. The internal portion of TED, flanked by long terminal repeats (LTRs), is composed of three long open reading frames comparable in size and location to the gag, pol, and env genes of the vertebrate retroviruses. Sequence similarity with the dipteran elements was the highest within individual domains of TED open reading frame 2 (pol region) that are also conserved among the retroviruses and encode protease, reverse transcriptase, and integrase functions, respectively. Mapping the 5' and 3' termini of TED RNAs indicated that the LTRs have a retroviral U3-R-U5 structural organization that is capable of directing the synthesis of transcripts that represent potential substrates for reverse transcription and intermediates in transposition. Abundant RNAs were also initiated from a site within the 5' LTR that matches the consensus motif for the promoter of late, hyperexpressed baculovirus genes. The presence of this viruslike promoter within TED and its subsequent activation only after integration within the viral genome suggest a possible symbiotic relationship with the baculovirus that could extend transposon host range. Images PMID:1692964

  19. 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

  20. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    PubMed

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  1. 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.

  2. 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.

  3. 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…

  4. Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming.

    PubMed

    Shikano, Ikkei; Hua, Kevin Ngoc; Cory, Jenny S

    2016-05-01

    Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to

  5. 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

  6. 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

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

    SciTech Connect

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

    1995-12-31

    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{sup {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{sup {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.

  8. 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.

  9. 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.

  10. 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.

  11. Towards an improved aerosol product from SCIAMACHY limb measurements

    NASA Astrophysics Data System (ADS)

    Rozanov, Alexei; Burrows, John; Hommel, Rene

    2015-04-01

    Stratospheric aerosols are of a great scientific interest because of their crucial role in the Earth's radiative budget as well as their contribution to chemical processes resulting in ozone depletion. While the permanent aerosol background in the stratosphere is determined by the tropical injection of SO2, COS and sulphate particles from the troposphere, major perturbations of the stratospheric aerosol layer result form an uplift of SO2 after strong volcanic eruptions. Satellite measurements in the visible spectral range represent one of the most important sources of information about the vertical distribution of the stratospheric aerosol on the global scale. This study employs measurements of the scattered solar light performed in the limb viewing geometry from the space borne spectrometer SCIAMACHY, which operated onboard the ENVISAT satellite from August 2002 to April 2012. A progress in the development of SCIAMACHY aerosol data product within the ROSA/ROMIC project including the improvements in the extinction coefficient data base and steps towards the retrieval of particle size distribution parameters is reported.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

    PubMed

    Xu, Xin-Gang; Tong, De-Wen; Wang, Zhi Sheng; Zhang, Qi; Li, Zhao-Cai; Zhang, Kuan; Li, Wei; Liu, Hung-Jen

    2011-06-01

    Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections. PMID:21793437

  18. 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

  19. Team Building: A Management Technology for Improving Productivity.

    ERIC Educational Resources Information Center

    White, Michael R.

    1987-01-01

    Team building and participatory strategies are techniques for increasing productivity in the workplace. Studies show that people often solve problems more effectively in groups, and they are more productive when they assume more responsibility for their work. (SK)

  20. 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.

  1. Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection.

    PubMed

    Rattanarojpong, Triwit; Khankaew, Suthiwat; Khunrae, Pongsak; Vanichviriyakit, Rapeepun; Poomputsa, Kanokwan

    2016-07-10

    White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future. PMID:27164257

  2. 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

  3. Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

    PubMed

    Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

    2016-03-01

    Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides. PMID:26787118

  4. 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

  5. Ecologically acceptable strategy for the use of genetically engineered baculovirus pesticides. Book chapter

    SciTech Connect

    Wood, H.A.; Hughes, P.R.; van Beek, N.; Hamblin, M.

    1990-01-01

    The basis for the first U.S. Environmental Protection Agency approval for the field release of a genetically-engineered virus has been to take advantage of the biological properties of baculovirus in such a way that an engineered virus could possess enhanced pesticidal properties but, at the same time, would pose no environmental or health hazards. The ultimate goal of these investigations is to reduce the agricultural requirement for synthetic chemical pesticides through the development of viral pesticides with enhanced pesticidal properties.

  6. 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. PMID:9018133

  7. Working for America: A Worker-Centered Approach to Productivity Improvement.

    ERIC Educational Resources Information Center

    Drewes, Donald W.

    This report examines worker-centered productivity and discusses the organizational and educational strategies for its improvement. 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…

  8. 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.

  9. 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 ...

  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 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...

  11. 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 ...

  12. 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…

  13. 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...

