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Sample records for expression systems yield

  1. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  2. High yield expression of catalytically active USP18 (UBP43) using a Trigger Factor fusion system

    PubMed Central

    2012-01-01

    Background Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens. Results High-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone. Conclusion The results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization. PMID:22916876

  3. 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

  4. 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

  5. High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

    PubMed

    Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

    2011-04-01

    Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs. PMID:20559869

  6. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  7. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  8. 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

  9. High yield exogenous protein HPL production in the Bombyx mori silk gland provides novel insight into recombinant expression systems

    PubMed Central

    Wang, Huan; Wang, Lu; Wang, Yulong; Tao, Hui; Yin, Weimin; SiMa, Yanghu; Wang, Yujun; Xu, Shiqing

    2015-01-01

    The silk gland of Bombyx mori (BmSG) has gained significant attention by dint of superior synthesis and secretion of proteins. However, the application of BmSG bioreactor is still a controversial issue because of low yields of recombinant proteins. Here, a 3057 bp full-length coding sequence of Hpl was designed and transformed into the silkworm genome, and then the mutant (Hpl/Hpl) with specific expression of Hpl in posterior BmSG (BmPSG) was obtained. In the mutants, the transcription level of Fib-L and P25, and corresponding encoding proteins, did not decrease. However, the mRNA level of Fib-H was reduced by 71.1%, and Fib-H protein in the secreted fibroin was decreased from 91.86% to 71.01%. The mRNA level of Hpl was 0.73% and 0.74% of Fib-H and Fib-L, respectively, while HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted silk protein. The exogenous protein was therefore very efficiently translated and secreted. Further analysis of differentially expressed gene (DEG) was carried out in the BmPSG cells and 891 DEGs were detected, of which 208 genes were related to protein metabolism. Reduced expression of endogenous silk proteins in the BmPSG could effectively improve the production efficiency of recombinant exogenous proteins. PMID:26370318

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

    PubMed

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

    2016-04-01

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

  11. Transformation yields of chlorinated ethenes by a methanotrophic mixed culture expressing particulate methane monooxygenase.

    PubMed Central

    Anderson, J E; McCarty, P L

    1997-01-01

    Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions. PMID:9023946

  12. [Prokaryotic expression systems].

    PubMed

    Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

    2013-01-01

    For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.  PMID:23475488

  13. Expressing and purifying membrane transport proteins in high yield.

    PubMed

    Hale, Calvin C; Hill, Chananada K; Price, Elmer M; Bossuyt, Julie

    2002-01-01

    Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic. PMID:11741710

  14. Direct laboratory tensile testing of select yielding rock bolt systems

    SciTech Connect

    VandeKraats, J.D.; Watson, S.O.

    1996-08-01

    Yielding rock bolt support systems have been developed to accommodate ground movement in shifting ground such as in coal operations; in creeping ground such as salt, trona, and potash; and in swelling ground associated with some clays. These systems, designed to remain intact despite ground movement, should enhance mine safety and help contain costs in areas where rebolting of rigid non-yielding systems is typically required. Four such systems were tested in straight tensile pulls in the laboratory. They include the Slip Nut System from Dywidag Systems International USA, Inc., Ischebeck`s bolt mounted Titan Load Indicator, Rocky Mountain Bolt Company`s Yielding Cable Bolt, and a rock bolt installed variation of the yielding steel post developed by RE/SPEC Inc. The first two systems are currently marketed products and the latter two are prototype systems. Each system responds to load and displacement by yielding in an unique manner. All are designed to yield at predetermined loads. A description of each system and its yield function is provided. Each system was tested over its prescribed yield range in a test machine. At least five tests were performed on each system. Each system yielded and continued to provide support according to its design. Each shows promise for ground control use in shifting or creeping rock. This work helps to illustrate the comparative differences in performance between these specialized systems and the applications where they may be most useful.

  15. High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

    PubMed

    Bis, Regina L; Stauffer, Tara M; Singh, Surinder M; Lavoie, Thomas B; Mallela, Krishna M G

    2014-07-01

    Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders. PMID:24794500

  16. High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

    PubMed Central

    Kuchenreuther, Jon M.; Grady-Smith, Celestine S.; Bingham, Alyssa S.; George, Simon J.; Cramer, Stephen P.; Swartz, James R.

    2010-01-01

    Background The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. Principal Findings In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms. Significance The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments. PMID:21124800

  17. Surprising yields with no-till cropping systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Producers using no-till systems have found that crop yields often exceed their expectation based on nutrient and water supply. For example, corn yields 7% higher in a no-till system in central South Dakota than in a tilled system in eastern South Dakota. This is surprising because rainfall is 5 in...

  18. Comparison of different Bacillus subtilis expression systems.

    PubMed

    Vavrová, Ludmila; Muchová, Katarína; Barák, Imrich

    2010-11-01

    Bacillus subtilis is considered to have great potential as a host for the production and secretion of recombinant proteins. Many different expression systems have been developed for B. subtilis. Here we compare two widely used expression systems, the IPTG-inducible derivative of spac system (hyper-spank) and the xylose-inducible (xyl) to the SURE (subtilin-regulated gene expression) system. Western blot analysis of the membrane protein SpoIISA together with its protein partner SpoIISB showed that the highest expression level of this complex is obtained using the SURE system. Measurement of β-galactosidase activities of the promoter-lacZ fusions in individual expression systems confirmed that the P(spaS) promoter of the SURE system is the strongest of those compared, although the induction/repression ratio reached only 1.84. Based on these results, we conclude that the SURE system is the most efficient of these three B. subtilis expression systems in terms of the amount of expressed product. Remarkably, the yield of the SpoIISA-SpoIISB complex obtained from B. subtilis was comparable to that normally obtained from the Escherichia coli arabinose-inducible expression system. PMID:20863884

  19. Identification of Crowding Stress Tolerance Co-Expression Networks Involved in Sweet Corn Yield.

    PubMed

    Choe, Eunsoo; Drnevich, Jenny; Williams, Martin M

    2016-01-01

    Tolerance to crowding stress has played a crucial role in improving agronomic productivity in field corn; however, commercial sweet corn hybrids vary greatly in crowding stress tolerance. The objectives were to 1) explore transcriptional changes among sweet corn hybrids with differential yield under crowding stress, 2) identify relationships between phenotypic responses and gene expression patterns, and 3) identify groups of genes associated with yield and crowding stress tolerance. Under conditions of crowding stress, three high-yielding and three low-yielding sweet corn hybrids were grouped for transcriptional and phenotypic analyses. Transcriptional analyses identified from 372 to 859 common differentially expressed genes (DEGs) for each hybrid. Large gene expression pattern variation among hybrids and only 26 common DEGs across all hybrid comparisons were identified, suggesting each hybrid has a unique response to crowding stress. Over-represented biological functions of DEGs also differed among hybrids. Strong correlation was observed between: 1) modules with up-regulation in high-yielding hybrids and yield traits, and 2) modules with up-regulation in low-yielding hybrids and plant/ear traits. Modules linked with yield traits may be important crowding stress response mechanisms influencing crop yield. Functional analysis of the modules and common DEGs identified candidate crowding stress tolerant processes in photosynthesis, glycolysis, cell wall, carbohydrate/nitrogen metabolic process, chromatin, and transcription regulation. Moreover, these biological functions were greatly inter-connected, indicating the importance of improving the mechanisms as a network. PMID:26796516

  20. Identification of Crowding Stress Tolerance Co-Expression Networks Involved in Sweet Corn Yield

    PubMed Central

    Choe, Eunsoo; Drnevich, Jenny; Williams, Martin M.

    2016-01-01

    Tolerance to crowding stress has played a crucial role in improving agronomic productivity in field corn; however, commercial sweet corn hybrids vary greatly in crowding stress tolerance. The objectives were to 1) explore transcriptional changes among sweet corn hybrids with differential yield under crowding stress, 2) identify relationships between phenotypic responses and gene expression patterns, and 3) identify groups of genes associated with yield and crowding stress tolerance. Under conditions of crowding stress, three high-yielding and three low-yielding sweet corn hybrids were grouped for transcriptional and phenotypic analyses. Transcriptional analyses identified from 372 to 859 common differentially expressed genes (DEGs) for each hybrid. Large gene expression pattern variation among hybrids and only 26 common DEGs across all hybrid comparisons were identified, suggesting each hybrid has a unique response to crowding stress. Over-represented biological functions of DEGs also differed among hybrids. Strong correlation was observed between: 1) modules with up-regulation in high-yielding hybrids and yield traits, and 2) modules with up-regulation in low-yielding hybrids and plant/ear traits. Modules linked with yield traits may be important crowding stress response mechanisms influencing crop yield. Functional analysis of the modules and common DEGs identified candidate crowding stress tolerant processes in photosynthesis, glycolysis, cell wall, carbohydrate/nitrogen metabolic process, chromatin, and transcription regulation. Moreover, these biological functions were greatly inter-connected, indicating the importance of improving the mechanisms as a network. PMID:26796516

  1. High yield and efficient expression and purification of the human 5-HT3A receptor

    PubMed Central

    Wu, Zhong-shan; Cui, Zhi-cheng; Cheng, Hao; Fan, Chen; Melcher, Karsten; Jiang, Yi; Zhang, Cheng-hai; Jiang, Hua-liang; Cong, Yao; Liu, Qian; Xu, H Eric

    2015-01-01

    Aim: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. Methods: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). Results: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. Conclusion: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor. PMID:26073329

  2. A High-Yield Co-Expression System for the Purification of an Intact Drs2p-Cdc50p Lipid Flippase Complex, Critically Dependent on and Stabilized by Phosphatidylinositol-4-Phosphate

    PubMed Central

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose; Fijalkowski, Frank; Jacquot, Aurore; Grønberg, Christina; López-Marqués, Rosa L.; Palmgren, Michael G.; Garrigos, Manuel; le Maire, Marc; Decottignies, Paulette; Gourdon, Pontus; Nissen, Poul; Champeil, Philippe; Lenoir, Guillaume

    2014-01-01

    P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and

  3. High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

    PubMed

    Subedi, Ganesh P; Johnson, Roy W; Moniz, Heather A; Moremen, Kelley W; Barb, Adam

    2015-01-01

    The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield. PMID:26779721

  4. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  5. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  6. The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis.

    PubMed

    Liebeton, Klaus; Lengefeld, Jette; Eck, Jürgen

    2014-12-10

    Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine-Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis. PMID:24997355

  7. Zilpaterol hydrochloride improves beef yield, changes palatability traits, and increases calpain-calpastatin gene expression in Nellore heifers.

    PubMed

    Cônsolo, Nara Regina Brandão; Ferrari, Viviane Borba; Mesquita, Ligia Garcia; Goulart, Rodrigo Silva; Silva, Luis Felipe Prada E

    2016-11-01

    This research aimed to evaluate the effects of the beta-agonist zilpaterol hydrochloride (ZH) on carcass traits, subprimal yield, meat quality, palatability traits, and gene expression in Nellore heifers. Zilpaterol increased Longissimus lumborum area and did not change back fat thickness, meat color, and cooking loss. Heifers fed ZH had greater hindquarter weight and carcass percentage. Muscles from hindquarter were heavier for animals fed ZH. Forequarter (% of carcass) decreased and brisket did not change with ZH supplementation. There were no differences between treatments for steak aroma, beef flavor, and off-flavor. However, tenderness and juiciness were reduced by ZH, depending on postmortem aging. Zilpaterol increased Calpain-1, Calpain-2, and calpastatin mRNA expression, with no effect of day of slaughter or ZH×Day interaction. In conclusion, ZH supplementation improved hypertrophy, meat production, and debone yield in Nellore heifers, which led to decreased tenderness and to increased mRNA expression in the calpain-calpastatin system. PMID:27427783

  8. Yield and yield gaps in central U.S. corn production systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The magnitude of yield gaps (YG) (potential yield – farmer yield) provides some indication of the prospects for increasing crop yield. Quantile regression analysis was applied to county maize (Zea mays L.) yields (1972 – 2011) from Kentucky, Iowa and Nebraska (irrigated) (total of 115 counties) to e...

  9. EarthSat spring wheat yield system test 1975

    NASA Technical Reports Server (NTRS)

    1976-01-01

    The results of an operational test of the EarthSat System during the period 1 June - 30 August 1975 over the spring wheat regions of North Dakota, South Dakota, and Minnesota are presented. The errors associated with each sub-element of the system during the operational test and the sensitivity of the complete system and each major functional sub-element of the system to the observed errors were evaluated. Evaluations and recommendations for future operational users of the system include: (1) changes in various system sub-elements, (2) changes in the yield model to affect improved accuracy, (3) changes in the number of geobased cells needed to develop an accurate aggregated yield estimate, (4) changes associated with the implementation of future operational satellites and data processing systems, and (5) detailed system documentation.

  10. Applicability and methodology of determining sustainable yield in groundwater systems

    NASA Astrophysics Data System (ADS)

    Kalf, Frans R. P.; Woolley, Donald R.

    2005-03-01

    There is currently a need for a review of the definition and methodology of determining sustainable yield. The reasons are: (1) current definitions and concepts are ambiguous and non-physically based so cannot be used for quantitative application, (2) there is a need to eliminate varying interpretations and misinterpretations and provide a sound basis for application, (3) the notion that all groundwater systems either are or can be made to be sustainable is invalid, (4) often there are an excessive number of factors bound up in the definition that are not easily quantifiable, (5) there is often confusion between production facility optimal yield and basin sustainable yield, (6) in many semi-arid and arid environments groundwater systems cannot be sensibly developed using a sustained yield policy particularly where ecological constraints are applied. Derivation of sustainable yield using conservation of mass principles leads to expressions for basin sustainable, partial (non-sustainable) mining and total (non-sustainable) mining yields that can be readily determined using numerical modelling methods and selected on the basis of applied constraints. For some cases there has to be recognition that the groundwater resource is not renewable and its use cannot therefore be sustainable. In these cases, its destiny should be the best equitable use.

  11. A harvester based calibration system for cotton yield monitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this work was to develop a system for measuring seed cotton weight on a cotton harvester to facilitate on-farm research efforts and provide information for use in semi-real-time calibration of yield monitors. The system tested in 2014 was improved from the original design developed...

  12. High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

    PubMed Central

    Sandee, Duanpen

    2011-01-01

    P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins. PMID:21586563

  13. ON-LINE PREDICTION OF YIELD GRADE, LONGISSIMUS MUSCLE AREA, PRELIMINARY YIELD GRADE, ADJUSTED PRELIMINARY YIELD GRADE, AND MARBLING SCORE USING THE MARC BEEF CARCASS IMAGE ANALYSIS SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present experiment was conducted to evaluate the ability of the MARC Beef Carcass Image Analysis System to predict calculated yield grade, longissimus area, preliminary yield grade, adjusted preliminary yield grade, and marbling score under commercial beef processing conditions. In two commerci...

  14. High-yield positron systems for linear colliders

    SciTech Connect

    Clendenin, J.E.

    1989-04-01

    Linear colliders, such as the SLC, are among those accelerators for which a high-yield positron source operating at the repetition rate of the accelerator is desired. The SLC, having electron energies up to 50 GeV, presents the possibility of generating positron bunches with useful charge even exceeding that of the initial electron bunch. The exact positron yield to be obtained depends on the particular capture, transport and damping system employed. Using 31 GeV electrons impinging on a W-type converter phase-space at the target to the acceptance of the capture rf section, the SLC source is capable of producing, for every electron, up to two positrons within the acceptance of the positron damping ring. The design of this source and the performance of the positron system as built are described. Also, future prospects and limitations for high-yield positron systems are discussed. 11 refs., 5 figs., 3 tabs.

  15. Leishmania-based expression systems.

    PubMed

    Taheri, Tahereh; Seyed, Negar; Mizbani, Amir; Rafati, Sima

    2016-09-01

    Production of therapeutic or medical recombinant proteins, such as monoclonal antibodies, proteins, or active enzymes, requires a highly efficient system allowing natural folding and perfect post-translation modifications of the expressed protein. These requirements lead to the generation of a variety of gene expression systems from bacteria to eukaryotes. To achieve the best form of eukaryotic proteins, two factors need to be taken into consideration: choosing a suitable organism to express the protein of interest, and selecting an efficient delivery system. For this reason, the expression of recombinant proteins in eukaryotic nonpathogenic Leishmania parasites is an interesting approach which meets both criteria. Here, new Leishmania-based expression systems are compared with current systems that have long histories in research and industry. PMID:27435294

  16. A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.

    PubMed

    Jones, Jesse A; Price, Emily; Miller, Donovan; Hevener, Kirk E

    2016-08-01

    Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity. PMID:27117979

  17. Milk yield of beef heifers from three calving systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a 2-yr study, first-calf heifers from three calving systems were used to study milk yield throughout a 190-d lactation. Calving occurred in late January to late February (Feb calving), mid-March to mid-April (Apr calving), and mid-May to mid-June (Jun calving). Milk production was measured by wei...

  18. Radiation yields of phenol derivatives in nitrobenzene-water systems

    NASA Astrophysics Data System (ADS)

    Čechová, S.; Macášek, F.; Čech, R.

    Radiolysis of nitrobenzene was studied in two-phase systems with water, sulfuric acid and nitric acid solutions. In kinetic regime of radiolysis the two-phase additivity rule was applied to calculate partial radiation yields of p-nitrophenol, m-nitrophenol, p-nitrosophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, o-nitroaniline and o-, m-, p-aminophenols in the systems of various volume ratios. Nitrobenzene phase was found to be several times more reactive than the aqueous one in respect of the phenols and aniline derivatives formation. In the absence of agitation the yields depend strongly on the thickness of the phases irradiated, as it ensues from the diffusion regime theory of radiolysis in biphasic systems, which was illustrated.

  19. Advanced software system for yield improvement on manufacturing fab

    NASA Astrophysics Data System (ADS)

    Recio, Miguel; Fernandez, Almudena; Martin Santamaria, Victorino; Peman, Maria J.; Gonzalez, Gerardo; Hoyer, J. R.; Whitlock, Steve; James, David; Hausen, Mark

    1996-09-01

    How can we optimize the data collection and data processing to provide us with the most significant information to drive the yield enhancement in a fast and efficient way? With the aim of facing this question this paper describes a software- system which is a part of the global yield enhancement strategy at our factory. The software developed correlates data gathered both in-line and at the end of the fabrication process. The description of the system is accompanied with descriptions of historical cases trying to show the capabilities of the type of correlation. The paper also discusses the natural expansion of the software-system to convert it into a more 'knowledge-based' tool.