  14. How Do We Improve Crop Production in a Warming World?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Global analysis of crop yields from 1981-2002, showed a negative response of wheat, maize and barley yields to rising temperature, costing an estimated $5 billion per year. An analysis of maize and soybean production in the northern Corn Belt region of the U.S. found that productivity was adversely ...

  15. 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…

  16. 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

  17. 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. PMID:26643933

  18. Cell line profiling to improve monoclonal antibody production.

    PubMed

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  19. 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

  20. 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

  1. 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.

  2. 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%.

  3. 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

  4. [Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus].

    PubMed

    Xu, Peng-Wei; Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Liu, Xiao-Yu; Zeng, Xian-Yin; Zhang, Zhi-Qing

    2012-05-01

    The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines. PMID:22764525

  5. Bioactive baculovirus nanohybrids for stent based rapid vascular re-endothelialization

    NASA Astrophysics Data System (ADS)

    Paul, Arghya; Elias, Cynthia B.; Shum-Tim, Dominique; Prakash, Satya

    2013-08-01

    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.

  6. Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

    PubMed Central

    Chernomordik, L; Leikina, E; Cho, M S; Zimmerberg, J

    1995-01-01

    We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion. PMID:7707532

  7. Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.

    PubMed

    Tan, Boon-Huan; Brown, Gaie; Sugrue, Richard J

    2007-01-01

    Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains. PMID:17502677

  8. 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

  9. Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity*

    PubMed Central

    Sugiura, Nobuo; Setoyama, Yuka; Chiba, Mie; Kimata, Koji; Watanabe, Hideto

    2011-01-01

    Chondroitin sulfate is a linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4–9) and temperature (30–60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism. PMID:21715327

  10. Targeting Solutions to Improve Crop Production and the Environment

    NASA Astrophysics Data System (ADS)

    West, P. C.; Gerber, J. S.; Mueller, N. D.; Brauman, K. A.; Cassidy, E. S.; Johnston, M.; Ray, D. K.; Foley, J. A.

    2012-12-01

    Several recent studies propose broad strategies for increasing food production and decreasing the environmental impacts of agriculture. Using data sets for crop production, land management practices, and a yield response model, we identify promising locations for reducing environmental effects of agriculture and boosting crop yields. We find that a small number of crops and countries account for over two-thirds of water use, excess nutrients, and greenhouse gas emissions from croplands. Similarly, a small set of crops and regions offers the best opportunities for boosting global production without expanding croplands. These findings can be used to direct policies to specific crops and regions to help meet food security while shifting to more sustainable practices.

  11. Morphology engineering of Aspergillus niger for improved enzyme production.

    PubMed

    Driouch, Habib; Sommer, Becky; Wittmann, Christoph

    2010-04-15

    Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher-value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision-induced disruption of conidia aggregates and probably also the hindrance of new spore-spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by-product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co-expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production. PMID:19953678

  12. 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.

  13. Avian influenza neuraminidase 1 (N1) ELISA using baculovirus expressed antigen and its application on DIVA vaccination strategy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISA was developed using baculovirus express N1 protein from the A/Chicken/Indonesia/11/03 (H5N1) virus. The objective of this study was to evaluate the specificity and sensitivity of the N1-ELISA. The specificity of the ELISA was tested with a chicken anti-sera panel raised against N1 to N9 v...

  14. Induction of an IAP antagonist in Culex quinquefasciatus larvae in response to infection by the baculovirus CuniNPV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CuniNPV is a member of the Dipteran–specific baculoviruses in the genus Deltabaculovirus that specifically infects mosquito larvae within the genus Culex while species of Aedes and Anopheles are refractory. Infections are restricted to the nuclei of larval midgut epithelial cells with transmission...

  15. 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

  16. Baculovirus infection of the armyworm (Lepidoptera:Noctuidae) feeding on spiny- or smooth-edged grass (Festuca spp.) leaf blades

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Susceptibility of the armyworm, Mythimna unipuncta (Haworth), to infection by a baculovirus isolated from a Kentucky armyworm population was compared on two suspected progenitors of tall fescue, Festuca mairei and Festuca arundinacea subsp. fenas, with spiny leaf margins intact or removed to test wh...

  17. Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.

    PubMed

    Kim, Yuna; Kim, Jinhyun; Kang, Kyoungsoo; Lyoo, Young S

    2002-03-01

    Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells. PMID:14614268

  18. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens

    PubMed Central

    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

  19. 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...

  20. Engineering Neurospora crassa for improved cellobiose and cellobionate production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 out of seven bgl genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of ex...