  20. Hydrodynamic Tail Vein Injection as a Simple Tool for Yielding Extended Transgene Expression in Solid Tumors.

    PubMed

    Takayama, Takuma; Ukawa, Masami; Kanazawa, Yuki; Ando, Hidenori; Shimizu, Taro; Ishida, Tatsuhiro

    2016-01-01

    Hydrodynamic tail vein injection was considered an in vivo transfection method that yields a higher level of gene expression mainly in the liver. This method has been applied to cancer gene therapy targeting both hepatic and non-hepatic cancers. However, intratumor transgene expression in non-hepatic tumors has not been well studied. In this study, we showed an extended transgene expression of β-galactosidase (LacZ), a nonsecretory protein, in a subcutaneously implanted murine solid tumor following the hydrodynamic injection of plasmid DNA (LacZ pDNA). Our result may indicate that the hydrodynamic injection method is a powerful tool that can be used to gain transgene expression not only in the liver but also in solid tumors. PMID:27582335

  1. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

    PubMed

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  2. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

    PubMed Central

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  3. Yield and Temporal Yield Variability Under Conventional and Alternative Management Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Year to year variation in yield is an inherit risk associated with crop production and many growers rely on intensive mechanical or chemical inputs to secure crop yields in the face of fluctuating environmental conditions. However, as interest in alternative approaches to crop management which are ...

  4. TaER Expression Is Associated with Transpiration Efficiency Traits and Yield in Bread Wheat

    PubMed Central

    Zheng, Jiacheng; Yang, Zhiyuan; Madgwick, Pippa J.; Carmo-Silva, Elizabete; Parry, Martin A. J.; Hu, Yin-Gang

    2015-01-01

    ERECTA encodes a receptor-like kinase and is proposed as a candidate for determining transpiration efficiency of plants. Two genes homologous to ERECTA in Arabidopsis were identified on chromosomes 6 (TaER2) and 7 (TaER1) of bread wheat (Triticum aestivum L.), with copies of each gene on the A, B and D genomes of wheat. Similar expression patterns were observed for TaER1 and TaER2 with relatively higher expression of TaER1 in flag leaves of wheat at heading (Z55) and grain-filling (Z73) stages. Significant variations were found in the expression levels of both TaER1 and TaER2 in the flag leaves at both growth stages among 48 diverse bread wheat varieties. Based on the expression of TaER1 and TaER2, the 48 wheat varieties could be classified into three groups having high (5 varieties), medium (27 varieties) and low (16 varieties) levels of TaER expression. Significant differences were also observed between the three groups varying for TaER expression for several transpiration efficiency (TE)- related traits, including stomatal density (SD), transpiration rate, photosynthetic rate (A), instant water use efficiency (WUEi) and carbon isotope discrimination (CID), and yield traits of biomass production plant-1 (BYPP) and grain yield plant-1 (GYPP). Correlation analysis revealed that the expression of TaER1 and TaER2 at the two growth stages was significantly and negatively associated with SD (P<0.01), transpiration rate (P<0.05) and CID (P<0.01), while significantly and positively correlated with flag leaf area (FLA, P<0.01), A (P<0.05), WUEi (P<0.05), BYPP (P<0.01) and GYPP (P<0.01), with stronger correlations for TaER1 than TaER2 and at grain-filling stage than at heading stage. These combined results suggested that TaER involved in development of transpiration efficiency -related traits and yield in bread wheat, implying a function for TaER in regulating leaf development of bread wheat and contributing to expression of these traits. Moreover, the results indicate

  5. Isolation of a new human scFv antibody recognizing a cell surface binding site to CEACAM1. Large yield production, purification and characterization in E. coli expression system.

    PubMed

    Moricoli, Diego; Laguardia, Maria Elena; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Serafini, Giordano; Flego, Michela; Cianfriglia, Maurizio; Magnani, Mauro

    2014-01-01

    The CEACAM1 cell adhesion molecule has recently received considerable interest as a tumour target antigen since its re-expression often occurs in the advanced stages of multiple malignancies including malignant melanoma, non-small cell lung cancer and other types of solid tumors. In this study, we describe the expression-purification and characterization of the new single chain variable fragment (scFv) antibody named DIATHIS1, that recognizes the N-terminal IgV-like domain present in CEACAM1. Three validation batches show that the production process is robust and reproducible. The scFv DIATHIS1 is formulated as a naturally occurring mixture of monomer and dimer. The antibody is biophysically stable at low temperature (-80°C), different concentrations and remains biologically active for at least 24months. The thermal stability of scFv DIATHIS1 at 37°C shows important features for its activity in vivo. The dimer behaves as a reservoir converting slowly into monomer. The monomer and dimer forms of scFv DIATHIS1 were isolated and characterized, showing high reactivity for CEACAM1. This new composition of antibody could have advantageous pharmacokinetics parameters over conventional scFv for in vivo applications. PMID:24184403

  6. Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai.

    PubMed

    Ait-ali, Tahar; Rands, Carley; Harberd, Nicholas P

    2003-09-01

    The growth of plants is repressed by DELLA proteins, nuclear regulators whose activities are opposed by the growth-promoting phytohormone gibberellin (GA). Mutations affecting DELLA protein function were previously used by plant breeders to create the high-yielding semidwarf wheat varieties of the green revolution. gai is an Arabidopsis mutant DELLA protein-encoding orthologue of the wheat semidwarfing genes. Here we describe the development of a transgene that confers ethanol-inducible gai expression. Transient induction of gai causes transient growth repression: growth prior to and after treatment is unaffected. Appropriate ethanol treatments result in dwarf plants that produce the same numbers of seeds as untreated controls. This new technology represents a substantial advance in the applicability of genes encoding mutant DELLA proteins to agricultural and horticultural improvement, enhancing the flexibity with which these genes can be used for the sustainable achievement of increased crop plant yields. PMID:17166132

  7. Pluto Express power system architecture

    SciTech Connect

    Carr, G.A.

    1996-12-31

    The Pluto Express power system must answer the challenge of the next generation spacecraft by reducing its power, mass and volume envelopes. Technology developed by the New Millennium Program will enable the power system to meet the stringent requirements for the Pluto Express mission without exceeding the spacecraft mass and volume budgets. Traditionally, there has been an increasing trend of the percentage of mass of the power system electronics with respect to the total spacecraft mass. With all of the previous technology focus on high density digital packaging, the power system electronics have not been keeping pace forcing the spacecraft to absorb a relative increase in the power system mass. The increasing trend can be reversed by using mixed signal ASICs and high density multi-chip-module (MCM) packaging techniques validated by the New Millennium Program. As the size of the spacecraft shrinks, the power system electronics must become tightly integrated with the spacecraft loads. The power system architecture needs the flexibility to accommodate the specific load requirements without sacrificing the capability for growth or reduction as the spacecraft requirements change throughout the development. Modularity is a key requirement that will reduce the overall power system cost. Although the focus has been on shrinking the power system volume and mass, the efficiency and functionality cannot be ignored. Increased efficiency and functionality will only enhance the power systems capability to reduce spacecraft power requirements. The combination of the New Millennium packaging technologies with the Pluto Express power system architecture will produce a product with the capability to meet a wide range of mission profiles while reducing system development costs.

  8. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  9. NSLS vacuum system operating experience conditioning and desorption yields

    SciTech Connect

    Halama, H.J.

    1991-01-01

    All straight sections in both the VUV and the X-Ray rings have been filled with various insertion devices, most of them fully operational. Beam lifetime in the VUV ring is limited by the Touschek effect to {approximately}100 minutes at 800 mA due to the small vertical beam size required by users. With no experiments running, X-Ray beam lifetime is >35 hours at 220 mA and is limited by beam gas scattering. During the past several years the U10 beam line was used to measure PSD yields from various metals to study their relative merits for light sources. These yields were also compared to those measured in X-Ray ring dipoles during initial commissioning when desorption was high. Despite the large differences in critical photon energies, agreement was quite good. Both rings are now fully conditioned and their pressures and lifetimes have reached equilibrium. Well established conditioning procedures are followed after every intervention into their vacuum systems.

  10. Improving starch yield in cereals by over-expression of ADPglucose pyrophosphorylase: expectations and unanticipated outcomes.

    PubMed

    Tuncel, Aytug; Okita, Thomas W

    2013-10-01

    Significant improvements in crop productivity are required to meet the nutritional requirements of a growing world population. This challenge is magnified by an increased demand for bioenergy as a means to mitigate carbon inputs into the environment. Starch is a major component of the harvestable organs of many crop plants, and various endeavors have been taken to improve the yields of starchy organs through the manipulation of starch synthesis. Substantial efforts have centered on the starch regulatory enzyme ADPglucose pyrophosphorylase (AGPase) due to its pivotal role in starch biosynthesis. These efforts include over-expression of this enzyme in cereal plants such as maize, rice and wheat as well as potato and cassava, as they supply the bulk of the staple food worldwide. In this perspective, we describe efforts to increase starch yields in cereal grains by first providing an introduction about the importance of source-sink relationship and the motives behind the efforts to alter starch biosynthesis and turnover in leaves. We then discuss the catalytic and regulatory properties of AGPase and the molecular approaches used to enhance starch synthesis by manipulation of this process during grain filling using seed-specific promoters. Several studies have demonstrated increases in starch content per seed using endosperm-specific promoters, but other studies have demonstrated an increase in seed number with only marginal impact on seed weight. Potential mechanisms that may be responsible for this paradoxical increase in seed number will also be discussed. Finally, we describe current efforts and future prospects to improve starch yield in cereals. These efforts include further enhancement of starch yield in rice by augmenting the process of ADPglucose transport into amyloplast as well as other enzymes involved in photoassimilate partitioning in seeds. PMID:23987811

  11. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  12. Systems Biophysics of Gene Expression

    PubMed Central

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  13. Surprising yields with no-till cropping systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Producers using no-till practices have observed that crop yields can greatly exceed expectations based on nutrient and water supply. For example, Ralph Holzwarth, who farms near Gettysburg, SD, has averaged 150 bu/ac of corn on his farm for the past 6 years. We were surprised with this yield, as c...

  14. Cotton yield assessment using plant height mapping system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant nitrogen status and yield potential are important factors for optimizing field management in cotton production. An experiment was conducted in 2011 at Stoneville, Mississippi to develop a method to assess plant N status and yield potential in cotton. The experimental plots were laid out in a s...

  15. HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

    PubMed

    Yadavalli, Raghavendra; Sam-Yellowe, Tobili

    2015-01-01

    Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be

  16. Production of serpins using yeast expression systems.

    PubMed

    Pemberton, Philip A; Bird, Phillip I

    2004-02-01

    Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered. PMID:14698631

  17. Gastrointestinal Spatiotemporal mRNA Expression of Ghrelin vs Growth Hormone Receptor and New Growth Yield Machine Learning Model Based on Perturbation Theory

    PubMed Central

    Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R.; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe

    2016-01-01

    The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R2 of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system. PMID:27460882

  18. Gastrointestinal Spatiotemporal mRNA Expression of Ghrelin vs Growth Hormone Receptor and New Growth Yield Machine Learning Model Based on Perturbation Theory.

    PubMed

    Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe

    2016-01-01

    The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R(2) of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system. PMID:27460882

  19. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    PubMed Central

    Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja; Scheuermann, Tina; Ebert, Lars; LaBaer, Joshua; Korn, Bernhard

    2007-01-01

    Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

  20. High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase.

    PubMed

    Liu, Xiaojuan; Wen, Mingjie; Li, Jing; Zhai, Fangli; Ruan, Jing; Zhang, Liqing; Li, Shentao

    2011-11-01

    We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = β = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities. PMID:21573940

  1. Identification of expressed genes in the mapped QTLs for yield related traits in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improvement of grain yield is a perpetual goal in rice breeding. Yield and its component traits are quantitatively inherited and controlled by many genes. To identify the Quantitative Trait Loci (QTL) involved in yield, a recombinant inbred line (RIL) population consisting of 259 progeny was devel...

  2. Enhancing Potato System Sustainability: Project Overview, Plant Growth, and Yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato yield in the Northeast U.S. has remained constant for over 50 years, despite increased inputs of pesticides, nutrients, and water. Consequently, research is needed to quantify and reduce the constraints to potato productivity. We established Status Quo, Soil Conserving, Soil Improving, and Di...

  3. High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins.

    PubMed

    Hildenbrand, Zacariah L; Molugu, Sudheer K; Paul, Atanu; Avila, Gustavo A; Herrera, Nadia; Xiao, Chuan; Cox, Marc B; Bernal, Ricardo A

    2010-10-15

    Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor-chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle. PMID:20829124

  4. High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli.

    PubMed

    Feng, Xingjun; Xu, Wenshan; Qu, Pei; Li, Xiaochong; Xing, Liwei; Liu, Di; Jiao, Jian; Wang, Jue; Li, Zhongqiu; Liu, Chunlong

    2015-01-01

    The antimicrobial peptide fowlicidin-2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin-2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin-2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin-2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET-32a(+), which features fusion protein thioredoxin at the N-terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria-Bertani (LB) medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction, the fowlicidin-2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse-phase high-performance liquid chromatography (RP-HPLC), ∼6.0 mg of fowlicidin-2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria, and even drug-resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large-scale production. PMID:25641948

  5. Maximal yields from multispecies fisheries systems: rules for systems with multiple trophic levels.

    PubMed

    Matsuda, Hiroyuki; Abrams, Peter A

    2006-02-01

    Increasing centralization of the control of fisheries combined with increased knowledge of food-web relationships is likely to lead to attempts to maximize economic yield from entire food webs. With the exception of predator-prey systems, we lack any analysis of the nature of such yield-maximizing strategies. We use simple food-web models to investigate the nature of yield- or profit-maximizing exploitation of communities including two types of three-species food webs and a variety of six-species systems with as many as five trophic levels. These models show that, for most webs, relatively few species are harvested at equilibrium and that a significant fraction of the species is lost from the web. These extinctions occur for two reasons: (1) indirect effects due to harvesting of species that had positive effects on the extinct species, and (2) intentional eradication of species that are not themselves valuable, but have negative effects on more valuable species. In most cases, the yield-maximizing harvest involves taking only species from one trophic level. In no case was an unharvested top predator part of the yield-maximizing strategy. Analyses reveal that the existence of direct density dependence in consumers has a large effect on the nature of the optimal harvest policy, typically resulting in harvest of a larger number of species. A constraint that all species must be retained in the system (a "constraint of biodiversity conservation") usually increases the number of species and trophic levels harvested at the yield-maximizing policy. The reduction in total yield caused by such a constraint is modest for most food webs but can be over 90% in some cases. Independent harvesting of species within the web can also cause extinctions but is less likely to do so. PMID:16705975

  6. Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions.

    PubMed

    Uga, Yusaku; Sugimoto, Kazuhiko; Ogawa, Satoshi; Rane, Jagadish; Ishitani, Manabu; Hara, Naho; Kitomi, Yuka; Inukai, Yoshiaki; Ono, Kazuko; Kanno, Noriko; Inoue, Haruhiko; Takehisa, Hinako; Motoyama, Ritsuko; Nagamura, Yoshiaki; Wu, Jianzhong; Matsumoto, Takashi; Takai, Toshiyuki; Okuno, Kazutoshi; Yano, Masahiro

    2013-09-01

    The genetic improvement of drought resistance is essential for stable and adequate crop production in drought-prone areas. Here we demonstrate that alteration of root system architecture improves drought avoidance through the cloning and characterization of DEEPER ROOTING 1 (DRO1), a rice quantitative trait locus controlling root growth angle. DRO1 is negatively regulated by auxin and is involved in cell elongation in the root tip that causes asymmetric root growth and downward bending of the root in response to gravity. Higher expression of DRO1 increases the root growth angle, whereby roots grow in a more downward direction. Introducing DRO1 into a shallow-rooting rice cultivar by backcrossing enabled the resulting line to avoid drought by increasing deep rooting, which maintained high yield performance under drought conditions relative to the recipient cultivar. Our experiments suggest that control of root system architecture will contribute to drought avoidance in crops. PMID:23913002

  7. Upper Limits for Power Yield in Thermal, Chemical, and Electrochemical Systems

    NASA Astrophysics Data System (ADS)

    Sieniutycz, Stanislaw

    2010-03-01

    We consider modeling and power optimization of energy converters, such as thermal, solar and chemical engines and fuel cells. Thermodynamic principles lead to expressions for converter's efficiency and generated power. Efficiency equations serve to solve the problems of upgrading or downgrading a resource. Power yield is a cumulative effect in a system consisting of a resource, engines, and an infinite bath. While optimization of steady state systems requires using the differential calculus and Lagrange multipliers, dynamic optimization involves variational calculus and dynamic programming. The primary result of static optimization is the upper limit of power, whereas that of dynamic optimization is a finite-rate counterpart of classical reversible work (exergy). The latter quantity depends on the end state coordinates and a dissipation index, h, which is the Hamiltonian of the problem of minimum entropy production. In reacting systems, an active part of chemical affinity constitutes a major component of the overall efficiency. The theory is also applied to fuel cells regarded as electrochemical flow engines. Enhanced bounds on power yield follow, which are stronger than those predicted by the reversible work potential.

  8. Transient expression systems for plant-derived biopharmaceuticals.

    PubMed

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems. PMID:20673010

  9. Overexpression of wheat gene TaMOR improves root system architecture and grain yield in Oryza sativa.

    PubMed

    Li, Bo; Liu, Dan; Li, Qiaoru; Mao, Xinguo; Li, Ang; Wang, Jingyi; Chang, Xiaoping; Jing, Ruilian

    2016-07-01

    Improved root architecture is an effective strategy to increase crop yield. We demonstrate that overexpression of transcription factor gene MORE ROOT (TaMOR) from wheat (Triticum aestivum L.) results in more roots and higher grain yield in rice (Oryza sativa). TaMOR, encoding a plant-specific transcription factor belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) protein family, is highly conserved in wheat and its wild relatives. In this study, tissue expression patterns indicated that TaMOR mainly localizes to root initiation sites. The consistent gene expression pattern suggests that TaMOR is involved in root initiation. Exogenous auxin treatment induced TaMOR expression without de novo protein biosynthesis. Both in vivo and in vitro experiments demonstrated that TaMOR interacts with TaMOR-related protein TaMRRP, which contains a four-tandem-pentatricopeptide repeat motif. Overexpression of TaMOR led to more lateral roots in Arabidopsis thaliana, and TaMOR-overexpressing rice plants had more crown roots, a longer main panicle, a higher number of primary branches on the main panicle, a higher grain number per plant, and higher yield per plant than the plants of wild type. In general, TaMOR-D-overexpressing lines had larger root systems in Arabidopsis and rice, and produce a higher grain yield per plant. TaMOR therefore offers an opportunity to improve root architecture and increase yield in crop plants. PMID:27229732

  10. Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

    PubMed

    Tata, Sandeep Kumar; Jung, Jihye; Kim, Yoon-Ha; Choi, Jun Young; Jung, Ji-Yul; Lee, In-Jung; Shin, Jeong Sheop; Ryu, Stephen Beungtae

    2016-01-01

    Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy. PMID:25644367

  11. Ectopic expression of SlAGO7 alters leaf pattern and inflorescence architecture and increases fruit yield in tomato.

    PubMed

    Lin, Dongbo; Xiang, Ya; Xian, Zhiqiang; Li, Zhengguo

    2016-08-01

    ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential. PMID:26847714

  12. The Effect of High Concentrations of Glufosinate Ammonium on the Yield Components of Transgenic Spring Wheat (Triticum aestivum L.) Constitutively Expressing the bar Gene

    PubMed Central

    Áy, Zoltán; Mihály, Róbert; Cserháti, Mátyás; Kótai, Éva; Pauk, János

    2012-01-01

    We present an experiment done on a bar+ wheat line treated with 14 different concentrations of glufosinate ammonium—an effective component of nonselective herbicides—during seed germination in a closed experimental system. Yield components as number of spikes per plant, number of grains per spike, thousand kernel weight, and yield per plant were thoroughly analysed and statistically evaluated after harvesting. We found that a concentration of glufosinate ammonium 5000 times the lethal dose was not enough to inhibit the germination of transgenic plants expressing the bar gene. Extremely high concentrations of glufosinate ammonium caused a bushy phenotype, significantly lower numbers of grains per spike, and thousand kernel weights. Concerning the productivity, we observed that concentrations of glufosinate ammonium 64 times the lethal dose did not lead to yield depression. Our results draw attention to the possibilities implied in the transgenic approaches. PMID:22649303

  13. Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome.