  1. Perpendicular cultivation for improved weed control in organic peanut production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Intensive cultivation in organic peanut is partially effective, but in-row weed control remains problematic. In an attempt to improve in-row weed control, trials were conducted to determine the feasibility of early-season cultivation perpendicular to row direction using a tine weeder when integrate...

  2. Using membrane transporters to improve crops for sustainable food production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With the global population predicted to grow by at least 25% by 2050, the need for sustainable production of nutritious foods is critical for human and environmental well-being. Recent advances show that specialized plant membrane transporters can be utilized to enhance yields of staple crops, incre...

  3. How to Improve Your Productivity. The Best of CASE Currents.

    ERIC Educational Resources Information Center

    Smith, Virginia Carter, Ed.; Said, Carolyn, Ed.

    Ideas on increasing personal and professional productivity for institutional advancement staff are provided in 25 articles. Techniques are outlined for getting organized, setting deadlines, delegating responsibilities, motivating staff members, and meeting the demands of one- or two-person compus offices. Some of the titles and authors of the…

  4. 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

  5. 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

  6. 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…

  7. Genetic improvement of drought tolerance for productivity and food safety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production losses in agriculture during extended or severe drought episodes can be significant. Among a number of important food crops in the U.S. and many regions of the world, peanut is an important legume that is a rich source for oil, proteins, and vitamins. Drought can negatively affect yield...

  8. Improving Biocatalysts for the Production of Biofuels from Lignocellulosic Feedstocks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Industrial-scale production of fuel ethanol from biomass continues to show promise for relieving dependence upon petroleum-based transportation fuels. The limited range of materials that can be practically converted, however, continues to be an obstacle to the lignocellulosic revolution. Therefore...

  9. 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

  10. 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.

  11. 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…

  12. 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.

  13. Safety and immunogenicity of H5N1 influenza vaccine based on baculovirus surface display system of Bombyx mori.

    PubMed

    Jin, Rongzhong; Lv, Zhengbing; Chen, Qin; Quan, Yanping; Zhang, Haihua; Li, Si; Chen, Guogang; Zheng, Qingliang; Jin, Lairong; Wu, Xiangfu; Chen, Jianguo; Zhang, Yaozhou

    2008-01-01

    Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans. PMID:19079592

  14. 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.

  15. Regulation of expression of a baculovirus ecdysteroid UDPglucosyltransferase gene.

    PubMed Central

    O'Reilly, D R; Miller, L K

    1990-01-01

    The Autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid UDPglucosyltransferase which catalyzes the transfer of glucose from UDPglucose to ecdysteroid insect molting hormones. Expression of this gene allows the virus to block molting and pupation of infected insect larvae. In this study, we present the nucleotide sequence of the A. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt was transcribed as two 5'-coterminal mRNAs early in infection. Transferase activity was detected in infected cells and in the extracellular fluid by 3 h after infection. The majority of the activity accumulated in the extracellular fluid. We show that the egt gene product is a 60-kilodalton protein which is secreted from the infected cell. The egt gene is located in a region of the A. californica nuclear polyhedrosis virus genome which exhibited hypervariability in serially passaged virus stocks. Nucleotide sequence analysis revealed that the most common deletion occurring in these serially passaged virus isolates is located in the egt gene. Images PMID:2106039

  16. 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.

  17. Induced Mutations for Improving Production on Bread and Durum Wheat

    NASA Astrophysics Data System (ADS)

    Stamo, Ilirjana; Ylli, Ariana; Dodbiba, Andon

    2007-04-01

    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.

  18. 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

  19. 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.

  20. Using membrane transporters to improve crops for sustainable food production

    PubMed Central

    Schroeder, Julian I.; Delhaize, Emmanuel; Frommer, Wolf B.; Guerinot, Mary Lou; Harrison, Maria J.; Herrera-Estrella, Luis; Horie, Tomoaki; Kochian, Leon V.; Munns, Rana; Nishizawa, Naoko K.; Tsay, Yi-Fang; Sanders, Dale

    2013-01-01

    With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land. PMID:23636397

  1. Scrubber and remote control system improves productivity and safety

    SciTech Connect

    Kost, J.

    1984-07-01

    The paper describes the development of a scrubber and remote control system for a continuous miner. Test results using the system and a greater depth of cut showed that productivity was increased without any sacrifice to health and safety. Approval has been obtained from MSHA for operation under a ventilation plan which allows a 40 aft cut and a face-to-brattice distance of 40 ft when the scrubber is in operation.