    PubMed

    Shi, Weibing; Moon, Christina D; Leahy, Sinead C; Kang, Dongwan; Froula, Jeff; Kittelmann, Sandra; Fan, Christina; Deutsch, Samuel; Gagic, Dragana; Seedorf, Henning; Kelly, William J; Atua, Renee; Sang, Carrie; Soni, Priya; Li, Dong; Pinares-Patiño, Cesar S; McEwan, John C; Janssen, Peter H; Chen, Feng; Visel, Axel; Wang, Zhong; Attwood, Graeme T; Rubin, Edward M

    2014-09-01

    Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation. PMID:24907284

  14. Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome

    PubMed Central

    Moon, Christina D.; Leahy, Sinead C.; Kang, Dongwan; Froula, Jeff; Kittelmann, Sandra; Fan, Christina; Deutsch, Samuel; Gagic, Dragana; Seedorf, Henning; Kelly, William J.; Atua, Renee; Sang, Carrie; Soni, Priya; Li, Dong; Pinares-Patiño, Cesar S.; McEwan, John C.; Janssen, Peter H.; Chen, Feng; Visel, Axel; Wang, Zhong; Attwood, Graeme T.

    2014-01-01

    Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation. PMID:24907284

  15. Pentopyranosyl Oligonucleotide Systems. Part 11: Systems with Shortened Backbones: D)-beta-Ribopyranosyl-(4 yields 3 )- and (L)-alpha - Lyxopyranosyl-(4 yields 3 )-oligonucleotides

    NASA Technical Reports Server (NTRS)

    Wippo, Harald; Reck, Folkert; Kudick, Rene; Ramaseshan, Mahesh; Ceulemans, Griet; Bolli, Martin; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2001-01-01

    The (L)-a-lyxopyranosyl-(4'yields 3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4' yields 3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the six-bonds-per-backbone-unit rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-a-lyxopyranosyl-(4' yields 3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system s capability to cross-pair with DNA.

  16. Plasmid-free T7-based Escherichia coli expression systems.

    PubMed

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  17. Construction and development of a novel expression system of Streptomyces.

    PubMed

    Guan, Chengran; Cui, Wenjing; He, Xiaotian; Hu, Xu; Xu, Jun; Du, Guocheng; Chen, Jian; Zhou, Zhemin

    2015-09-01

    Streptomyces is well known to be an attractive host for producing large amounts of proteins with potent biological activities into the culture supernatant. To expand its expression system, we constructed a novel expression plasmid for gene expression in Streptomyces by inserting the promoter (P(tg)) and the signal peptide (SP(tg)) of transglutaminase (TGase) from Streptomyces hygroscopicus WSH03-13 into vector pIJ86, followed by multiple cloning sites and a transcriptional terminator fd (fd-ter). The secretion capacity of the vector was further enhanced by optimizing the signal peptidase cleavage site and a rare codon of SP(tg), yielding expression vector pSG02. Using this vector, TGase was actively and greatly expressed in the supernatant in several Streptomyces strains. In addition, the heterologous proteins aminopeptidase from Bacillus subtilis Zj016 (BSAP) and phenylalanine ammonia-lyase from Rhodotorula glutinis (PAL) were also expressed in various Streptomyces strains by this vector. This expression system should be useful for the expression of other proteins. PMID:25956536

  18. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    PubMed Central

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

  19. EarthSat spring wheat yield system test 1975, appendix 4

    NASA Technical Reports Server (NTRS)

    1976-01-01

    A computer system is presented which processes meteorological data from both ground observations and meteorologic satellites to define plant weather aspects on a four time per day basis. Plant growth stages are calculated and soil moisture profiles are defined by the system. The EarthSat system assesses plant stress and prepares forecasts of end-of-year yields. The system was used to forecast spring wheat yields in the upper Great Plains states. Hardware and software documentation is provided.

  20. Topical imiquimod yields systemic effects due to unintended oral uptake.

    PubMed

    Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N; Vandenbroucke, Roosmarijn E; Libert, Claude

    2016-01-01

    Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models. PMID:26818707

  1. Topical imiquimod yields systemic effects due to unintended oral uptake

    PubMed Central

    Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N.; Vandenbroucke, Roosmarijn E.; Libert, Claude

    2016-01-01

    Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models. PMID:26818707

  2. Identification of crowding stress tolerance co-expression networks involved in sweet corn yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tolerance to crowding stress has played a crucial role in improving agronomic productivity in field corn; however, commercial sweet corn hybrids vary greatly in crowding stress tolerance. The objectives were to 1) explore transcriptional changes among sweet corn hybrids with differential yield under...

  3. Controlled expression of functional miR-122 with a ligand inducible expression system

    PubMed Central

    2010-01-01

    Background To study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression. Results We present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed. Conclusion We established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired

  4. Quartz crystal microbalance-based system for high-sensitivity differential sputter yield measurements

    SciTech Connect

    Rubin, B.; Topper, J. L.; Farnell, C. C.; Yalin, A. P.

    2009-10-15

    We present a quartz crystal microbalance-based system for high sensitivity differential sputter yield measurements of different target materials due to ion bombardment. The differential sputter yields can be integrated to find total yields. Possible ion beam conditions include ion energies in the range of 30-350 eV and incidence angles of 0 deg. - 70 deg. from normal. A four-grid ion optics system is used to achieve a collimated ion beam at low energy (<100 eV) and a two-grid ion optics is used for higher energies (up to 750 eV). A complementary weight loss approach is also used to measure total sputter yields. Validation experiments are presented that confirm high sensitivity and accuracy of sputter yield measurements.

  5. Quartz crystal microbalance-based system for high-sensitivity differential sputter yield measurements.

    PubMed

    Rubin, B; Topper, J L; Farnell, C C; Yalin, A P

    2009-10-01

    We present a quartz crystal microbalance-based system for high sensitivity differential sputter yield measurements of different target materials due to ion bombardment. The differential sputter yields can be integrated to find total yields. Possible ion beam conditions include ion energies in the range of 30-350 eV and incidence angles of 0 degrees-70 degrees from normal. A four-grid ion optics system is used to achieve a collimated ion beam at low energy (<100 eV) and a two-grid ion optics is used for higher energies (up to 750 eV). A complementary weight loss approach is also used to measure total sputter yields. Validation experiments are presented that confirm high sensitivity and accuracy of sputter yield measurements. PMID:19895063

  6. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    PubMed

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (∼7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. PMID:26627201

  7. Expression of Vitreoscilla hemoglobin in Bacillus thuringiensis improve the cell density and insecticidal crystal proteins yield.

    PubMed

    Liang, Feng; Shouwen, Chen; Ming, Sun; Ziniu, Yu

    2007-02-01

    The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb- strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb- strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition. PMID:17089120

  8. High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

    PubMed Central

    Swain, Monalisa; Slomiany, Mark G.; Rosenzweig, Steven A.; Atreya, Hanudatta S.

    2010-01-01

    The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-IR). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1–6; 22–31 kDa) that via high affinity binding to the IGFs (KD ~ 300–700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in E. coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP. PMID:20541521

  9. Influence of an oxic settling anoxic system on biomass yield, protozoa and filamentous bacteria.

    PubMed

    Rodriguez-Perez, Santiago; Fermoso, Fernando G

    2016-01-01

    An oxic settling anoxic system coupled with an activated sludge process has been studied to reduce sewage sludge production. The reduction of sludge yield, excess sludge production and active biomass yield were 51.7%, 52.9% and 67.1%, respectively, compared with the control system. The oxic reactor of the oxic settling anoxic system, even with a lower active biomass concentration than the oxic reactor of control system, showed a higher metabolic activity in their active biomass. Diversity and crawling ciliates group have been shown as promising bioindicators of active biomass yield reduction. The identification of floc-forming bacteria in the control system suggested that oxic settling anoxic system will improve settling properties compared to a Conventional Activated Sludge process. PMID:26479432

  10. Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

    PubMed Central

    2010-01-01

    Background Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. Results Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. Conclusion The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in

  11. Greenhouse tomato limited cluster production systems: crop management practices affect yield

    NASA Technical Reports Server (NTRS)

    Logendra, L. S.; Gianfagna, T. J.; Specca, D. R.; Janes, H. W.

    2001-01-01

    Limited-cluster production systems may be a useful strategy to increase crop production and profitability for the greenhouse tomato (Lycopersicon esculentum Mill). In this study, using an ebb-and-flood hydroponics system, we modified plant architecture and spacing and determined the effects on fruit yield and harvest index at two light levels. Single-cluster plants pruned to allow two leaves above the cluster had 25% higher fruit yields than did plants pruned directly above the cluster; this was due to an increase in fruit weight, not fruit number. Both fruit yield and harvest index were greater for all single-cluster plants at the higher light level because of increases in both fruit weight and fruit number. Fruit yield for two-cluster plants was 30% to 40% higher than for single-cluster plants, and there was little difference in the dates or length of the harvest period. Fruit yield for three-cluster plants was not significantly different from that of two-cluster plants; moreover, the harvest period was delayed by 5 days. Plant density (5.5, 7.4, 9.2 plants/m2) affected fruit yield/plant, but not fruit yield/unit area. Given the higher costs for materials and labor associated with higher plant densities, a two-cluster crop at 5.5 plants/m2 with two leaves above the cluster was the best of the production system strategies tested.

  12. Develop a field grid system for yield mapping and machine control. Final report, Invention 544

    SciTech Connect

    1995-12-15

    The objective of this project was to build and test the Field Grid Sense system for yield mapping and machine control during harvesting. Secondly, to use Field Grid Sense system with chemical application equipment to demonstrate a workable in-field system. This document contains summarized quarterly reports.

  13. High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

    PubMed

    Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey

    2015-07-01

    Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza. PMID:25740321

  14. Ecological intensification of cereal production systems: Yield potential, soil quality, and precision agriculture

    PubMed Central

    Cassman, Kenneth G.

    1999-01-01

    Wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) provide about two-thirds of all energy in human diets, and four major cropping systems in which these cereals are grown represent the foundation of human food supply. Yield per unit time and land has increased markedly during the past 30 years in these systems, a result of intensified crop management involving improved germplasm, greater inputs of fertilizer, production of two or more crops per year on the same piece of land, and irrigation. Meeting future food demand while minimizing expansion of cultivated area primarily will depend on continued intensification of these same four systems. The manner in which further intensification is achieved, however, will differ markedly from the past because the exploitable gap between average farm yields and genetic yield potential is closing. At present, the rate of increase in yield potential is much less than the expected increase in demand. Hence, average farm yields must reach 70–80% of the yield potential ceiling within 30 years in each of these major cereal systems. Achieving consistent production at these high levels without causing environmental damage requires improvements in soil quality and precise management of all production factors in time and space. The scope of the scientific challenge related to these objectives is discussed. It is concluded that major scientific breakthroughs must occur in basic plant physiology, ecophysiology, agroecology, and soil science to achieve the ecological intensification that is needed to meet the expected increase in food demand. PMID:10339523

  15. Gene expression analysis and SNP/InDel discovery to investigate yield heterosis of two rubber tree F1 hybrids.

    PubMed

    Li, Dejun; Zeng, Rizhong; Li, Yan; Zhao, Manman; Chao, Jinquan; Li, Yu; Wang, Kai; Zhu, Lihuang; Tian, Wei-Min; Liang, Chengzhi

    2016-01-01

    As an important industrial material, natural rubber is mainly harvested from the rubber tree. Rubber tree breeding is inefficient, expensive and time-consuming, whereas marker-assisted selection is a feasible method for early selection of high-yield hybrids. We thus sequenced and analyzed the transcriptomes of two parent rubber trees (RRIM 600 and PR 107) and their most productive hybrids (RY 7-33-97 and RY 7-20-59) to understand their gene expression patterns and genetic variations including single nucleotide polymorphisms (SNPs) and small insertions/deletions (InDels). We discovered >31,000 genetic variations in 112,702 assembled unigenes. Our results showed that the higher yield in F1 hybrids was positively associated with their higher genome heterozygosity, which was further confirmed by genotyping 10 SNPs in 20 other varieties. We also showed that RY 7-33-97 and RY 7-20-59 were genetically closer to RRIM 600 and PR 107, respectively, in agreement with both their phenotypic similarities and gene expression profiles. After identifying ethylene- and jasmonic acid-responsive genes at the transcription level, we compared and analyzed the genetic variations underlying rubber biosynthesis and the jasmonic acid and ethylene pathways in detail. Our results suggest that genome-wide genetic variations play a substantive role in maintaining rubber tree heterosis. PMID:27108962

  16. Gene expression analysis and SNP/InDel discovery to investigate yield heterosis of two rubber tree F1 hybrids

    PubMed Central

    Li, Dejun; Zeng, Rizhong; Li, Yan; Zhao, Manman; Chao, Jinquan; Li, Yu; Wang, Kai; Zhu, Lihuang; Tian, Wei-Min; Liang, Chengzhi

    2016-01-01

    As an important industrial material, natural rubber is mainly harvested from the rubber tree. Rubber tree breeding is inefficient, expensive and time-consuming, whereas marker-assisted selection is a feasible method for early selection of high-yield hybrids. We thus sequenced and analyzed the transcriptomes of two parent rubber trees (RRIM 600 and PR 107) and their most productive hybrids (RY 7-33-97 and RY 7-20-59) to understand their gene expression patterns and genetic variations including single nucleotide polymorphisms (SNPs) and small insertions/deletions (InDels). We discovered >31,000 genetic variations in 112,702 assembled unigenes. Our results showed that the higher yield in F1 hybrids was positively associated with their higher genome heterozygosity, which was further confirmed by genotyping 10 SNPs in 20 other varieties. We also showed that RY 7-33-97 and RY 7-20-59 were genetically closer to RRIM 600 and PR 107, respectively, in agreement with both their phenotypic similarities and gene expression profiles. After identifying ethylene- and jasmonic acid–responsive genes at the transcription level, we compared and analyzed the genetic variations underlying rubber biosynthesis and the jasmonic acid and ethylene pathways in detail. Our results suggest that genome-wide genetic variations play a substantive role in maintaining rubber tree heterosis. PMID:27108962

  17. Fertilizer source and tillage effects on yield-scaled nitrous oxide emissions in a corn cropping system.

    PubMed

    Venterea, Rodney T; Bijesh, Maharjan; Dolan, Michael S

    2011-01-01

    Management practices such as fertilizer or tillage regime may affect nitrous oxide (N₂O) emissions and crop yields, each of which is commonly expressed with respect to area (e.g., kg N ha or Mg grain ha). Expressing N₂O emissions per unit of yield can account for both of these management impacts and might provide a useful metric for greenhouse gas inventories by relating N₂O emissions to grain production rates. The objective of this study was to examine the effects of long-term (>17 yr) tillage treatments and N fertilizer source on area- and yield-scaled N₂O emissions, soil N intensity, and nitrogen use efficiency for rainfed corn ( L.) in Minnesota over three growing seasons. Two different controlled-release fertilizers (CRFs) and conventional urea (CU) were surface-applied at 146 kg N ha(-1) several weeks after planting to conventional tillage (CT) and no-till (NT) treatments. Yield-scaled emissions across all treatments represented 0.4 to 1.1% of the N harvested in the grain. Both CRFs reduced soil nitrate intensity, but not N₂O emissions, compared with CU. One CRF, consisting of nitrification and urease inhibitors added to urea, decreased N₂O emissions compared with a polymer-coated urea (PCU). The PCU tended to have lower yields during the drier years of the study, which increased its yield-scaled N₂O emissions. The overall effectiveness of CRFs compared with CU in this study may have been reduced because they were applied several weeks after corn was planted. Across all N treatments, area-scaled N₂O emissions were not significantly affected by tillage. However, when expressed per unit yield of grain, grain N, or total aboveground N, N₂O emissions with NT were 52, 66, and 69% greater, respectively, compared with CT. Thus, in this cropping system and climate regime, production of an equivalent amount of grain using NT would generate substantially more N₂O compared with CT. PMID:21869514

  18. Quantum statistical thermodynamics of hot finite nuclear systems: Temperatures and isotopic yield ratios

    SciTech Connect

    Majka, Z.; Staszel, P.; Cibor, J.; Natowitz, J.B.; Hagel, K.; Li, J.; Mdeiwayeh, N.; Wada, R.; Zhao, Y.

    1997-06-01

    We investigate the importance of the quantum statistics and deexcitation of primary fragments on the isotope yield ratio temperature determination. A phenomenological formula is presented which allows derivation of the temperature of the decaying nuclear system at the freeze-out time from the measured double yield ratios of two isotope pairs. This prescription is applied to the recent ALADIN and EOS Collaboration data. {copyright} {ital 1997} {ital The American Physical Society}

  19. Longer-term potato cropping system effects on soilborne diseases and tuber yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In field trials established in 2004, different 3-yr potato cropping systems focused on specific crop management goals of (SC) soil conservation, (SI) soil improvement, and (DS) disease-suppression were evaluated for their effects on soilborne diseases and tuber yield. These systems were compared to ...

  20. Statistical modeling of yield and variance instability in conventional and organic cropping systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cropping systems research was undertaken to address declining crop diversity and verify competitiveness of alternatives to the predominant conventional cropping system in the northern Corn Belt. To understand and capitalize on temporal yield variability within corn and soybean fields, we quantified ...

  1. Milk Yield of Primiparous Beef Cows From Three Calving Systems and Varied Weaning Ages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a 2-yr study, primiparous beef cows produced in 3 calving systems were used to study milk yield throughout a 190-d lactation (2002, n = 20; 2003, n = 24 per calving system). Calving occurred in late winter (average calving date = February 4 ± 2 d), early spring (average calving date = March 30 ± ...

  2. Vermont Finance Reform: The Implementation of a Block Grant and Guaranteed Yield System.

    ERIC Educational Resources Information Center

    Mathis, William J.