  2. CLAES Product Improvement by use of GSFC Data Assimilation System

    NASA Technical Reports Server (NTRS)

    Kumer, J. B.; Douglass, Anne (Technical Monitor)

    2001-01-01

    Recent development in chemistry transport models (CTM) and in data assimilation systems (DAS) indicate impressive predictive capability for the movement of airparcels and the chemistry that goes on within these. This project was aimed at exploring the use of this capability to achieve improved retrieval of geophysical parameters from remote sensing data. The specific goal was to improve retrieval of the CLAES CH4 data obtained during the active north high latitude dynamics event of 18 to 25 February 1992. The model capabilities would be used: (1) rather than climatology to improve on the first guess and the a-priori fields, and (2) to provide horizontal gradients to include in the retrieval forward model. The retrieval would be implemented with the first forward DAS prediction. The results would feed back to the DAS and a second DAS prediction for first guess, a-priori and gradients would feed to the retrieval. The process would repeat to convergence and then proceed to the next day.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Why "Working Smarter" Isn't Working: White-Collar Productivity Improvement.

    ERIC Educational Resources Information Center

    Shaw, Edward

    2001-01-01

    Discusses the productivity and work days of white collar workers. Topics include productivity improvement; task analysis; the amount of time spent reading, and how to reduce it by improving writing skills; time spent in meetings; empowered time management; and sustaining a climate for change. (LRW)

  8. Compressed Air System Optimization Project Improves Production at a Metal Forging Plant (Modern Forge, TN, Plant)

    SciTech Connect

    2000-12-01

    In 1995, Modern Forge of Tennessee implemented a compressed air system improvement project at its Piney Flats, Tennessee, forging plant. Due to the project’s implementation, the plant was able to operate with fewer compressors and improve its product quality, thus allowing it to increase productivity. The project also resulted in considerable energy and maintenance savings.

  9. Programming for Preventive Disease and Improved Production in Sheep

    PubMed Central

    Boundy, T.

    1981-01-01

    Sheep losses each year are high in all types of rearing systems throughout the United Kingdom. Losses could well be reduced by a closer relationship between the sheep owner and the veterinarian. A closer relationship should involve the development of a simple disease preventive programme requiring a number of regular farm visits each year. Any such programme could attempt to control disease mainly by preventive methods, establish a complete parasite control programme and incorporate a high degree of instruction. There is probably a need for many veterinarians to attend specialist sheep courses, and suggestions are offered whereby the situation could be improved. PMID:7340921

  10. 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.

  11. Optimization of some additives to improve protease production under SSF.

    PubMed

    Tunga, R; Banerjee, R; Bhattacharyya, B C

    2001-11-01

    In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation. PMID:11906108

  12. Scrubber and remote control system improves productivity and safety

    SciTech Connect

    Kust, J.

    1984-07-01

    The test program conducted at NREC No. 1 Mine clearly indicated that methane levels did not increase with greater depths of cut (up to 40 ft) and corresponding increases in the face-to-brattice distance and methane and respirable dust levels remained well within acceptable levels. By using the scrubber, remote control, and greater depth of cut, productivity is increased without any sacrifice to miner health and safety. Based on the test results, NREC recently received approval from MSHA to operate under a ventilation plan which allows a 40-ft cut and face-to-brattice distance of 40 ft when the scrubber is in operation.

  13. 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. PMID:27233128

  14. Improvements in FCC catalyst technology for light hydrocarbon production

    SciTech Connect

    Ritter, R.E.; Habib, E.T.; Peters, A.W.; Rheaume, L.; Thiel, P.G.; Wallace, D.N.; Wormsbecher, R.F.

    1985-03-01

    As the refining industry continues to undergo dramatic changes due to crude pricing, environmental demands, and product demand, cracking catalyst manufacturers continue to make significant strides in the understanding of the processes and products to meet the refiners' needs. This paper builds on information reported to the industry in previous articles, with the aim of keeping the industry informed of the many dramatic changes taking place in cracking catalyst technology. From our vantage point, the key refining industry concerns today are gasoline octane quality due to the lead phase-down, atmospheric emissions due to increasingly strict EPA and local limits on SOx emissions, and catalytic upgrading of resid feeds due to an expected long term price differential between high and low quality crudes. Hence, we focus here on the significant changes in cracking catalyst technology which will help the refiner handle these issues. In particular, we discuss the catalytic enhancement of gasoline octane with Davison's Octacat and GXO catalyst families, the reduction of FCC SOx emissions with Davison's Additive R SOx catalyst, and the passivation of vanadium with Davison's DVT, so that catalyst deactivation is minimized.

  15. 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.

  16. 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.

  17. 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.

  18. 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…

  19. 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.

  20. 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. PMID:10228965

  1. 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.

  2. 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

  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. 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. PMID:27017378

  5. Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells.

    PubMed

    Laukkanen, M L; Oker-Blom, C; Keinänen, K

    1996-09-24

    Trichoplusia ni (High Five) and Spodoptera frugiperda (Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase of Autographa californica nuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications. PMID:8831686

  6. Identification of a single-nucleocapsid baculovirus isolated from Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

    PubMed

    Wang, Liqun; Yi, Jianping; Zhu, Shanying; Li, Bing; Chen, Yan; Shen, Weide; Wang, Wenbing

    2008-01-01

    A nucleopolyhedrovirus isolated from infected larvae of Clanis bilineata tsingtauica was characterized. Electron microscopical studies on the ultrastructure of C. bilineata nucleopolyhedrovirus (ClbiSNPV) occlusion bodies (OBs) showed several virions (up to 16) with a single nucleocapsid packaged within a single viral envelope. The diameter of the OBs was 0.77-1.7 mum with a mean of 1.13 +/- 0.19 mum. The complete sequence of the ClbiSNPV polyhedrin (polh) gene contained 741 nucleotides, predicting a protein of 246 amino acids. Phylogenetic analyses using the complete sequence of the polh genes indicated that ClbiSNPV clusters with Group II NPVs. This is the first record of a baculovirus from C. bilineata. PMID:18584114

  7. 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

  8. Selection for uterine capacity improves lifetime productivity of sows.

    PubMed

    Freking, B A; Lents, C A; Vallet, J L

    2016-04-01

    Selection for 11 generations for uterine capacity (UC) increased litter size in gilts by 1.6 more fully formed pigs at birth compared to an unselected control line (CO) despite averaging one less ovulation. The objective of the present study was to quantify line by parity interactions and characterize litter performance traits of sows in each line at later parities. Gilts farrowed in contemporary groups of 19 litters and were maintained through four parities if successfully mated in that contemporary group. A total of 243 litters and 2639 piglets were analyzed. Fixed effects of farrowing group, line, parity (1-4), and two-way interactions involving line were fitted. Sire (n=57) of the sow within farrowing group and line was fitted as a random effect. No significant line by parity interactions were observed. Parity effects were detected (P<0.01) for individual piglet birth weight, pre-weaning gain, and weaning weight. Parity effects were also detected (P<0.05) for total number born, average and total litter birth weight, and average and total litter weaning weight. Selection line differences for litter traits were detected (P<0.05) for number stillborn piglets and approached significance (P=0.06) for number of piglets weaned. Retention of sows in the herd was greater (P<0.05) with an average of 2.33 parities for the UC line females compared to 1.87 parities for the CO line. This resulted in favorable cumulative lifetime productivity of the UC line for total number of piglets born, number of piglets born alive, litter birth weight, number of piglets weaned and litter weaning weight. PMID:26869209

  9. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    PubMed

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine. PMID:27015979

  10. 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.

  11. 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

  12. Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

    PubMed

    Theilmann, D A; Chantler, J K; Stweart, S; Flipsen, H T; Vlak, J M; Crook, N E

    1996-04-01

    A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins. PMID:8615018

  13. 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. PMID:23536684

  14. Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

    PubMed

    Sugiura, T; Sugita, S; Imagawa, H; Kanaya, T; Ishiyama, S; Saeki, N; Uchiyama, A; Tanigawa, M; Kuwano, A

    2001-10-01

    The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses. PMID:11543878

  15. Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp.

    PubMed

    Liu, Bo; Gong, Yongchang; Li, Zhen; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2016-06-01

    Grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in China. This study was aimed to develop a novel oral vaccine against grass carp reovirus (GCRV). GCRV vp6 and vp7 genes with β-actin promoter of Megalobrama amblycephala and polyhedrin promoter (Ph10) of baculovirus, respectively, were cloned into plasmid pFast™-Dual to construct a vector pFast-PHVP7-AVP6, which was used to generate a recombinant baculovirus BacFish-vp6/vp7 via Bac-to-Bac system. The VP7 expression was analyzed from freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae by western blotting, and VP6 expression was analyzed from orally vaccinated fish with the freeze-dried powder by RT-PCR. The VP6 expression was also analyzed from both CIK cells transduced with BacFish-vp6/vp7 and tissues of vaccinated fish by immunofluorescence analysis. Recombinant VP7 could be detected from the BacFish-vp6/vp7-infected silkworm pupae. Pathological changes were not observed in CIK cells transduced with BacFish-vp6/vp7, and VP6 expression was found in CIK cells. When the grass carps were orally administrated with the freeze-dried powder, vp6 gene transcription was found in blood of the vaccinated fishes and VP6 protein was observed in liver and kidney of the vaccinated fish by immunofluorescence analysis. These results indicated that vp7 gene was expressed in the BacFish-vp6/vp7-infected silkworm and vp6 gene was expressed in orally vaccinated fish with freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae, suggesting the possibility to use the powder as an orally administrated vaccine. PMID:27085857

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. NGS for the Masses: Empowering Biologists to Improve Bioinformatics Productivity ( 7th Annual SFAF Meeting, 2012)

    ScienceCinema

    Qaadri, Kashef [Biomatters

    2013-03-22

    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.