    This report examines tax reform in Vermont. A unanimous state supreme court decision in 1997 ruled that the state's educational finance system was unconstitutional. In response to this ruling, the legislature passed a new statewide property-tax system with two tiers: a block grant of $5,010 per pupil and a guaranteed yield for spending above the…

  3. Development of a telemetry and yield-mapping system of olive harvester.

    PubMed

    Castillo-Ruiz, Francisco J; Pérez-Ruiz, Manuel; Blanco-Roldán, Gregorio L; Gil-Ribes, Jesús A; Agüera, Juan

    2015-01-01

    Sensors, communication systems and geo-reference units are required to achieve an optimized management of agricultural inputs with respect to the economic and environmental aspects of olive groves. In this study, three commercial olive harvesters were tracked during two harvesting seasons in Spain and Chile using remote and autonomous equipment that was developed to determine their time efficiency and effective based on canopy shaking for fruit detachment. These harvesters work in intensive/high-density (HD) and super-high-density (SHD) olive orchards. A GNSS (Global Navigation Satellite System) and GSM (Global System for Mobile Communications) device was installed to track these harvesters. The GNSS receiver did not affect the driver's work schedule. Time elements methodology was adapted to the remote data acquisition system. The effective field capacity and field efficiency were investigated. In addition, the field shape, row length, angle between headland alley and row, and row alley width were measured to determinate the optimum orchard design parameters value. The SHD olive harvester showed significant lower effective field capacity values when alley width was less than 4 m. In addition, a yield monitor was developed and installed on a traditional olive harvester to obtain a yield map from the harvested area. The hedge straddle harvester stood out for its highly effective field capacity; nevertheless, a higher field efficiency was provided by a non-integral lateral canopy shaker. All of the measured orchard parameters have influenced machinery yields, whether effective field capacity or field efficiency. A saving of 40% in effective field capacity was achieved with a reduction from 4 m or higher to 3.5 m in alley width for SHD olive harvester. A yield map was plotted using data that were acquired by a yield monitor, reflecting the yield gradient in spite of the larger differences between tree yields. PMID:25675283

  4. Development of a Telemetry and Yield-Mapping System of Olive Harvester

    PubMed Central

    Castillo-Ruiz, Francisco J.; Pérez-Ruiz, Manuel; Blanco-Roldán, Gregorio L.; Gil-Ribes, Jesús A.; Agüera, Juan

    2015-01-01

    Sensors, communication systems and geo-reference units are required to achieve an optimized management of agricultural inputs with respect to the economic and environmental aspects of olive groves. In this study, three commercial olive harvesters were tracked during two harvesting seasons in Spain and Chile using remote and autonomous equipment that was developed to determine their time efficiency and effective based on canopy shaking for fruit detachment. These harvesters work in intensive/high-density (HD) and super-high-density (SHD) olive orchards. A GNSS (Global Navigation Satellite System) and GSM (Global System for Mobile Communications) device was installed to track these harvesters. The GNSS receiver did not affect the driver’s work schedule. Time elements methodology was adapted to the remote data acquisition system. The effective field capacity and field efficiency were investigated. In addition, the field shape, row length, angle between headland alley and row, and row alley width were measured to determinate the optimum orchard design parameters value. The SHD olive harvester showed significant lower effective field capacity values when alley width was less than 4 m. In addition, a yield monitor was developed and installed on a traditional olive harvester to obtain a yield map from the harvested area. The hedge straddle harvester stood out for its highly effective field capacity; nevertheless, a higher field efficiency was provided by a non-integral lateral canopy shaker. All of the measured orchard parameters have influenced machinery yields, whether effective field capacity or field efficiency. A saving of 40% in effective field capacity was achieved with a reduction from 4 m or higher to 3.5 m in alley width for SHD olive harvester. A yield map was plotted using data that were acquired by a yield monitor, reflecting the yield gradient in spite of the larger differences between tree yields. PMID:25675283

  5. Study on paddy rice yield estimation based on multisource data and the Grey system theory

    NASA Astrophysics Data System (ADS)

    Deng, Wensheng; Wang, Wei; Liu, Hai; Li, Chen; Ge, Yimin; Zheng, Xianghua

    2009-10-01

    The paddy rice is our important crops. In study of the paddy rice yield estimation, compared with the scholars who usually only take the remote sensing data or meteorology as the influence factors, we combine the remote sensing and the meteorological data to make the monitoring result closer reality. Although the gray system theory has used in many aspects, it is applied very little in paddy rice yield estimation. This study introduces it to the paddy rice yield estimation, and makes the yield estimation model. This can resolve small data sets problem that can not be solved by deterministic model. It selects some regions in Jianghan plain for the study area. The data includes multi-temporal remote sensing image, meteorological and statistic data. The remote sensing data is the 16-day composite images (250-m spatial resolution) of MODIS. The meteorological data includes monthly average temperature, sunshine duration and rain fall amount. The statistical data is the long-term paddy rice yield of the study area. Firstly, it extracts the paddy rice planting area from the multi-temporal MODIS images with the help of GIS and RS. Then taking the paddy rice yield as the reference sequence, MODIS data and meteorological data as the comparative sequence, computing the gray correlative coefficient, it selects the yield estimation factor based on the grey system theory. Finally, using the factors, it establishes the yield estimation model and does the result test. The result indicated that the method is feasible and the conclusion is credible. It can provide the scientific method and reference value to carry on the region paddy rice remote sensing estimation.

  6. Auto-induction for high yield expression of recombinant novel isoallergen tropomyosin from King prawn (Melicertus latisulcatus) for improved diagnostics and immunotherapeutics.

    PubMed

    Koeberl, Martina; Kamath, Sandip D; Saptarshi, Shruti R; Smout, Michael J; Rolland, Jennifer M; O'Hehir, Robyn E; Lopata, Andreas L

    2014-12-15

    Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-D-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens. PMID:25450004

  7. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

    DOE PAGESBeta

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang -Jun

    2016-06-28

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens’ lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in themore » yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Furthermore, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.« less

  8. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

    PubMed Central

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

    2016-01-01

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications. PMID:27349324

  9. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase.

    PubMed

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

    2016-01-01

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications. PMID:27349324

  10. Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

    2016-03-01

    Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis. PMID:26803502

  11. A quality assessment of the MARS crop yield forecasting system for the European Union

    NASA Astrophysics Data System (ADS)

    van der Velde, Marijn; Bareuth, Bettina

    2015-04-01

    Timely information on crop production forecasts can become of increasing importance as commodity markets are more and more interconnected. Impacts across large crop production areas due to (e.g.) extreme weather and pest outbreaks can create ripple effects that may affect food prices and availability elsewhere. The MARS Unit (Monitoring Agricultural ResourceS), DG Joint Research Centre, European Commission, has been providing forecasts of European crop production levels since 1993. The operational crop production forecasting is carried out with the MARS Crop Yield Forecasting System (M-CYFS). The M-CYFS is used to monitor crop growth development, evaluate short-term effects of anomalous meteorological events, and provide monthly forecasts of crop yield at national and European Union level. The crop production forecasts are published in the so-called MARS bulletins. Forecasting crop yield over large areas in the operational context requires quality benchmarks. Here we present an analysis of the accuracy and skill of past crop yield forecasts of the main crops (e.g. soft wheat, grain maize), throughout the growing season, and specifically for the final forecast before harvest. Two simple benchmarks to assess the skill of the forecasts were defined as comparing the forecasts to 1) a forecast equal to the average yield and 2) a forecast using a linear trend established through the crop yield time-series. These reveal a variability in performance as a function of crop and Member State. In terms of production, the yield forecasts of 67% of the EU-28 soft wheat production and 80% of the EU-28 maize production have been forecast superior to both benchmarks during the 1993-2013 period. In a changing and increasingly variable climate crop yield forecasts can become increasingly valuable - provided they are used wisely. We end our presentation by discussing research activities that could contribute to this goal.

  12. The systemic control of circadian gene expression.

    PubMed

    Gerber, A; Saini, C; Curie, T; Emmenegger, Y; Rando, G; Gosselin, P; Gotic, I; Gos, P; Franken, P; Schibler, U

    2015-09-01

    The mammalian circadian timing system consists of a central pacemaker in the brain's suprachiasmatic nucleus (SCN) and subsidiary oscillators in nearly all body cells. The SCN clock, which is adjusted to geophysical time by the photoperiod, synchronizes peripheral clocks through a wide variety of systemic cues. The latter include signals depending on feeding cycles, glucocorticoid hormones, rhythmic blood-borne signals eliciting daily changes in actin dynamics and serum response factor (SRF) activity, and sensors of body temperature rhythms, such as heat shock transcription factors and the cold-inducible RNA-binding protein CIRP. To study these systemic signalling pathways, we designed and engineered a novel, highly photosensitive apparatus, dubbed RT-Biolumicorder. This device enables us to record circadian luciferase reporter gene expression in the liver and other organs of freely moving mice over months in real time. Owing to the multitude of systemic signalling pathway involved in the phase resetting of peripheral clocks the disruption of any particular one has only minor effects on the steady state phase of circadian gene expression in organs such as the liver. Nonetheless, the implication of specific pathways in the synchronization of clock gene expression can readily be assessed by monitoring the phase-shifting kinetics using the RT-Biolumicorder. PMID:26332965

  13. Crop yield network and its response to changes in climate system

    NASA Astrophysics Data System (ADS)

    Yokozawa, M.

    2013-12-01

    Crop failure (reduction in crop yield) due to extreme weather and climate change could lead to unstable food supply, reflecting the recent globalization in world agricultural production. Specifically, in several major production countries producing large amount of main cereal crops, wheat, maize, soybean and rice, abrupt crop failures in wide area are significantly serious for world food supply system. We examined the simultaneous changes in crop yield in USA, China and Brazil, in terms of the changes in climate system such as El Nino, La nina and so on. In this study, we defined a crop yield networks, which represent the correlation between yearly changes in crop yields and climate resources during the crop growing season in two regions. The climate resources during the crop growing season represents here the average temperature and the accumulated precipitation during the crop growing season of a target crop. As climate data, we used a reanalysis climate data JRA-25 (Japan Meteorological Agency). The yearly changes in crop yields are based on a gridded crop productivity database with a resolution of 1.125 degree in latitude/longitude (Iizumi et al. 2013). It is constructed from the agriculture statistics issued by local administrative bureau in each country, which covers the period during 1982 to 2006 (25 years). For the regions being lack of data, the data was interpolated referring to NPP values estimated by satellite data. Crop yield network is constructed as follows: (1) let DY(i,y) be negative difference in crop yield of year y from the trend yield at grid i; (2) define the correlation of the differences Cij(y) = DY(i, y) DY(j, y); (3) if Cij(y) > Q, then grids i and j are mutually linked for a threshold value Q. Links between grids make a crop yield network. It is here noted that only negative differences are taken into account because we focused on the lean year cases (i.e. yields of both grids were lower than those in the long-term trend). The arrays of

  14. Tillage and forage system effects on forage yields and nutrient uptake under broiler litter amended soils.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Planting and harvesting high yielding forage grasses may remove phosphorus (P), copper (Cu) and zinc (Zn) from surface soils with a long history of broiler litter application. A study was conducted in Alabama’s Sand Mountain region from 1998 to 2000 to determine tillage and forage systems best suite...

  15. Sweet Corn, Southern Pea, and Watermelon Yields following Winter Annual Grazing across Tillage Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vegetable grower income may be supplemented by winter annual grazing of stockers, but excess soil compaction can decrease vegetable yields. We initiated a study to determine the optimal tillage system for sweet corn (Zea mays, L.) cv. ‘Silver Queen’, southern pea (Vigna unguiculata L.) cv. ‘Pinkeye...

  16. SOIL WATER USE AND GRAIN YIELD OF THREE DRYLAND CROPS UNDER DIFFERING TILLAGE SYSTEMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Combining the use of drought-adapted and early maturing crops with reduced tillage practices in dryland cropping systems can increase soil water storage, water-use efficiency and crop yields. The objective of this study was to evaluate soil water use by cowpeas (Vigna unguiculata), grain sorghum [So...

  17. Sheep Grazing Effect on Dryland Soil Properties and Wheat Yield in the Wheat-Fallow System

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheep (Ovis aries L.) grazing during fallow is an effective method of controlling weeds and pests in the wheat (Triticum aestivum L.)-fallow system. Little is known about the effect of sheep grazing on dryland soil properties and wheat yield. We evaluated the effects of fallow management for weed co...

  18. Sheep Grazing in the Wheat-Fallow System Affects Dryland Soil Properties and Grain Yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheep (Ovis aries L.) grazing during fallow is an effective method of controlling weeds and pests in the wheat (Triticum aestivum L.)-fallow system. Little is known about the effect of sheep grazing on dryland soil properties and wheat yield. We evaluated the effects of fallow management for weed co...

  19. Destiny-yield relationship for channel catfish reared in a biofloc technology production system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of stocking density on yield of stocker channel catfish and water quality in a biofloc technology production system was studied in this completely randomized design experiment. Fingerling channel catfish (Ictalurus punctatus; 48.0 g/fish, 17.8 cm/fish) were stocked into nine continuously ...

  20. Does increasing milk yield per cow reduce greenhouse gas emissions? A system approach.

    PubMed

    Zehetmeier, M; Baudracco, J; Hoffmann, H; Heißenhuber, A

    2012-01-01

    Milk yield per cow has continuously increased in many countries over the last few decades. In addition to potential economic advantages, this is often considered an important strategy to decrease greenhouse gas (GHG) emissions per kg of milk produced. However, it should be considered that milk and beef production systems are closely interlinked, as fattening of surplus calves from dairy farming and culled dairy cows play an important role in beef production in many countries. The main objective of this study was to quantify the effect of increasing milk yield per cow on GHG emissions and on other side effects. Two scenarios were modelled: constant milk production at the farm level and decreasing beef production (as co-product; Scenario 1); and both milk and beef production kept constant by compensating the decline in beef production with beef from suckler cow production (Scenario 2). Model calculations considered two types of production unit (PU): dairy cow PU and suckler cow PU. A dairy cow PU comprises not only milk output from the dairy cow, but also beef output from culled cows and the fattening system for surplus calves. The modelled dairy cow PU differed in milk yield per cow per year (6000, 8000 and 10 000 kg) and breed. Scenario 1 resulted in lower GHG emissions with increasing milk yield per cow. However, when milk and beef outputs were kept constant (Scenario 2), GHG emissions remained approximately constant with increasing milk yield from 6000 to 8000 kg/cow per year, whereas further increases in milk yield (10 000 kg milk/cow per year) resulted in slightly higher (8%) total GHG emissions. Within Scenario 2, two different allocation methods to handle co-products (surplus calves and beef from culled cows) from dairy cow production were evaluated. Results showed that using the 'economic allocation method', GHG emissions per kg milk decreased with increasing milk yield per cow per year, from 1.06 kg CO2 equivalents (CO2eq) to 0.89 kg CO2eq for the 6000 and

  1. The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems.

    PubMed

    Wilson, Erica L; Kim, Younggy

    2016-05-01

    In conventional wastewater treatment, waste sludge management and disposal contribute the major cost for wastewater treatment. Bioelectrochemical systems, as a potential alternative for future wastewater treatment and resources recovery, are expected to produce small amounts of waste sludge because exoelectrogenic bacteria grow on anaerobic respiration and form highly populated biofilms on bioanode surfaces. While waste sludge production is governed by the yield and decay coefficient, none of previous studies have quantified these kinetic constants for exoelectrogenic bacteria. For yield coefficient estimation, we modified McCarty's free energy-based model by using the bioanode potential for the free energy of the electron acceptor reaction. The estimated true yield coefficient ranged 0.1 to 0.3 g-VSS (volatile suspended solids) g-COD(-1) (chemical oxygen demand), which is similar to that of most anaerobic microorganisms. The yield coefficient was sensitively affected by the bioanode potential and pH while the substrate and bicarbonate concentrations had relatively minor effects on the yield coefficient. In lab-scale experiments using microbial electrolysis cells, the observed yield coefficient (including the effect of cell decay) was found to be 0.020 ± 0.008 g-VSS g-COD(-1), which is an order of magnitude smaller than the theoretical estimation. Based on the difference between the theoretical and experimental results, the decay coefficient was approximated to be 0.013 ± 0.002 d(-1). These findings indicate that bioelectrochemical systems have potential for future wastewater treatment with reduced waste sludge as well as for resources recovery. Also, the found kinetic information will allow accurate estimation of wastewater treatment performance in bioelectrochemical systems. PMID:26963605

  2. Yielding of the clamped-wire system in the Ilizarov external fixator.

    PubMed

    Watson, M A; Matthias, K J; Maffulli, N; Hukins, D W L

    2003-01-01

    This study demonstrates that the clamped-wire system used to suspend bones within an Ilizarov external fixator yields when the perpendicular load exceeds 50 N per wire. Cyclic loading was applied to tensioned wires clamped within an Ilizarov ring component, with steadily increasing load amplitude. Wires were tested at four initial tension settings. The amount of energy lost within the clamped-wire system per load cycle was calculated for every test. The results showed that there was a consistent trend to increasing non-recoverable energy loss per load cycle when peak loads exceed 50 N for all initial tension settings. A finite element (FE) model replicating the experimental conditions was performed to investigate the levels of stress within the loaded wires. The FE model analyses showed that high stresses were generated in the wires close to the clamping sites, and that the stress levels could reasonably be expected to exceed the material yield stress when loaded to about 55 N, for all initial tension settings. The results show that material yield, accompanied by some wire slippage through the clamps, is responsible for system yield, in agreement with previous studies. Although the initial wire tension has an appreciable effect on the wire stiffness, it did not affect the elastic load range of the clamped-wire system. To prevent yield of the clamped-wire system in practice, the fixator should be assembled with sufficient wires to ensure that the load transmitted to each wire by the patient does not exceed 50 N. PMID:14558649

  3. Modeling and characterization of X-ray yield in a polychromatic, lab-scale, X-ray computed tomography system

    NASA Astrophysics Data System (ADS)

    Mertens, J. C. E.; Chawla, Nikhilesh

    2015-05-01

    A modular X-ray computed micro-tomography (μXCT) system is characterized in terms of X-ray yield resulting both from the generated X-ray spectrum and from X-ray detection with an energy-sensitive detector. The X-ray computed tomography system is composed of a commercially available cone-beam microfocus X-ray source and a modular optically-coupled-CCD-scintillator X-ray detector. The X-ray yield is measured and reported in units independent from exposure time, X-ray tube beam target current, and cone-beam-to-detector geometry. The polychromatic X-ray source is modeled as a broad Bremsstrahlung X-ray spectrum in order to understand the effect of the controllable parameters, that is, X-ray tube accelerating voltage and X-ray beam filtering. An approach is adopted which expresses the absolute number of emitted X-rays. The response of the energy-sensitive detector to the modeled spectrum is modeled as a function of scintillator composition and thickness. The detection efficiency model for the polychromatic X-ray detector considers the response of the light collection system and the electronic imaging array in order to predict absolute count yield under the studied conditions. The modeling approach is applied to the specific hardware implemented in the current μXCT system. The model's predictions for absolute detection rate are in reasonable agreement with measured values under a range of conditions applied to the system for X-ray microtomography imaging, particularly for the LuAG:Ce scintillator material.