  3. Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66

    PubMed Central

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Onishi, Momo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitu

    2012-01-01

    Baculovirus envelope protein ODV-E66 (67–704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67–704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å3 Da−1. PMID:22297996

  4. 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. PMID:25215537

  5. 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.

  6. 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. PMID:12507483

  7. 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.

  8. INVESTIGATION OF ADVANCED THERMAL-CHEMICAL CONCEPTS FOR OBTAINING IMPROVED MSW-DERIVED PRODUCTS

    EPA Science Inventory

    Although a number of resource recovery from refuse programs have been effective, the quality of the products recovered could be enhanced to improve their marketability. The purpose of this study was to investigate the potential of known processes that could improve the quality of...

  9. Present and future applications of DNA technologies to improve beef production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Substantial improvements in production efficiency and quality of beef and dairy products have been made possible through manipulation of bovine genetics. The advent of modern breeds in the last two centuries, the institution of phenotypic selection practices and quantitative genetics, even the proce...

  10. 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

  11. 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...

  12. Automated Performance Tracking and Productivity Improvement Project. Annual Report: Innovations in Protective Services.

    ERIC Educational Resources Information Center

    Dennis-Small, Lucretia

    Findings are reported of a process evaluation of the Automated Performance Tracking and Productivity Improvement Project, a project developed to enhance the productivity and efficiency of child protective services (CPS) staff and to enable Region 10 of the Texas Department of Human Services to better meet federal and state performance guidelines.…

  13. 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…

  14. Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation▿ †

    PubMed Central

    Wang, Manli; Tuladhar, Era; Shen, Shu; Wang, Hualin; van Oers, Monique M.; Vlak, Just M.; Westenberg, Marcel

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly. PMID:20519380

  15. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    SciTech Connect

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  16. Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

    PubMed

    Rueda, P; Fominaya, J; Langeveld, J P; Bruschke, C; Vela, C; Casal, J I

    2000-11-22

    We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure. PMID:11115693

  17. Improvement of xylanase production by Cochliobolus sativus in solid state fermentation

    PubMed Central

    Bakri, Yasser; Jawhar, Mohammed; Arabi, Mohammed Imad Eddin

    2008-01-01

    The xylanase production by Cochliobolus sativus strain Cs6 was improved under solid state fermentation (SSF). High xylanase activity (1079 U/g) was obtained when wheat straw was used after 8 days of incubation. Combinations of sodium nitrate with peptone or yeast extract resulted in an increased xylanase production (1543 and 1483 U/g, respectively). The Cs6 strain grown in SSF in a simple medium, proved to be a promising microorganism for xylanase production. PMID:24031273

  18. Improved exponential product cum dual to product type estimator of population mean

    NASA Astrophysics Data System (ADS)

    Singh, B. K.; Choudhury, Sanjib; Kumar, Abhishek

    2013-09-01

    In the present paper, an efficient exponential product cum dual to product type estimator has been proposed to estimate the population mean of the study variable by using simple random sampling scheme. The bias and mean squared error of the proposed estimator have been obtained up to the first order of approximation. A comparison has been made with existing similar estimators. The estimator has shown its efficiency over other estimators in terms of mean squared error (MSE). The numerical demonstrations have been made to show the gain in the estimator under study.

  19. Compressed Air System Optimization Project Saves Energy and Improves Production at a Citation Forging Plant

    SciTech Connect

    2003-05-01

    In the 1990s, a subsidiary of the Citation Corporation, Interstate Forging, implemented a compressed air system improvement project at its Milwaukee, Wisconsin, forging plant. This improvement enabled the plant to maintain an adequate and stable pressure level using fewer compressors, which led to improved product quality and lower production downtime. The project also yielded annual energy savings of 820,000 kWh and $45,000. With a total project cost of $67,000, the plant achieved a simple payback of just 1.5 years.

  20. Citation Corporation: Compressed Air System Optimization Project Saves Energy and Improves Production at Forging Plant

    SciTech Connect

    Not Available

    2003-05-01

    In the 1990s, a subsidiary of the Citation Corporation, Interstate Forging, implemented a compressed air system improvement project at its Milwaukee, Wisconsin, forging plant. This improvement enabled the plant to maintain an adequate and stable pressure level using fewer compressors, which led to improved product quality and lower production downtime. The project also yielded annual energy savings of 820,000 kWh and$45,000. With a total project cost of$67,000, the plant achieved a simple payback of just 1.5 years.

  1. Technology for Improving Production, Economic Efficiency, Quality and Sustainability in Peanut Production and Handling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proper crop rotation is essential to maintaining high peanut yield and quality. However, the economic considerations of maintaining or altering crop rotation sequences must incorporate the commodity prices, production costs, and yield responses of all crops in, or potentially in, the crop rotation ...

  2. 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

  3. 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.

  4. 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.

  5. Improved heterologous erythromycin A production through expression plasmid re-design.

    PubMed

    Jiang, Ming; Fang, Lei; Pfeifer, Blaine A

    2013-01-01

    The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli-derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species-specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5-fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. PMID:23804312

  6. Application of kaolin to improve citric acid production by a thermophilic Aspergillus niger.

    PubMed

    Ali, Sikander

    2006-12-01

    Citric acid production by a thermophilic strain of the filamentous fungus Aspergillus niger IIB-6 in a medium containing blackstrap cane molasses was improved by the addition of kaolin to the fermentation medium. The fermentation was run in a 7.5-l stirred bioreactor (60% working volume). The optimal sugar concentration was found to be 150 g/l. Kaolin (1.0 ml) was added to the fermentation medium to enhance volumetric production. The best results in terms of product formation were observed when 15 parts per million (ppm) kaolin was added 24 h after inoculation. With added kaolin, citric acid production was enhanced 2.34-fold, compared to a control fermentation without added kaolin. The length of incubation to attain this product yield was shortened from 168 to 96 h. The comparison of kinetic parameters showed improved citrate synthase activity of the culture (Y (p/x)=7.046 g/g). When the culture grown at various kaolin concentrations was monitored for Q (p), Q (s), and q (p), there was significant improvement in these variables over the control. Specific production by the culture (q (p)=0.073 g/g cells/h) was improved several fold. The addition of kaolin substantially improved the enthalpy (DeltaH (D)=74.5 kJ/mol) and entropy of activation (DeltaS=-174 J/mol/K) for citric acid production, free energies for transition state formation, and substrate binding for sucrose hydrolysis. The performance of fuzzy logic control of the bioreactor was found to be very promising for an improvement ( approximately 4.2-fold) in the production of citric acid (96.88 g/l), which is of value in commercial applications. PMID:16871375

  7. 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

  8. Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes.

    PubMed

    Sindelar, Georg; Wendisch, Volker F

    2007-09-01

    For the biotechnological production of L: -lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis. PMID:17364200

  9. Bayreuth Productivity Analysis-a method for ascertaining and improving the holistic service productivity of acute care hospitals.

    PubMed

    Pfannstiel, Mario Alexander

    2016-01-01

    The healthcare sector is lacking a method with which hospitals can measure the extent to which they achieve their goals in terms of aggregate productivity from both patients' and employees' perspectives. The Bayreuth Productivity Analysis (BPA) provides a solution to this problem because it uses two standardized questionnaires-one for patients and one for employees-to ascertain productivity at hospitals. These questionnaires were developed in several steps according to the principles of classical test theory, and they consist of six dimensions (information, organization, climate, methods, infrastructure and equipment) of five items each. One item describes a factual situation relevant to productivity and services so that it makes a contribution to the overall productivity of a hospital. After individualized evaluation of these items, the dimensions are subjectively weighted in the two questionnaires. The productivity index thus ascertained can be considered "holistic" when all patients and employees in a hospital make a differentiated assessment and weigh off each of the dimensions. In conclusion, the BPA constitutes a simple yet practicable method to ascertain and improve the holistic service productivity of hospitals. Copyright © 2014 John Wiley & Sons, Ltd. PMID:24839174

  10. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used. PMID:19737754

  11. 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

  12. Assembly-defective point mutants of the human immunodeficiency virus type 1 Gag precursor phenotypically expressed in recombinant baculovirus-infected cells.