  4. Effect of tillage system on yield and weed populations of soybean ( Glycin Max L.).

    PubMed

    Hosseini, Seyed Z; Firouzi, Saeed; Aminpanah, Hashem; Sadeghnejhad, Hamid R

    2016-03-01

    Field experiment was conducted at Agricultural and Natural Resources Research Center of Golestan Province, Iran, to determine the effects of tillage system and weed management regime on yield and weed populations in soybean ( Glycin max L.). The experimental design was a split plot where the whole plot portion was a randomized complete block with three replicates. Main plots were tillage system: 1- No-till row crop seeding, 2- No-till seed drilling, 3- Tillage with disc harrow and drill planting, 4- Tillage with chisel packer and drill planting. The subplots were weed management regimes: 1-Weed control with herbicide application, 2- Hand weeding, 3- Herbicide application plus hand weeding, and 4- Non-weeding. Results indicated that the main effects of tillage system and weed management regime were significant for seed yield, pod number per plant, seed number per pod, weed density and biomass, while their interaction were significant only for weed density, weed biomass, and seed number per pod. The highest grain yields (3838 kg ha-1) were recorded for No-till row crop seeding. The highest seed yield (3877 kg ha-1) also was recorded for weed control with herbicide and hand weeding treatment, followed by hand weeding (3379 kg ha-1). PMID:26871488

  5. Influence of kid rearing systems on milk composition and yield of Murciano-Granadina dairy goats.

    PubMed

    Peris, S; Caja, G; Such, X; Casals, R; Ferret, A; Torre, C

    1997-12-01

    One-hundred eight lactations of Murciano-Granadina goats from different years were used to compare two kid rearing systems. Goats were separated into two groups: suckling and milking. Dams in the suckling group were milked once daily until kids were weaned (wk 0 to 7) and then were milked twice daily. Dams in the milking group were separated from their kids at 48 h after birth; then, kids were raised artificially, and goats were milked twice daily. Total milk yield was estimated according to the oxytocin method during suckling. Stage of lactation, parity, prolificacy, and year effects on milk yield and composition were also studied. As expected, during the first 7 wk of lactation, marketable milk was higher for dams that were milked than for dams that were suckled. Neither milk yield nor milk composition throughout the entire lactation was affected by group or prolificacy with the exception of the percentage of milk CP. The lactation curve peaked at wk 4 or 5 and declined slowly afterward. First parity goats had the lowest milk yield but the highest fat and protein percentages. Third parity goats had the highest milk yield. The separation of kids from their dams after birth did not affect total lactation performance because of the minimal importance of the neuroendocrine milk ejection reflex in goats compared with that of other ruminants. PMID:9436106

  6. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

    PubMed Central

    Mahmoud, Soheil S.; Croteau, Rodney B.

    2001-01-01

    Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition. PMID:11427737

  7. Homologous haplotypes, expression, genetic effects and geographic distribution of the wheat yield gene TaGW2

    PubMed Central

    2014-01-01

    Background TaGW2-6A, cloned in earlier research, strongly influences wheat grain width and TKW. Here, we mainly analyzed haplotypes of TaGW2-6B and their effects on TKW and interaction with haplotypes at TaGW2-6A. Results About 2.9 kb of the promoter sequences of TaGW2-6B and TaGW2-6D were cloned in 34 bread wheat cultivars. Eleven SNPs were detected in the promoter region of TaGW2-6B, forming 4 haplotypes, but no divergence was detected in the TaGW2-6D promoter or coding region. Three molecular markers including CAPS, dCAPS and ACAS, were developed to distinguish the TaGW2-6B haplotypes. Haplotype association analysis indicated that TaGW2-6B has a stronger influence than TaGW2-6A on TKW, and Hap-6B-1 was a favored haplotype increasing grain width and weight that had undergone strong positive selection in global wheat breeding. However, clear geographic distribution differences for TaGW2-6A haplotypes were found; Hap-6A-A was favored in Chinese, Australian and Russian cultivars, whereas Hap-6A-G was preferred in European, American and CIMMYT cultivars. This difference might be caused by a flowering and maturity time difference between the two haplotypes. Hap-6A-A is the earlier type. Haplotype interaction analysis between TaGW2-6A and TaGW2-6B showed additive effects between the favored haplotypes. Hap-6A-A/Hap-6B-1 was the best combination to increase TKW. Relative expression analysis of the three TaGW2 homoeologous genes in 22 cultivars revealed that TaGW2-6A underwent the highest expression. TaGW2-6D was the least expressed during grain development and TaGW2-6B was intermediate. Diversity of the three genes was negatively correlated with their effect on TKW. Conclusions Genetic effects, expression patterns and historic changes of haplotypes at three homoeologous genes of TaGW2 influencing yield were dissected in wheat cultivars. Strong and constant selection to favored haplotypes has been found in global wheat breeding during the past century. This research

  8. 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.

  9. Recombinant production of mecasermin in E. coli expression system

    PubMed Central

    Jafari, S.; Babaeipour, V.; Seyedi, H.A. Eslampanah; Rahaie, M.; Mofid, M.R.; Haddad, L.; Namvaran, M.M.; Fallah, J.

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  10. Recombinant production of mecasermin in E. coli expression system.

    PubMed

    Jafari, S; Babaeipour, V; Seyedi, H A Eslampanah; Rahaie, M; Mofid, M R; Haddad, L; Namvaran, M M; Fallah, J

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  11. CHO-S Antibody Titers >1 Gram/Liter Using Flow Electroporation-Mediated Transient Gene Expression followed by Rapid Migration to High-Yield Stable Cell Lines

    PubMed Central

    Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-01-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities. PMID:25520372

  12. Ectopic expression of a hot pepper bZIP-like transcription factor in potato enhances drought tolerance without decreasing tuber yield.

    PubMed

    Moon, Seok-Jun; Han, Se-Youn; Kim, Dool-Yi; Yoon, In Sun; Shin, Dongjin; Byun, Myung-Ok; Kwon, Hawk-Bin; Kim, Beom-Gi

    2015-11-01

    Over-expression of group A bZIP transcription factor genes in plants improves abiotic stress tolerance but usually reduces yields. Thus, there have been several efforts to overcome yield penalty in transgenic plants. In this study, we characterized that expression of the hot pepper (Capsicum annuum) gene CaBZ1, which encodes a group S bZIP transcription factor, was induced by salt and osmotic stress as well as abscisic acid (ABA). Transgenic potato (Solanum tuberosum) plants over-expressing CaBZ1 exhibited reduced rates of water loss and faster stomatal closure than non transgenic potato plants under drought and ABA treatment conditions. CaBZ1 over-expression in transgenic potato increased the expression of ABA- and stress-related genes (such as CYP707A1, CBF and NAC-like genes) and improved drought stress tolerance. Interestingly, over-expression of CaBZ1 in potato did not produce undesirable growth phenotypes in major agricultural traits such as plant height, leaf size and tuber formation under normal growth conditions. The transgenic potato plants also had higher tuber yields than non transgenic potato plants under drought stress conditions. Thus, CaBZ1 may be useful for improving drought tolerance in tuber crops. This might be the first report of the production of transgenic potato with improved tuber yields under drought conditions. PMID:26394867

  13. Orbital Express fluid transfer demonstration system

    NASA Astrophysics Data System (ADS)

    Rotenberger, Scott; SooHoo, David; Abraham, Gabriel

    2008-04-01

    Propellant resupply of orbiting spacecraft is no longer in the realm of high risk development. The recently concluded Orbital Express (OE) mission included a fluid transfer demonstration that operated the hardware and control logic in space, bringing the Technology Readiness Level to a solid TRL 7 (demonstration of a system prototype in an operational environment). Orbital Express (funded by the Defense Advanced Research Projects Agency, DARPA) was launched aboard an Atlas-V rocket on March 9th, 2007. The mission had the objective of demonstrating technologies needed for routine servicing of spacecraft, namely autonomous rendezvous and docking, propellant resupply, and orbital replacement unit transfer. The demonstration system used two spacecraft. A servicing vehicle (ASTRO) performed multiple dockings with the client (NextSat) spacecraft, and performed a variety of propellant transfers in addition to exchanges of a battery and computer. The fluid transfer and propulsion system onboard ASTRO, in addition to providing the six degree-of-freedom (6 DOF) thruster system for rendezvous and docking, demonstrated autonomous transfer of monopropellant hydrazine to or from the NextSat spacecraft 15 times while on orbit. The fluid transfer system aboard the NextSat vehicle was designed to simulate a variety of client systems, including both blowdown pressurization and pressure regulated propulsion systems. The fluid transfer demonstrations started with a low level of autonomy, where ground controllers were allowed to review the status of the demonstration at numerous points before authorizing the next steps to be performed. The final transfers were performed at a full autonomy level where the ground authorized the start of a transfer sequence and then monitored data as the transfer proceeded. The major steps of a fluid transfer included the following: mate of the coupling, leak check of the coupling, venting of the coupling, priming of the coupling, fluid transfer, gauging

  14. PERUN system and its application for assessing the crop yield potential of the Czech Republic

    NASA Astrophysics Data System (ADS)

    Dubrovsky, M.; Zalud, Z.; Eitzinger, J.; Trnka, M.; Semeradova, D.

    2003-04-01

    The main purpose of the first version of the computer system PERUN, which has been developed in 2001-2002 (presented in EGS 2002), is the probabilistic seasonal crop yield forecasting for a given site. The system is based on the crop growth model WOFOST (version 7, slightly modified) and the six-variate version of the stochastic weather generator Met&Roll. The system is now being enhanced to allow assessment of the crop yield potential of a larger area. As this assessment requires a great amount of meteorological, pedological and crop data to be gathered, but these data are not yet all available to the authors, the presentation will rather focus on the methodological aspects and the results of the sensitivity analysis. The presentation will consist of the following points: (i) Overview of the PERUN system. The results of the validation experiments (spring barley and winter wheat at selected Czech locations) will be presented, too. (ii) Methodology used for a spatial assessment. The assessment is based on integrating model crop yields simulated at multiple sub-regions with region-specific climatic and pedological conditions. The input daily weather series are produced by the stochastic generator. The multi-year crop model simulation is performed for each sub-region to assess the mean and variability of the model yields. (iii) Sensitivity of the regional crop production potential to uncertainties in selected input characteristics: crop cultivar, soil type, hydrological characteristics (e.g. amount of available water at the beginning of the simulation), and climatic conditions (e.g temperature, precipitation). In assessing sensitivity to climate, the climatic characteristics will be varied within the range of values typical for the territory of the Czech Republic. The crops applied in the analysis are spring barley and winter wheat. Acknowledgement: The system PERUN has been developed within the frame of project QC1316 sponsored by the Czech National Agency for

  15. Hydraulic conductivity, specific yield, and pumpage--High Plains aquifer system, Nebraska

    USGS Publications Warehouse

    Pettijohn, Robert A.; Chen, Hsiu-Hsiung

    1983-01-01

    Hydrologic data used to evalute the ground-water potential of the High Plains aquifer system in Nebraska are presented on maps showing the hydraulic conductivity and specific yield of the aquifer system and the volume and distribution of water pumped for irrigation from the aquifer system during 1980. The High Plains aquifer system underlies 177,000 square miles in parts of eight states, including 64,770 square miles in Nebraska. It consists of the Ogallala Formation and Tertiary and Quaternary deposits that are saturated and hydraulically connected to the Ogallala. The hydraulic conductivity of the aquifer system varies from greater than 200 feet per day in parts of the North Platte, Platte, Elkhorn, and Republican River valleys to less than 25 feet per day in the northwestern part of the state. Specific yield of the aquifer system ranges from 10 to 20 percent in most of the state and averages 16 percent. The estimated volume of water recoverable from the aquifer system in Nebraska is 2,237 million acre-feet. Inches of water withdrawn from the aquifer system during 1980 varied from less than 1.5 in the sandhills of north-central Nebraska to more than 12 in the Platte River and Blue River basins. This withdrawal represents about 6,703,000 acre-feet of ground water. (USGS)

  16. Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

    PubMed

    Tanghe, Magali; Danneels, Barbara; Camattari, Andrea; Glieder, Anton; Vandenberghe, Isabel; Devreese, Bart; Stals, Ingeborg; Desmet, Tom

    2015-12-01

    The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate. PMID:26285758

  17. High yield expression and purification of Chikungunya virus E2 recombinant protein and its evaluation for serodiagnosis.

    PubMed

    Verma, Anil; Chandele, Anmol; Nayak, Kaustuv; Kaja, Murali Krishna; Arulandu, Arockiasamy; Lodha, Rakesh; Ray, Pratima

    2016-09-01

    Disease caused by Chikungunya virus (CHIKV) is clinically characterized by sudden-onset of fever and severe arthralgia, which may persist for weeks, months, or years after acute phase of the infection. CHIKV is spreading globally; in India it first appeared in the 1960s followed by a quiescent period and then a full-blown remergence in 2006 and sporadic persistence since then. Despite a large number of commercially available diagnostic kits for CHIKV, clinical preparedness and diagnostics suffer from sub-optimal assays. An international diagnostic laboratory survey suggested that there is a critical need for improved CHIKV diagnostics especially in the early acute phase of illness. With the recent studies indicating that a vast majority of human humoral response in CHIKV infection is directed against E2 protein, this supports strong interest to develop CHIKV E2 based serological tests. However, methods to produce large amounts of CHIKV protein are limited. Here we report cloning, expression and purification methods for obtaining a truncated 37kDa Chikungunya E2 protein at a high yield of 65-70mg/l. We found that this purified protein can be reliably used in ELISA and western blot to detect CHIKV specific antibodies in sera from patients who were PCR or IgM positive. Thus, using this protocol, laboratories can make large quantities of purified protein that can be potentially used in CHIKV serological analysis. PMID:27180040

  18. Genome-Wide Identification of Differentially Expressed Genes Associated with the High Yielding of Oleoresin in Secondary Xylem of Masson Pine (Pinus massoniana Lamb) by Transcriptomic Analysis

    PubMed Central

    Liu, Qinghua; Zhou, Zhichun; Wei, Yongcheng; Shen, Danyu; Feng, Zhongping; Hong, Shanping

    2015-01-01

    Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen

  19. Enhancing the expressiveness of structured reporting systems.

    PubMed

    Langlotz, C P

    2000-05-01

    The overall goal of this research is to build a structured reporting system that reduces the cost, delays, and inconvenience associated with conventional dictation and speech recognition systems. We have implemented such a structured reporting system for radiology that replaces current dictation and transcription processes by allowing radiologists and other imaging professionals to select imaging findings from a medical lexicon. The system uses an imaging-specific information model, called a "description set,' to organize selected terms in a relational database. Unique features of the knowledge representation that enhance its expressiveness include its ability to codify uncertainty about an imaging observation and to represent explicitly the logical relationships among imaging findings. In addition, the system does not require the user to fill in "blanks' in a static text template. Instead, it allows entry of terms in arbitrary order and uses automated text-generation techniques to create a text report that referring physicians are accustomed to receiving. In parallel, the system also produces a multimedia report that the referring physician can use as a quick reference. Unlike the results of conventional dictation or speech recognition, each finding is coded in a relational database for later information processing. Thus, the structured report database can be used to index images by content, to provide real-time decision support, to enhance radiologists' performance, to conduct exploratory clinical research, and to transmit imaging report data to computer-based patient record systems. PMID:10847362

  20. [Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

    PubMed

    Zheng, Hai-Xue; Jin, Ye; Yin, Shuang-Hui; Guo, Hui-Chen; Shang, You-Jun; Bai, Xing-Wen; Liu, Xiang-Tao; Xie, Qing-Ge

    2007-09-01

    To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system. PMID:18051880

  1. Detailed analysis of the energy yield of systems with covered sheet-and-tube PVT collectors

    SciTech Connect

    Santbergen, R.; Rindt, C.C.M.; van Zolingen, R.J.Ch.; Zondag, H.A.

    2010-05-15

    Solar cells have a typical efficiency in the range of 5-20%, implying that 80% or more of the incident solar energy can be harvested in the form of heat and applied for low-temperature heating. In a PVT collector one tries to collect this heat. In this work, the electrical and thermal yield of solar domestic hot water systems with one-cover sheet-and-tube PVT collectors were considered. Objectives of the work were to understand the mechanisms determining these yields, to investigate measures to improve these yields and to investigate the yield consequences if various solar cell technologies are being used. The work was carried out using numerical simulations. A detailed quantitative understanding of all loss mechanisms was obtained, especially of those being inherent to the use of PVT collectors instead of PV modules and conventional thermal collectors. The annual electrical efficiencies of the PVT systems investigated were up to 14% (relative) lower compared to pure PV systems and the annual thermal efficiencies up to 19% (relative) lower compared to pure thermal collector systems. The loss of electrical efficiency is mainly caused by the relatively high fluid temperature. The loss of thermal efficiency is caused both by the high emissivity of the absorber and the withdrawal of electrical energy. However, both the loss of electrical and thermal efficiency can be reduced further by the application of anti-reflective coatings. The thermal efficiency can be improved by the application of a low-emissivity coating on the absorber, however at the cost of a reduced electrical efficiency. (author)

  2. An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

    PubMed

    Ta, Duy Tien; Redeker, Erik Steen; Billen, Brecht; Reekmans, Gunter; Sikulu, Josephine; Noben, Jean-Paul; Guedens, Wanda; Adriaensens, Peter

    2015-10-01

    In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications. PMID:26243885

  3. Silkworm expression system as a platform technology in life science.

    PubMed

    Kato, Tatsuya; Kajikawa, Mizuho; Maenaka, Katsumi; Park, Enoch Y

    2010-01-01

    Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3-6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the "Silkroad," we expect that the "Bioroad" from Asia to Europe will be established by the silkworm expression system. PMID:19830419

  4. The Potato Systems Planner: Integrating Cropping System Impacts on Crop Yield and Quality, Soil Biology, Nutrient Cycling, Diseases, and Economics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Finding and developing profitable cropping systems is a high priority for the potato industry. Consequently, an interdisciplinary team of ARS scientists from the New England Plant, Soil, & Water Laboratory evaluated 14 different rotations for their impacts on crop yield and quality, nutrient availa...