    PubMed Central

    Hong, S S; Boulanger, P

    1993-01-01

    Two substitution mutants of the human immunodeficiency virus type 1 gag gene product were isolated after nitrous acid mutagenesis of a recombinant baculovirus expressing a non-N-myristylated, p6-deleted Gag precursor (Pr49). Both mutants failed to assemble intracellular Gag virus-like particles, as does the parental recombinant, and therefore expressed a self-assembly defective (Sad) phenotype in insect cells. The mutations consisted of nonconservative changes involving highly conserved hydrophobic residues in the p24 domain, Leu to Pro at position 268 (L268P) and Leu to Ser at amino acid 322 (L322S). Experimental data suggested that the two mutated residues belonged to functionally different regions of the Gag precursor. (i) A partial complementation effect between the two mutants for Gag precursor assembly was observed in coinfection experiments. (ii) The two mutations showed different phenotypes when placed in the N-myristylated context, of which only the L268P mutation abolished extracellular budding and release of Gag particles at the plasma membrane. Both L268P and L322S mutants had a trans-dominant negative effect on the intracellular assembly of a non-N-myristylated, full-length (Pr55) Gag precursor expressed by a coinfecting recombinant. None of the mutants, however, showed any detectable effect in trans on membrane targeting and budding of the coexpressed N-myristylated wild-type Gag precursor. Images PMID:8474175

  13. 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. PMID:25151072

  14. Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Kim, Ji-Ye; Seul, Hee-Jeong; Lee, Hee-Soo; Kwon, Hyuk-Moo; Sung, Haan-Woo

    2014-03-01

    Avian paramyxovirus 2 (APMV-2) infections are associated with respiratory diseases in poultry worldwide. The hemagglutination inhibition (HI) test is a useful tool for surveillance and monitoring of this virus. In this study, full-length hemagglutinin (HN) gene of APMV-2 was chemically synthesized based on its published sequence, cloned and expressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. The biological, antigenic and immunogenic properties of the expressed protein were evaluated to assess its ability to produce diagnostic reagents for HI testing. Recombinant APMV-2 HN protein showed two distinct bands with molecular masses of 64 and 75kDa, which showed hemagglutination (HA) and neuraminidase activities, respectively. The recombinant HN (rHN) protein extracted from infected cells produced high HA titers (2(13) per 25μL). HA activity of the protein was inhibited by APMV-2 antiserum, although there were weak cross reactions with other APMV serotype antisera. The rHN protein induced high titers of APMV-2-specific antibodies in immunized chickens based on the HI test. These results indicated that recombinant APMV-2 HN protein is a useful alternative to the APMV-2 antigen in HI assays. PMID:24374124

  15. A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats.

    PubMed

    Ebling, Peter M; Holmes, Stephen B

    2002-12-01

    A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (OB) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. OBs, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5 g of lyophilized L, F-H and mineral soil horizons, and 1.0 ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively. PMID:12476994

  16. 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

  17. A baculovirus anti-apoptosis gene homolog of the Trichoplusia ni granulovirus.

    PubMed

    Bideshi, D K; Anwar, A T; Federici, B A

    1999-01-01

    An inhibitor of apoptosis (iap) gene homolog (Tn-iap) of the Trichoplusia ni granulovirus (TnGV) was cloned, sequenced and mapped on the genome of TnGV. Tn-iap encoded a protein (Tn-IAP) of 301 amino acids with a predicted molecular mass of 35 kDa. The Tn-IAP contained the two sequence motifs, BIRs and RING finger, characteristic of IAP proteins, and shared identities of 21-27% and similarities of 28-53% with IAP proteins of Cydia pomonella GV (Cp-IAP), Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (MNPV) (Op-IAP1, 3), Autographa californica MNPV (Ac-IAP1), Bombyx mori NPV (Bm-IAP1), Lymantria dispar MNPV (Ld-IAP3) and Buzura suppressaria single nucleocapsid NPV (Bs-IAP1). However, Tn-IAP shared no significant homology with baculovirus IAP2 proteins. Using an antisense Tn-iap probe, two major transcripts of approximately 800 nt and 1600 nt were detected by Northern blot analysis of RNA extracted from the fat body of T. ni larvae infected with the TnGV. Unlike Cp-IAP and Op-IAP3, however, Tn-IAP did not rescue virion occlusion in SF21 cells infected with a p35-deficient AcMNPV mutant. Tn-IAP's synthesis in vivo but failure to rescue p35-deficient AcMNPV in SF21 cells suggests it is a functional IAP that is only effective in certain cell types. PMID:10541013

  18. Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

    PubMed

    Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

    2016-01-01

    Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171-177, 2016. PMID:26519022

  19. Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens.

    PubMed

    Breitenbach, Jonathan E; Shelby, Kent S; Popham, Holly J R

    2011-11-01

    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

  20. Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

    PubMed

    Katsuma, S; Deng, D X; Zhou, C L; Iwanaga, M; Noguchi, Y; Kobayashi, M; Maeda, S

    2000-10-01

    A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin. PMID:11129641