  5. Integrating remote sensing, geographic information system and modeling for estimating crop yield

    NASA Astrophysics Data System (ADS)

    Salazar, Luis Alonso

    This thesis explores various aspects of the use of remote sensing, geographic information system and digital signal processing technologies for broad-scale estimation of crop yield in Kansas. Recent dry and drought years in the Great Plains have emphasized the need for new sources of timely, objective and quantitative information on crop conditions. Crop growth monitoring and yield estimation can provide important information for government agencies, commodity traders and producers in planning harvest, storage, transportation and marketing activities. The sooner this information is available the lower the economic risk translating into greater efficiency and increased return on investments. Weather data is normally used when crop yield is forecasted. Such information, to provide adequate detail for effective predictions, is typically feasible only on small research sites due to expensive and time-consuming collections. In order for crop assessment systems to be economical, more efficient methods for data collection and analysis are necessary. The purpose of this research is to use satellite data which provides 50 times more spatial information about the environment than the weather station network in a short amount of time at a relatively low cost. Specifically, we are going to use Advanced Very High Resolution Radiometer (AVHRR) based vegetation health (VH) indices as proxies for characterization of weather conditions.

  6. Holographic lens spectrum splitting photovoltaic system for increased diffuse collection and annual energy yield

    NASA Astrophysics Data System (ADS)

    Vorndran, Shelby D.; Wu, Yuechen; Ayala, Silvana; Kostuk, Raymond K.

    2015-09-01

    Concentrating and spectrum splitting photovoltaic (PV) modules have a limited acceptance angle and thus suffer from optical loss under off-axis illumination. This loss manifests itself as a substantial reduction in energy yield in locations where a significant portion of insulation is diffuse. In this work, a spectrum splitting PV system is designed to efficiently collect and convert light in a range of illumination conditions. The system uses a holographic lens to concentrate shortwavelength light onto a smaller, more expensive indium gallium phosphide (InGaP) PV cell. The high efficiency PV cell near the axis is surrounded with silicon (Si), a less expensive material that collects a broader portion of the solar spectrum. Under direct illumination, the device achieves increased conversion efficiency from spectrum splitting. Under diffuse illumination, the device collects light with efficiency comparable to a flat-panel Si module. Design of the holographic lens is discussed. Optical efficiency and power output of the module under a range of illumination conditions from direct to diffuse are simulated with non-sequential raytracing software. Using direct and diffuse Typical Metrological Year (TMY3) irradiance measurements, annual energy yield of the module is calculated for several installation sites. Energy yield of the spectrum splitting module is compared to that of a full flat-panel Si reference module.

  7. Yield stresses and flow curves in metallic glass formers and granular systems.

    PubMed

    Voigtmann, Th

    2011-09-01

    We discuss the concept of a glass transition line in the temperature-shear-stress plane in the context of recent simulation data for a metallic melt and dense-packed granular systems. Analyzing these data within a schematic model of the mode-coupling theory for dense glass formers under shear, values for the critical dynamic yield stress (the stress resulting in the limit of arbitrarily slow shear, at the glass transition) are estimated. We discuss two possible scenarios, that of a continuous rise in the dynamic yield stress at the transition, and that of a discontinuous transition, and discuss the data range that needs to be covered to decide between the two cases. A connection is made to the two commonly drawn versions of the jamming diagram, one convex and one concave regarding to the shape of the solid region. PMID:21959546

  8. Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities

    PubMed Central

    Boosani, Chandra S; Varma, Ashok K; Sudhakar, Akulapalli

    2010-01-01

    The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails. PMID:20824118

  9. Impact of land management system on crop yields and soil fertility in Cameroon

    NASA Astrophysics Data System (ADS)

    Tsozué, D.; Nghonda, J. P.; Mekem, D. L.

    2015-09-01

    The impact of direct-seeding mulch-based cropping systems (DMC), direct seeding (DS) and tillage seeding (TS) on Sorghum yields, soil fertility and the rehabilitation of degraded soils was evaluated in northern Cameroon. Field work consisted of visual examination, soil sampling, yield and rainfall data collection. Three fertilization rates (F1: 100 kg ha-1 NPK + 25 kg ha-1 of urea in DMC, F2: 200 kg ha-1 NPK + 50 kg ha-1 of urea in DMC and F3: 300 kg ha-1 NPK + 100 kg ha-1 of urea in DMC) were applied to each cropping system (DS, TS and DMC), resulting in nine experimental plots. Two types of chemical fertilizer were used (NPK 22.10.15 and urea) and applied each year from 2002 to 2012. Average Sorghum yields were 1239, 863 and 960 kg ha-1 in DMC, DS and TS, respectively, at F1, 1658, 1139 and 1192 kg ha-1 in DMC, DS and TS, respectively, at F2, and 2270, 2138 and 1780 kg ha-1 in DMC, DS and TS, respectively, at F3. pH values were 5.2-5.7 under DMC, 4.9-5.3 under DS and TS and 5.6 in the control sample. High values of cation exchange capacity were recorded in the control sample, TS system and F1 of DMC. Base saturation rates, total nitrogen and organic matter contents were higher in the control sample and DMC than in the other systems. All studied soils were permanently not suitable for Sorghum due to the high percentage of nodules. F1 and F2 of the DS were currently not suitable, while F1 and F3 of DMC, F3 of DS and F1, F2 and F3 of TS were marginally suitable for Sorghum due to low pH values.

  10. Impact of land management system on crop yields and soil fertility in Cameroon

    NASA Astrophysics Data System (ADS)

    Tsozué, D.; Nghonda, J. P.; Mekem, D. L.

    2015-06-01

    The impact of direct-seeding mulch-based cropping systems (DMC), direct seeding (DS) and tillage seeding (TS) on Sorghum yields, soil fertility and the rehabilitation of degraded soils was evaluated in northern Cameroon. Field work consisted of visual examination, soil sampling, yield and rainfall data collection. Three fertilization rates (F1: 100 kg ha-1 NPK + 25 kg ha-1 of urea in DMC, F2: 200 kg ha-1 NPK + 50 kg ha-1 of urea in DMC and F3: 300 kg ha-1 NPK + 100 kg ha-1 of urea in DMC) were applied to each cropping system (DS, TS and DMC), resulting in nine experimental plots. Two types of chemical fertilizer were used (NPK 22.10.15 and urea) and applied each year from 2002 to 2012. Average Sorghum yields were 1239, 863 and 960 kg ha-1 respectively in DMC, DS and TS at F1, 1658, 1139 and 1192 kg ha-1 respectively in DMC, DS and TS at F2, and 2270, 2138 and 1780 kg ha-1 respectively in DMC, DS and TS at F3. pH values were 5.2 to 5.7 under DMC, 4.9 to 5.3 under DS and TS, and 5.6 in the control sample. High values of cation exchange capacity were recorded in the control sample, TS system and F1 of DMC. Base saturation rates, total nitrogen and organic matter contents were high in the control sample and the DMC than in the others systems. All studied soils were permanently not suitable for Sorghum due to the high percentage of nodules. F1 and F2 of the DS were currently not suitable, while F1 and F3 of DMC, F3 of DS and F1, F2 and F3 of TS were marginally suitable for Sorghum due to low soil pH values.

  11. Yield and gas exchange ability of sweetpotato plants cultured in a hydroponic system

    NASA Astrophysics Data System (ADS)

    Kitaya, Y.; Hirai, H.; Saiful Islam, A. F. M.; Yamamoto, M.

    Life support of crews in space is greatly dependent on the amounts of food atmospheric O 2 and clean water produced by plants Therefore the space farming systems with scheduling of crop production obtaining high yields with a rapid turnover rate converting atmospheric CO 2 to O 2 and purifying water should be established with employing suitable plant species and varieties and precisely controlling environmental variables around plants grown at a high density in a limited space In this study three sweetpotato varieties were cultured in a newly developed hydroponic system and the yield the photosynthetic rate and the transpiration rate were compared on the earth as a fundamental study for establishing the space farming systems The varieties were Elegant summer Koukei 14 and Beniazuma The hydroponic system mainly consisted of water channels and rockwool boards A growing space for roots was made between the rockwool board and nutrient solution in the water channel Storage roots were developed on the lower surface of the rockwool plates Fresh weights of the storage roots were 1 6 1 2 and 0 6 kg plant for Koukei 14 Elegant summer and Beniazuma respectively grown for five months from June to October under the sun light in Osaka Japan Koukei 14 and Elegant summer produced greater total phytomass than Beniazuma There were positive correlations among the total phytomass the net photosynthetic rate and the transpiration rate Young stems and leaves as well as storage roots of Elegant summer are edible Therefore Elegant-summer

  12. Online evaluation of a commercial video image analysis system (Computer Vision System) to predict beef carcass red meat yield and for augmenting the assignment of USDA yield grades. United States Department of Agriculture.

    PubMed

    Cannell, R C; Belk, K E; Tatum, J D; Wise, J W; Chapman, P L; Scanga, J A; Smith, G C

    2002-05-01

    Objective quantification of differences in wholesale cut yields of beef carcasses at plant chain speeds is important for the application of value-based marketing. This study was conducted to evaluate the ability of a commercial video image analysis system, the Computer Vision System (CVS) to 1) predict commercially fabricated beef subprimal yield and 2) augment USDA yield grading, in order to improve accuracy of grade assessment. The CVS was evaluated as a fully installed production system, operating on a full-time basis at chain speeds. Steer and heifer carcasses (n = 296) were evaluated using CVS, as well as by USDA expert and online graders, before the fabrication of carcasses into industry-standard subprimal cuts. Expert yield grade (YG), online YG, CVS estimated carcass yield, and CVS measured ribeye area in conjunction with expert grader estimates of the remaining YG factors (adjusted fat thickness, percentage of kidney-pelvic-heart fat, hot carcass weight) accounted for 67, 39, 64, and 65% of the observed variation in fabricated yields of closely trimmed subprimals. The dual component CVS predicted wholesale cut yields more accurately than current online yield grading, and, in an augmentation system, CVS ribeye measurement replaced estimated ribeye area in determination of USDA yield grade, and the accuracy of cutability prediction was improved, under packing plant conditions and speeds, to a level close to that of expert graders applying grades at a comfortable rate of speed offline. PMID:12019606

  13. 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

  14. Milk yield responses to changes in milking frequency during early lactation are associated with coordinated and persistent changes in mammary gene expression

    PubMed Central

    2013-01-01

    Background The lactating mammary gland responds to changes in milking frequency by modulating milk production. This response is locally regulated and, in dairy cows, the udder is particularly sensitive during early lactation. Relative to cows milked twice-daily throughout lactation, those milked four-times-daily for just the first 3 weeks of lactation produce more milk throughout that lactation. We hypothesized that the milk yield response would be associated with increased mammary cell turnover and changes in gene expression during frequent milking and persisting thereafter. Cows were assigned to unilateral frequent milking (UFM; left udder halves milked twice-daily; right udder halves milked four-times daily) on days 1 to 21 of lactation, followed by twice-daily milking for the remainder of lactation. Relative to udder halves milked twice-daily, those milked four-times produced more milk during UFM; the difference in milk yield declined acutely upon cessation of UFM after day 21, but remained significantly elevated thereafter. We obtained mammary biopsies from both udder halves on days 21, 23, and 40 of lactation. Results Mammary cell proliferation and apoptosis were not affected by milking frequency. We identified 75 genes that were differentially expressed between paired udder halves on day 21 but exhibited a reversal of differential expression on day 23. Among those genes, we identified four clusters characterized by similar temporal patterns of differential expression. Two clusters (11 genes) were positively correlated with changes in milk yield and were differentially expressed on day 21 of lactation only, indicating involvement in the initial milk yield response. Two other clusters (64 genes) were negatively correlated with changes in milk yield. Twenty-nine of the 75 genes were also differentially expressed on day 40 of lactation. Conclusions Changes in milking frequency during early lactation did not alter mammary cell population dynamics, but were

  15. Economics of fertility in high-yielding dairy cows on confined TMR systems.

    PubMed

    Cabrera, V E

    2014-05-01

    The objective of this review paper was to summarise the latest findings in dairy cattle reproductive economics with an emphasis on high yielding, confined total mixed ration systems. The economic gain increases as the reproductive efficiency improves. These increments follow the law of diminishing returns, but are still positive even at high reproductive performance. Reproductive improvement results in higher milk productivity and, therefore, higher milk income over feed cost, more calf sales and lower culling and breeding expenses. Most high-yielding herds in the United States use a combination of timed artificial insemination (TAI) and oestrous detection (OD) reproductive programme. The ratio of achievable pregnancies between OD and TAI determines the economic value difference between both and their combinations. Nonetheless, complex interactions between reproductive programme, herd relative milk yield, and type of reproductive programme are reported. For example, higher herd relative milk yield would favour programme relying more on TAI. In addition, improved reproductive efficiency produces extra replacements. The availability of additional replacements could allow more aggressive culling policies (e.g. less services for non-pregnant cows) to balance on-farm supply and demand of replacements. Balancing heifer replacement availability in an efficient reproductive programme brings additional economic benefits. New technologies such as the use of earlier chemical tests for pregnancy diagnosis could be economically effective depending on the goals and characteristics of the farm. Opportunities for individual cow reproductive management within defined reproductive programme exist. These decisions would be based on economic metrics derived from the value of a cow such as the value of a new pregnancy, the cost of a pregnancy loss, or the cost of an extra day open. PMID:24679357

  16. Method for improving product yields in an anionic metalloporphyrin-based artificial photosynthesis system

    DOEpatents

    Shelnutt, J.A.

    1984-11-29

    A method is disclosed improving product yields in an anionic metalloporphyrin-based artificial photosynthesis system for hydrogen generation. The method comprises forming an aqueous solution comprising an electron donor, methylviologen, and certain metalloporphyrins and metallochlorins, and irradiating said aqueous solution with light in the presence of a catalyst. In the photosynthesis process, solar energy is collected and stored in the form of a hydrogen. Ligands attached above and below the metalloporphyrin and metallochlorin plane are capable of sterically blocking photochemically inactive electrostatically bound ..pi..-..pi.. complexes which can develop.

  17. Method for improving product yields in an anionic metalloporphyrin-based artificial photosynthesis system

    DOEpatents

    Shelnutt, John A.

    1986-01-01

    A method for improving product yields in an anionic metalloporphyrin-based artificial photosynthesis system for hydrogen generation which comprises forming an aqueous solution comprising an electron donor, methylviologen, and certain metalloporphyrins and metallochlorins, and irradiating said aqueous solution with light in the presence of a catalyst. In the photosynthesis process, solar energy is collected and stored in the form of a gas hydrogen. Ligands attached above and below the metalloporphyrin and metallochlorin plane are capable of sterically blocking photochemically inactive electrostatically bound .pi.--.pi. complexes which can develop.

  18. Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System

    PubMed Central

    Pirdel, Leila; Hosseini, Ahmad Zavaran; Kazemi, Bahram; Rasouli, Manoochehr; Bandehpour, Mojgan; Soudi, Sara

    2012-01-01

    Background Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). Methods The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Results Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. Conclusion These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis. PMID:23407850

  19. Biosurfactant yields and nutrient consumption of Pseudomonas fluorescens 378 studied in a microcomputer controlled multifermentation system.

    PubMed

    Persson, A; Molin, G; Andersson, N; Sjöholm, J

    1990-07-01

    Production of biosurfactant AP-6 and consumption of carbon (succinic acid) and nitrogen (ammonium ions) by Pseudomonas fluorescens 378 were studied under different growth conditions. The study was performed in a microcomputer controlled multibatch fermentation system which enabled simultaneous running of 10 fermentors. The fermentors were mantled glass vessels, temperature controlled by circulated water, and mixing was arranged by magnetic stirrers. They were connected to the computer system (pH measurement and control) via signal conditioning cards. The microcomputer had a 128 kbytes RAM, two 800-kbyte floppy disc drives, a graphic terminal, and expansion cards. Biosurfactant production was independent of the carbon-to-nitrogen ratio and the phosphorus content in the medium. Omitting the Fe(III) supplement to the medium increased the product yield by 120%. Changes in oxygen transfer rate and pH in the iron deficient cultures did not have any effect on the product yield. Iron deficiency increased the cell consumption of carbon source. Consumption of carbon source in relation to nitrogen uptake (carbon/nitrogen quotient) increased with increasing quotient in the growth medium. The uptake of carbon and nitrogen changed in the intervals of 1.2-1.5 g/g biomass and 0.09-0.16 g/g biomass, respectively. The consumption of carbon increased from 1.5 g/g biomass to 2.0 g/g biomass when the medium concentration of phosphorus was decreased from 0.18 to 0.027 g/L. PMID:18595075

  20. Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions.

    PubMed

    Nuccio, Michael L; Wu, Jeff; Mowers, Ron; Zhou, Hua-Ping; Meghji, Moez; Primavesi, Lucia F; Paul, Matthew J; Chen, Xi; Gao, Yan; Haque, Emdadul; Basu, Shib Sankar; Lagrimini, L Mark

    2015-08-01

    Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls. PMID:26473199

  1. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  2. Perun: The System For Seasonal Crop Yield Forecasting Based On The Crop Model and Weather Generator

    NASA Astrophysics Data System (ADS)

    Dubrovsky, M.; Zalud, Z.; Trnka, M.; Haberle, J.; Pesice, P.

    The main purpose of the computer system PERUN, which is now being developed, is the probabilistic seasonal crop yield forecasting. The crop yields (winter wheat and spring barley in the first step) are simulated by crop model WOFOST. The input daily weather series consist of observed data, which are available in the date of forecast issuance, and synthetic data, which follow up with the observed data till the end of the crop model simulation. The synthetic weather series are generated by stochastic generator Met&Roll conditionally on the seasonal weather forecast. The probabilis- tic forecast is based on multiple crop model runs. To provide the six daily weather characteristics required for crop model simulation (precipitation, solar radiation, max- imum and minimum temperatures, air humidity, wind speed), the previous WGEN- like four-variate version of Met&Roll generator was supplemented by a new module. This module adds wind speed and air humidity (necessary to calculate evapotranspi- ration) using the nearest neighbours resampling from the observed data. Because of the problems with availability and/or accuracy of wind and humidity data, the source code of the WOFOST model was modified and allows now to switch between Penman and Makkink methods of calculating the evapotranspiration (the daily values of wind speed and humidity are not required in the Makkink method). The contribution will address following items: 1) Structure of the PERUN system: components and their inputs and outputs. Modifications to WOFOST crop model and Met&Roll generator will be discussed. 2) Validation of the WOFOST crop model. The accuracy obtained using the Penman and Makkink methods will be compared. 3) Demonstration of the forecast accuracy in dependence on the date of issuance. Acknowledgement: The system PERUN is being developed within the frame of project QC1316 sponsored by the Czech National Agency for Agricultural Research (NAZV).

  3. A meta-analysis of maize and wheat yields in low-input vs. conventional and organic systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Organic and low-input systems are proposed as ways to reduce the environmental impacts of agriculture. Previous studies have shown that yields of organic systems can be ~19-25% lower than conventional systems. An intermediary, low-input system could be less damaging for the environment than conventi...

  4. ROI on yield data analysis systems through a business process management strategy

    NASA Astrophysics Data System (ADS)

    Rehani, Manu; Strader, Nathan; Hanson, Jeff

    2005-05-01

    The overriding motivation for yield engineering is profitability. This is achieved through application of yield management. The first application is to continually reduce waste in the form of yield loss. New products, new technologies and the dynamic state of the process and equipment keep introducing new ways to cause yield loss. In response, the yield management efforts have to continually come up with new solutions to minimize it. The second application of yield engineering is to aid in accurate product pricing. This is achieved through predicting future results of the yield engineering effort. The more accurate the yield prediction, the more accurate the wafer start volume, the more accurate the wafer pricing. Another aspect of yield prediction pertains to gauging the impact of a yield problem and predicting how long that will last. The ability to predict such impacts again feeds into wafer start calculations and wafer pricing. The question then is that if the stakes on yield management are so high why is it that most yield management efforts are run like science and engineering projects and less like manufacturing? In the eighties manufacturing put the theory of constraints1 into practice and put a premium on stability and predictability in manufacturing activities, why can't the same be done for yield management activities? This line of introspection led us to define and implement a business process to manage the yield engineering activities. We analyzed the best known methods (BKM) and deployed a workflow tool to make them the standard operating procedure (SOP) for yield managment. We present a case study in deploying a Business Process Management solution for Semiconductor Yield Engineering in a high-mix ASIC environment. We will present a description of the situation prior to deployment, a window into the development process and a valuation of the benefits.

  5. Improvement in the yield and quality of kalmegh (Andrographis paniculata Nees) under the sustainable production system.

    PubMed

    Verma, Rajesh Kumar; Verma, Sanjeet K; Pankaj, Umesh; Gupta, Anand K; Khan, Khushboo; Shankar, Karuna

    2015-02-01

    Andrographis paniculata Nees is an annual erect herb with wide medicinal and pharmacological applications due to the presence of andrographolide and other active chemical constituents. The large-scale cultivation of the kalmegh is not in practice. The aim of this study was to establish sustainable production systems of A. paniculata cv CIM-Megha with the application of different bioinoculants and chemical fertilisers. A. paniculata herb and andrographolide yield in the dried leaves was found to be highest (218% and 61.3%, respectively) in treatment T3 (NPK+Bacillus sp.) compared with T1 (control). The soil organic carbon, soil microbial respiration, soil enzymes activity and available nutrients improved significantly with combined application of bioinoculants and chemical fertilisers. PMID:25348874

  6. Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

    PubMed

    Yin, Hua; Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Zhao, Junfeng; Dong, Jianjun; Yu, Junhong; Chang, Zongming

    2016-08-01

    Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering. PMID:27302037

  7. Expression of UNC-5 in the developing Xenopus visual system.

    PubMed

    Anderson, Richard B; Holt, Christine E

    2002-10-01

    Netrin-1 and its receptors deleted in colorectal cancer (DCC) and neogenin are expressed within the developing visual system and have been implicated in mediating axon guidance and cell migration. However, the expression of the netrin-1 receptor UNC-5 has not yet been addressed. Here we clone Xenopus UNC-5 (XUNC-5) and describe its expression within the developing visual system. Xunc-5 transcripts are expressed within the developing optic vesicles and later become restricted to the dorsal ciliary marginal zone, a site of retinoblast proliferation and differentiation. This highly restricted expression pattern could make XUNC-5 an excellent marker of dorsal retinal precursor cells. PMID:12351179

  8. Estimation of yield capacity of fractured rock aquifer for multi-well groundwater heat pump system

    NASA Astrophysics Data System (ADS)

    Bak, Hyeongmin; Yeo, In Wook

    2015-04-01

    Geothermal heat pump system is classified as closed loop and open loop. Closed loop uses a refrigerant as a heat source. For the reason, when using it for a long time, there is a possibility that the refrigerant pipe is corroded. Accordingly, soil and groundwater can be contaminated. Whereas the open loop system uses a eco-friendly groundwater as a heat source. Thermal circulation of standing column well (SCW) occurs in one well. In contrast, thermal circulation of multi-well groundwater heat pump system (MGHP) occurs through fractured rock aquifer between extraction and injection wells. Therefore, temperature efficiency of MGHP appears to be better than that of SCW. However, the MGHP has problems such as the overflowing in the injection well and the clogging, which restricts the wide use of MGHP. This study aims at how to to array the extraction and injection wells for stable circulating of groundwater and at evaluating the sustainable yield capacity of groundwater circulation between the two wells. The study site is located in Chuncheon, Republic of Korea. Pumping tests were conducted to estimate transmissivity of the two wells (W3, W4). In addition, the step-circulation tests were conducted to estimate the sustainable yield capacity. Transmissivity of W3 and W4 was estimated to be 5.81 x 10^-5 m^2/s and 2.57 x 10^-5 m^2/s, respectively. Preliminary groundwater circulation tests were conducted to figure out the array of the extraction and injection wells. Circulation tests were performed for two cases: first, extraction well was set at the well with higher transmissivity and injection well set at the well with lower transmissivity, and the opposite array was set for the second case. In the first case, when flow rate was set at 70.47 m^3/day, the water level of W3 fell 0.61m and that of W4 rose 1.89m. In the second case, when flow rate was set at 67.70 m^3/day, the water level of W4 fell 2.17m and that of W3 rose 0.5m. Preliminary groundwater

  9. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    PubMed

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis. PMID:24096450

  10. The Expression of TaRca2-α Gene Associated with Net Photosynthesis Rate, Biomass and Grain Yield in Bread Wheat (Triticum aestivum L.) under Field Conditions

    PubMed Central

    Saeed, Iqbal; Bachir, Daoura Goudia; Chen, Liang; Hu, Yin-Gang

    2016-01-01

    Improvement in activation of Rubisco by Rubisco activase can potentially enhance CO2 assimilation and photosynthetic efficiency in plants. The three homoeologous copies of TaRca2-α were identified on chromosomes 4AL, 4BS and 4DS (TaRca2-α-4AL, TaRca2-α-4BS, and TaRca2-α-4DS) in bread wheat. Expression patterns of the three copies at heading (Z55), anthesis (Z67) and grain-filling (Z73) stages were investigated through qRT-PCR analyses in a panel of 59 bread wheat genotypes and their effects on net photosynthesis rate (Pn), biomass plant-1 (BMPP) and grain yield plant-1 (GYPP) were further explored. Different but similar expression patterns were observed for the three copies of TaRca2-α at the three growth stages with highest expression at grain-filling stage. TaRca2-α-4BS expressed higher at the three stages than TaRca2-α-4AL and TaRca2-α-4DS. The 59 genotypes could be clustered into three groups as high (7 genotypes), intermediate (41 genotypes) and low (11 genotypes) expression based on the expression of the three copies of TaRca2-α at three growth stages. Significant variations (P<0.01) were observed among the three groups of bread wheat genotypes for Pn, BMPP and GYPP. Generally, the genotypes with higher TaRca2-α expression also showed higher values for Pn, BMPP and GYPP. The expressions of the three copies of TaRca2-α at heading, anthesis and grain-filling stages were positively correlated with Pn, BMPP and GYPP (P<0.01) with stronger association for TaRca2-α-4BS at grain-filling stage. These results revealed that the expression of TaRca2-α contribute substantially to Pn, BMPP and GYPP, and suggested that manipulating TaRca-α expression may efficiently improve Pn, BMPP and GYPP in bread wheat and detecting TaRca-α expression levels with emphasis on TaRca2-α-4BS may be a positive strategy for selection in improving photosynthetic efficiency and grain yield of bread wheat. PMID:27548477

  11. The Expression of TaRca2-α Gene Associated with Net Photosynthesis Rate, Biomass and Grain Yield in Bread Wheat (Triticum aestivum L.) under Field Conditions.

    PubMed

    Saeed, Iqbal; Bachir, Daoura Goudia; Chen, Liang; Hu, Yin-Gang

    2016-01-01

    Improvement in activation of Rubisco by Rubisco activase can potentially enhance CO2 assimilation and photosynthetic efficiency in plants. The three homoeologous copies of TaRca2-α were identified on chromosomes 4AL, 4BS and 4DS (TaRca2-α-4AL, TaRca2-α-4BS, and TaRca2-α-4DS) in bread wheat. Expression patterns of the three copies at heading (Z55), anthesis (Z67) and grain-filling (Z73) stages were investigated through qRT-PCR analyses in a panel of 59 bread wheat genotypes and their effects on net photosynthesis rate (Pn), biomass plant-1 (BMPP) and grain yield plant-1 (GYPP) were further explored. Different but similar expression patterns were observed for the three copies of TaRca2-α at the three growth stages with highest expression at grain-filling stage. TaRca2-α-4BS expressed higher at the three stages than TaRca2-α-4AL and TaRca2-α-4DS. The 59 genotypes could be clustered into three groups as high (7 genotypes), intermediate (41 genotypes) and low (11 genotypes) expression based on the expression of the three copies of TaRca2-α at three growth stages. Significant variations (P<0.01) were observed among the three groups of bread wheat genotypes for Pn, BMPP and GYPP. Generally, the genotypes with higher TaRca2-α expression also showed higher values for Pn, BMPP and GYPP. The expressions of the three copies of TaRca2-α at heading, anthesis and grain-filling stages were positively correlated with Pn, BMPP and GYPP (P<0.01) with stronger association for TaRca2-α-4BS at grain-filling stage. These results revealed that the expression of TaRca2-α contribute substantially to Pn, BMPP and GYPP, and suggested that manipulating TaRca-α expression may efficiently improve Pn, BMPP and GYPP in bread wheat and detecting TaRca-α expression levels with emphasis on TaRca2-α-4BS may be a positive strategy for selection in improving photosynthetic efficiency and grain yield of bread wheat. PMID:27548477

  12. A Proposed Non-Intrusion Method for Estimating the Specific Yield for a Regional Groundwater System

    NASA Astrophysics Data System (ADS)

    Chen, Y.; Cheng, C.; Chang, L.; Hwang, C.; Tsai, J.; Yeh, W.

    2013-12-01

    spatial distribution of the values of specific yield for the aquifer can be further refined. The proposed method is a cost-saving and accuracy alternative for the estimation of specific yield in a regional groundwater system.

  13. A high loading overland flow system: Impacts on soil characteristics, grass constituents, yields and nutrient removal.

    PubMed

    Wen, C G; Chen, T H; Hsu, F H; Lu, C H; Lin, J B; Chang, C H; Chang, S P; Lee, C S

    2007-04-01

    The objectives of this paper are to determine effects of different grass species and their harvests on pollutant removal, elucidate impacts on soil characteristics and grass constituents, observe grass yield and quantify nutrient uptake by vegetation in an overland flow system (OLFS). Polluted creek water was applied to eight channels in the OLFS, which were planted with Paragrass, Nilegrass, Cattail, and Vetiver, with each two channels being randomly planted with a given grass species. The grass in one channel was harvested while that in the other channel was not. At a high rate of 27.8 m d(-1) hydraulic loading, the removal efficiencies of conventional pollutants such as BOD, COD, suspended solids (SS), and total coliforms in wastewater are not affected by the type of the grasses species, but those of nitrogen and phosphorus are affected by different species. Overall average removal efficiencies of BOD, COD, SS, ammonia, total nitrogen, total phosphorus and total coliforms through the OLFS are 42%, 48%, 78%, 47%, 40%, 33% and 89%, respectively. The concentration of nitrate, however, increases due to nitrification. Soil characteristics in OLFS have been changed significantly; specific conductivity, organic matter, exchangeable magnesium, extractable copper and zinc in soils all increase with time while pHs decrease. During the winter season, there is a significant accumulation of nitrate in grass with the subsequent reduction during the active growing season (Spring). The contents of nitrate and phosphorus in grass tissue are higher than those of grass in general pastureland, probably due to nutrient luxury uptake by grass. The overall grass yield, growth rate and nutrient uptake are quantified and implication of such high rate OLFS discussed. PMID:17234253

  14. Subplots facilitate assessment of corn yield losses from weed competition in a long-term systems experiment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Weeds can potentially limit crop yield, particularly in organic systems where herbicide technologies are unavailable. Weedy and weed-free subplots were established within full plots of a long-term cropping systems experiment, the Farming Systems Project, at Beltsville, Maryland, USA, to determine t...

  15. Crop yield and light / energy efficiency in a closed ecological system: two laboratory biosphere experiments

    NASA Astrophysics Data System (ADS)

    Nelson, M.; Dempster, W. F.; Silverstone, S.; Alling, A.; Allen, J. P.; van Thillo, M.

    field results but somewhat below greenhouse trials at comparable light levels, and the best portion of the crop at 0.22g/mole was inbetween those values. Sweet potato production was overall close to 50% higher than trials using hydroponic methods with TU-82-155 at NASA JSC. Compared to projected yields for the Mars on Earth life support system, these wheat yields were about 15% higher, and the sweet potato yields averaged over 80% higher

  16. Regulated Expression of an Isopentenyltransferase Gene (IPT) in Peanut Significantly Improves Drought Tolerance and Increases Yield Under Field Conditions.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isopentenyltransferase (IPT) is a critical enzyme in the cytokinin biosynthetic pathway. The expression of IPT under the control of a maturation- and stress-induced promoter was shown to delay stress-induced plant senescence that resulted in an enhanced drought tolerance in both monocot and dicot p...

  17. Thrombomodulin Expression in Tissues From Dogs With Systemic Inflammatory Disease.

    PubMed

    Kim, S D; Baker, P; DeLay, J; Wood, R D

    2016-07-01

    Thrombomodulin (TM) is a membrane glycoprotein expressed on endothelial cells, which plays a major role in the protein C anticoagulation pathway. In people with inflammation, TM expression can be down-regulated on endothelial cells and a soluble form released into circulation, resulting in increased risk of thrombosis and disseminated intravascular coagulation. TM is present in dogs; however, there has been minimal investigation of its expression in canine tissues, and the effects of inflammation on TM expression in canine tissues have not been investigated. The objective of this study was to evaluate endothelial TM expression in tissues from dogs with systemic inflammatory diseases. A retrospective evaluation of tissue samples of lung, spleen, and liver from dogs with and without systemic inflammatory diseases was performed using immunohistochemistry (IHC) and a modified manual IHC scoring system. TM expression was significantly reduced in all examined tissues in dogs diagnosed with septic peritonitis or acute pancreatitis. PMID:26926084

  18. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

    PubMed Central

    Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  19. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

    PubMed

    Binder, Dennis; Probst, Christopher; Grünberger, Alexander; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  20. Assessment of cotton plant nitrogren status and yield potential using plant height mapping system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant nitrogen status and yield potential are important factors for optimizing field management in cotton production. An experiment was conducted in 2011 at Stoneville, Mississippi to develop a method to assess plant N status and yield potential in cotton. The experimental plots were laid out in a s...

  1. Biomass yield and quality of reed canarygrass under five harvest management systems for bioenergy production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reed canarygrass, Phalaris arundinacea L., produces high biomass yields in cool climates and wetlands. The number and timing of harvests during a growing season directly affect biomass yield and biofuel quality. In order to determine optimum harvest management, seven cultivars of reed canarygrass we...

  2. Soil and rainfall factors influencing yields of a dryland cropping system in Colorado

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The semi-arid Great Plains of the United States experience a large variation in crop yields due to variability in rainfall, soil, and other factors. We analyzed crop yields (24-year period) from a no-till rotation of wheat(Triticum aestivum)-corn (Zea mays L.) or sorghum[Sorghum bicolor (L.) Moench]...

  3. Impact of Varying Planting Dates and Tillage Systems on Cotton Growth and Lint Yield Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As economic conditions have deteriorated, cotton (Gossypium hirsutum L.) producers have looked to improve profit margins by reducing inputs while maintaining yields. Pairing the yield benefits from early planting with the input reductions from conservation tillage might be a strategy to accomplish ...

  4. Yield estimation in commercial cranberry systems using physiological, environmental, and genetic variables

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study sought to improve the current understanding of yield by measuring the effects of genetic, physiological, and environmental variables on yield. Sixty-six variables representing ‘Stevens’ and ‘Ben Lear’ cultivars were evaluated from samples collected from eight commercial cranberry marshes ...

  5. An IPTG Inducible Conditional Expression System for Mycobacteria

    PubMed Central

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K.; Sambandamurthy, Vasan K.; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  6. An IPTG Inducible Conditional Expression System for Mycobacteria.

    PubMed

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K; Sambandamurthy, Vasan K; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  7. Effect of initial biomass on channel catfish yield and water quality in a biofloc technology production system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofloc technology (BFT) production systems are being used more commonly to produce high yields of fish or shrimp because very high feed rates are possible. In an outdoor BFT production system, a complex of living organisms is closely associated with particulate organic matter and is maintained in ...

  8. System approach for evaluating the potential yield and plantation of Jatropha curcas L. on a global scale.

    PubMed

    Li, Zhengguo; Lin, Bin-Le; Zhao, Xiaofeng; Sagisaka, Masayuki; Shibazaki, Ryosuke

    2010-03-15

    Many Jatropha curcas Linnaeus (JCL) plantations have been established in tropical and subtropical regions worldwide. To assess the potential of JCL for biofuel production, the potential areas for JCL plantations, and the yields of JCL must be estimated as accurately as possible. Here, we present a system approach to estimate JCL yields, classify yield levels, and estimate productivity of future JCL plantations. We used a process-based net primary productivity (NPP) model to estimate potential JCL yields. The model estimated that the potential yield of JCL dry seed will vary from 0 to 7.62 ton ha(-1) y(-1), in contrast to estimates of 1.50-7.80 ton ha(-1) y(-1) from previous assessments. We formulated a zoning scheme that takes into account land cover status and potential yield levels. This scheme was used to evaluate the potential area and production of future plantations at the global, regional, and national levels. The estimated potential area of JCL plantations is 59-1486 million hectares worldwide, and the potential production is 56-3613 million ton dry seed y(-1). This study provides scientific information on global patterns of potential plantation areas and yields, which can be used to support bioenergy policy makers to plan commercial-scale JCL plantations. PMID:20141103

  9. Comparative Proteomic Analysis of saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield

    PubMed Central

    2011-01-01

    Background Saccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. Results In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Conclusions The present study is the first comprehensive and

  10. Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

    PubMed

    Bosworth, A H; Williams, M K; Albrecht, K A; Kwiatkowski, R; Beynon, J; Hankinson, T R; Ronson, C W; Cannon, F; Wacek, T J; Triplett, E W

    1994-10-01

    The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that

  11. Interspecific Differential Expression Analysis of RNA-Seq Data Yields Insight into Life Cycle Variation in Hydractiniid Hydrozoans

    PubMed Central

    Sanders, Steven M.; Cartwright, Paulyn

    2015-01-01

    Hydrozoans are known for their complex life cycles, which can alternate between an asexually reproducing polyp stage and a sexually reproducing medusa stage. Most hydrozoan species, however, lack a free-living medusa stage and instead display a developmentally truncated form, called a medusoid or sporosac, which generally remains attached to the polyp. Although evolutionary transitions in medusa truncation and loss have been investigated phylogenetically, little is known about the genes involved in the development and loss of this life cycle stage. Here, we present a new workflow for evaluating differential expression (DE) between two species using short read Illumina RNA-seq data. Through interspecific DE analyses between two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, we identified genes potentially involved in the developmental, functional, and morphological differences between the fully developed medusa of P. carnea and reduced sporosac of H. symbiolongicarpus. A total of 10,909 putative orthologs of H. symbiolongicarpus and P. carnea were identified from de novo assemblies of short read Illumina data. DE analysis revealed 938 of these are differentially expressed between P. carnea developing and adult medusa, when compared with H. symbiolongicarpus sporosacs, the majority of which have not been previously characterized in cnidarians. In addition, several genes with no corresponding ortholog in H. symbiolongicarpus were expressed in developing medusa of P. carnea. Results presented here show interspecific DE analyses of RNA-seq data to be a sensitive and reliable method for identifying genes and gene pathways potentially involved in morphological and life cycle differences between species. PMID:26251524

  12. The response of a high sediment yield depositional system to episodic rises in sea level: The record from the Brazos fluvial system, central Texas coast

    SciTech Connect

    Bartek, L.R.; Anderson, J.B.; Abdulah, K.C. )

    1991-03-01

    High resolution seismic data, cores, and platform borings have been utilized to investigate the response of the Brazos fluvial system, a high sediment yield depositional system, to late Pleistocene-Holocene episodic rises in sea level. This investigation was conducted in order to provide control for a related study of the Trinity-Sabine fluvial system, a low sediment yield depositional system. The Brazos incised valley, which was carved during the Wisconsin eustatic lowstand, was abandoned during an interval of rapid eustatic rise. The fluvial-deltaic system occupied the adjacent interfluve following abandonment of the incised valley. This behavior contrasts sharply with the responses of the Trinity-Sabine low sediment yield depositional system to rapid base level rises. Stream piracy, triggered by the episodic eustatic rises, played an important role in diverting the fluvial system of the high sediment yield system out of the lowstand entrenched valley and onto the interfluve. The research presented in this paper suggests that exploration geologists working with up-dip portions of high sediments yield depositional systems in the transgressive systems tract may find prospects (slightly modified wave-dominated delta deposits encased in marine shales) by searching along strike, away from the abandoned incised valley, at positions along depositional dip that correspond to eustatic stillstands. Accretionary structures observed in recently acquired seismic data indicate that the down-dip portion of the high sediment yield incised valley also contains a large volume of sand.

  13. Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors*S⃞

    PubMed Central

    Qin, Dajiang; Gan, Yi; Shao, Kaifeng; Wang, Hao; Li, Wen; Wang, Tao; He, Wenzhi; Xu, Jianyong; Zhang, Yu; Kou, Zhaohui; Zeng, Lingwen; Sheng, Guoqing; Esteban, Miguel A.; Gao, Shaorong; Pei, Duanqing

    2008-01-01

    Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application. PMID:18826945

  14. Relationships between Written Expression Achievement and the Cognitive Assessment System.

    ERIC Educational Resources Information Center

    Johnson, Judy A.; Bardos, Achilles N.; Tayebi, Kandi A.

    The purpose of this study was to explore the relationships between the Cognitive Assessment System (CAS) and writing achievement in junior high students (aged 11-15 years) with and without written expression disabilities. Ninety-six students with (n=48) and without (n=48) written expression disabilities were administered the Das-Naglieri:…

  15. Local and Systemic Cytokine Expression in Patients with Postherpetic Neuralgia

    PubMed Central

    Üçeyler, Nurcan; Valet, Michael; Kafke, Waldemar; Tölle, Thomas R.; Sommer, Claudia

    2014-01-01

    Background Postherpetic neuralgia (PHN) is the painful complication of a varicella zoster virus reactivation. We investigated the systemic and local gene expression of pro- and anti-inflammatory cytokine expression in patients with PHN. Methods Thirteen patients with PHN at the torso (Th4-S1) were recruited. Skin punch biopsies were obtained from the painful and the contralateral painless body area for intraepidermal nerve fiber density (IENFD) and cytokine profiling. Additionally, blood was withdrawn for systemic cytokine expression and compared to blood values of healthy controls. We analyzed the gene expression of selected pro- and anti-inflammatory cytokines (tumor necrosis factor-alpha [TNF] and interleukins [IL]-1β, IL-2, and IL-8). Results IENFD was lower in affected skin compared to unaffected skin (p<0.05), while local gene expression of pro- and anti-inflammatory cytokines did not differ except for two patients who had 7fold higher IL-6 and 10fold higher IL-10 gene expression in the affected skin compared to the contralateral unaffected skin sample. Also, the systemic expression of cytokines in patients with PHN and in healthy controls was similar. Conclusion While the systemic and local expression of the investigated pro- and anti-inflammatory cytokines was not different from controls, this may have been influenced by study limitations like the low number of patients and different disease durations. Furthermore, other cytokines or pain mediators need to be considered. PMID:25127283

  16. The Facial Expression Coding System (FACES): Development, Validation, and Utility

    ERIC Educational Resources Information Center

    Kring, Ann M.; Sloan, Denise M.

    2007-01-01

    This article presents information on the development and validation of the Facial Expression Coding System (FACES; A. M. Kring & D. Sloan, 1991). Grounded in a dimensional model of emotion, FACES provides information on the valence (positive, negative) of facial expressive behavior. In 5 studies, reliability and validity data from 13 diverse…

  17. Solid-phase fermentation and juice expression systems for sweet sorghum

    SciTech Connect

    Bryan, W.L.; Monroe, G.E.; Caussariel, P.M.

    1985-01-01

    Two systems to recover fermented juice from variety M 81E sweet sorghum stalks that contained about 11% fermentable sugar were compared. (a) Stalks with leaves and tops removed were chopped and inoculated with 0.2% yeast in a forage harvester, stored under anaerobic conditions for 75 hours in insulated fermentors and pressed in a screw press to recover fermented juice (5-6% ethanol). (b) Mechanically harvested sweet sorghum billets (30 cm length) without leaves or seed heads were shredded and milled in a 3-roll mill; and bagasse was inoculated with 0.2% yeast, fermented for 100 h and pressed to recover fermented juice (4 to 5% ethanol). Potential ethanol yields were 75% of theoretical for the forage harvest system and 78% for the shredder mill system, based on 95% of theoretical ethanol yield from juice expressed during milling and no loss of ethanol during fermentation, handling and pressing in the screw press. 20 references.

  18. Tradeoffs between vigor and yield for crops grown under different management systems

    NASA Astrophysics Data System (ADS)

    Simic Milas, Anita; Keller Vincent, Robert; Romanko, Matthew; Feitl, Melina; Rupasinghe, Prabha

    2016-04-01

    Remote sensing can provide an effective means for rapid and non-destructive monitoring of crop status and biochemistry. Monitoring pattern of traditional vigor algorithms generated from Landsat 8 OLI satellite data represents a robust method that can be widely used to differentiate the status of crops, as well as to monitor nutrient uptake functionality of differently treated seeds grown under different managements. This study considers 24 factorial parcels of winter wheat in 2013, corn in 2014, and soybeans in 2015, grown under four different types of agricultural management. The parcels are located at the Kellogg Biological Station, Long-Term Ecological Research site in the State of Michigan USA. At maturity, the organic crops exhibit significantly higher vigor and significantly lower yield than conventionally managed crops under different treatments. While organic crops invest in their metabolism at the expense of their yield, the conventional crops manage to increase their yield at the expense of their vigor. Landsat 8 OLI is capable of 1) differentiating the biochemical status of crops under different treatments at maturity, and 2) monitoring the tradeoff between crop yield and vigor that can be controlled by the seed treatments and proper conventional applications, with the ultimate goal of increasing food yield and food availability, and 3) distinguishing between organic and conventionally treated crops. Timing, quantity and types of herbicide applications have a great impact on early and pre-harvest vigor, maturity and yield of conventionally treated crops. Satellite monitoring using Landsat 8 is an optimal tool for coordinating agricultural applications, soil practices and genetic coding of the crop to produce higher yield as well as have early crop maturity, desirable in northern climates.

  19. Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

    PubMed Central

    Kant, Surya; Burch, David; Badenhorst, Pieter; Palanisamy, Rajasekaran; Mason, John; Spangenberg, German

    2015-01-01

    Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions. PMID:25602960

  20. Establishment of a transient expression system for Dictyostelium discoideum.

    PubMed Central

    Howard, P K; Ahern, K G; Firtel, R A

    1988-01-01

    We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes. PMID:3362676

  1. Regulation of cadherin expression in nervous system development

    PubMed Central

    Paulson, Alicia F; Prasad, Maneeshi S; Thuringer, Amanda Henke; Manzerra, Pasquale

    2014-01-01

    This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. While the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. With the growing understanding of the broader roles of cadherins in cell–cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context. PMID:24526207

  2. Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system.

    PubMed

    DiFranco, Marino; Neco, Patricia; Capote, Joana; Meera, Pratap; Vergara, Julio L

    2006-05-01

    The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement. PMID:16325422

  3. Genotype x environment interactions in milk yield and quality in Angus, Brahman, and reciprocal-cross cows on different forage systems.

    PubMed

    Brown, M A; Brown, A H; Jackson, W G; Miesner, J R

    2001-07-01

    Milk yield and quality were observed on 93 Angus, Brahman, and reciprocal-cross cows over 3 yr to evaluate the interactions of direct and maternal breed effects and heterosis with forage environment. Forage environments were common bermudagrass (BG), endophyte-infected tall fescue (E+), and a rotational system (ROT) of both forages, in which each forage (BG or E+) was grazed during its appropriate season, usually June through October for BG and November through May for E+. Milk yield was estimated each of 6 mo (April through September) via milking machine and converted to a 24-h basis. Milk fat, milk protein, and somatic cell count were analyzed by a commercial laboratory. Heterosis for milk yield was similar among forages, averaging 2.4 kg (P < 0.01). Expressed as percentages of purebred means, heterosis for milk yield was largest on E+ (52.8%), intermediate on ROT (39.3%), and smallest on BG (23.7%). Direct breed effects for milk yield favored Brahman, and they were similar among forages but tended to be larger for E+ (2.5 kg) and ROT (2.8 kg) than for BG (1.3 kg). Direct breed effects for milk fat favored Brahman and were similar among forages but tended to be larger for E+ (1.0%) and ROT (1.0%) than for BG (0.6%). Purebred cows exceeded crossbreds in milk protein by 0.1% on ROT (P < 0.10). Crossbred cows had lower somatic cell counts than purebreds on BG (P < 0.05), E+ (P < 0.01), or ROT (P > 0.30). Heterosis for somatic cell counts as percentages of purebred means was similar for BG (-68.3%) and E+ (-68.9%) and less favorable for ROT (-31.6%). Maternal breed effects for somatic cell count favored Angus on ROT (P < 0.10) with a similar nonsignificant trend on BG and E+. Direct breed effects for somatic cell count favored Brahman on ROT (P < 0.10) with similar nonsignificant trends on BG and E+. These results suggested that a rotation of cows from E+ to BG in the summer can partially alleviate negative effects of E+ on milk yield. Conclusions also indicated an

  4. Radiation use efficiency, forage yield and quality of sorghum-legume intercropping systems in the Southern High Plains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interest in intercropping of non-legume with legume crops to increase resources use efficiency is increasing. Increasing in radiation interception and its subsequent radiation use efficiency (RUE) by forage sorghum-legume intercropping systems can improve forage yield and quality. However, there is ...

  5. Effect of channel catfish stocking rate on yield and water quality in an intensive, mixed suspended-growth production system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to determine the effect of channel catfish (Ictalurus punctatus) stocking rate on yield and water quality in a mixed suspended-growth production system (bio-floc) with zero water exchange. Channel catfish (National Warmwater Aquaculture Center 103 strain; average weight = 13...

  6. Assimilating remote sensing observations of leaf area index and soil moisture for wheat yield estimates: An observing system simulation experiment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We develop a robust understanding of the effects of assimilating remote sensing observations of leaf area index and soil moisture (in the top 5 cm) on DSSAT-CSM CropSim-Ceres wheat yield estimates. Synthetic observing system simulation experiments compare the abilities of the Ensemble Kalman Filter...

  7. Impact of liquid fertilizers on plant growth, yield, fruit quality and fertigation management in an organic processing blackberry production system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The impact of organic fertilizer source on the growth, fruit quality, and yield of blackberry cultivars (‘Marion’ and ‘Black Diamond’) grown in machine-harvested, organic production systems for the processed market was evaluated from 2011-13. The planting was established in spring 2010 using approve...

  8. 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

  9. Disease severity and yield potential of rice cultivars in organic production systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The market demand for organically produced rice has driven the steady increase in the acreage of organic rice in the U. S., with Texas and California being the largest states. Yield potential and disease management are among the principal challenges associated with organic rice production. We evalua...

  10. Comparison of disease severity and yield potential of rice varieties in two organic cropping systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The market demand for organically produced rice has driven the steady increase in acreage of organic rice in the U. S., with Texas and California having the most acreage. Yield potential and disease management are among the principal challenges associated with organic rice production. Organic rice c...

  11. Compost and Biological Amendments in Potato Systems: Effects on Soilborne Diseases and Yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of compost and biological amendments on soil fertility, soil microbial communities, soil-borne diseases, insect pests and potato yield quality and quantity were assessed at two farm sites in Northern Maine in 2007. The two sites were: Aroostook Farm (AF), a research farm using convention...

  12. Increasing plant density in eastern United States broccoli production systems to maximize marketable head yields

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased demand for fresh market broccoli (Brassica oleracea L. var. italica) has led to increased production along the eastern seaboard of the United States. Maximizing broccoli yields is a primary concern for quickly expanding eastern commercial markets. Thus, a plant density study was carried ...

  13. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    SciTech Connect

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  14. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.

    PubMed

    Livet, Jean; Weissman, Tamily A; Kang, Hyuno; Draft, Ryan W; Lu, Ju; Bennis, Robyn A; Sanes, Joshua R; Lichtman, Jeff W

    2007-11-01

    Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale. PMID:17972876

  15. Effect of chemical and mechanical weed control on cassava yield, soil quality and erosion under cassava cropping system

    NASA Astrophysics Data System (ADS)

    Islami, Titiek; Wisnubroto, Erwin; Utomo, Wani

    2016-04-01

    Three years field experiments were conducted to study the effect of chemical and mechanical weed control on soil quality and erosion under cassava cropping system. The experiment were conducted at University Brawijaya field experimental station, Jatikerto, Malang, Indonesia. The experiments were carried out from 2011 - 2014. The treatments consist of three cropping system (cassava mono culture; cassava + maize intercropping and cassava + peanut intercropping), and two weed control method (chemical and mechanical methods). The experimental result showed that the yield of cassava first year and second year did not influenced by weed control method and cropping system. However, the third year yield of cassava was influence by weed control method and cropping system. The cassava yield planted in cassava + maize intercropping system with chemical weed control methods was only 24 t/ha, which lower compared to other treatments, even with that of the same cropping system used mechanical weed control. The highest cassava yield in third year was obtained by cassava + peanuts cropping system with mechanical weed control method. After three years experiment, the soil of cassava monoculture system with chemical weed control method possessed the lowest soil organic matter, and soil aggregate stability. During three years of cropping soil erosion in chemical weed control method, especially on cassava monoculture, was higher compared to mechanical weed control method. The soil loss from chemical control method were 40 t/ha, 44 t/ha and 54 t/ha for the first, second and third year crop. The soil loss from mechanical weed control method for the same years was: 36 t/ha, 36 t/ha and 38 t/ha. Key words: herbicide, intercropping, soil organic matter, aggregate stability.

  16. Whole intact rapeseeds or sunflower oil in high-forage or high-concentrate diets affects milk yield, milk composition, and mammary gene expression profile in goats.

    PubMed

    Ollier, S; Leroux, C; de la Foye, A; Bernard, L; Rouel, J; Chilliard, Y

    2009-11-01

    This study aimed to ascertain the response of goat mammary metabolic pathways to concentrate and lipid feeding in relation to milk fatty acid (FA) composition and secretion. Sixteen midlactation multiparous goats received diets differing in forage-to-concentrate ratio [high forage (HF) 64:36, and low forage (LF) 43:57] supplemented or not with lipids [HF with 130 g/d of oil from whole intact rapeseeds (RS) and LF with 130 g/d of sunflower oil (SO)] in a 4 x 4 Latin square design. Milk yield, milk composition, FA profile, and FA secretion were measured, as well as the expression profiles of key genes in mammary metabolism and of 8,382 genes, using a bovine oligonucleotide microarray. After 3 wk of treatment, milk, lactose, and protein yields were lower with HF-RS than with the other diets, whereas treatment had no effect on milk protein content. Milk fat content was higher with the HF-RS and LF-SO diets than with the HF and LF diets, and SO supplementation increased milk fat yield compared with the LF diet. Decreasing the forage-to-concentrate ratio from 64:36 to 43:57 had a limited effect on goat milk FA concentrations and secretions. Supplementing the LF diet with SO changed almost all the FA concentrations, including decreases in medium-chain saturated FA and large increases in trans C18:1 and C18:2 isomers (particularly trans-11 C18:1 and cis-9, trans-11 conjugated linoleic acid), without significant changes in C18:0 and cis-9 C18:1, whereas supplementing the HF diet with RS led to a strong decrease in short- and medium-chain saturated FA and a very strong increase in C18:0 and cis-9 C18:1, without significant changes in trans C18:1 and conjugated linoleic acid. Despite the decreases in milk lactose and protein yields observed with HF-RS, and despite the decrease in milk medium-chain FA and the increase in C18 FA secretion with RS or SO supplementation, none of the dietary treatments had any effect on mammary mRNA expression of the key genes involved in lactose

  17. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    PubMed

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  18. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    PubMed Central

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  19. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. PMID:25851716

  20. Nematode Numbers and Crop Yield in a Fenamiphos-Treated Sweet Corn-Sweet Potato-Vetch Cropping System

    PubMed Central

    Johnson, A. W.; Dowler, C. C.; Glaze, N. C.; Chalfant, R. B.; Golden, A. M.

    1992-01-01

    Nematode population densities and yield of sweet corn and sweet potato as affected by the nematicide fenamiphos, in a sweet corn-sweet potato-vetch cropping system, were determined in a 5-year test (1981-85). Sweet potato was the best host of Meloidogyne incognita of these three crops. Fenamiphos 15G (6.7 kg a.i./ha) incorporated broadcast in the top 15 cm of the soil layer before planting of each crop increased (P ≤ 0.05) yields of sweet corn in 1981 and 1982 and sweet potato number 1 grade in 1982 and 1983. Yield of sweet corn and numbers of M. incognita second-stage juveniles (J2) in the soil each month were negatively correlated from planting (r = - 0.47) to harvest (r = -0.61) in 1982. Yield of number 1 sweet potato was inversely related to numbers of J2 in the soil in July-October 1982 and July-September 1983. Yield of cracked storage roots was positively related to the numbers of J2 in the soil on one or more sampling dates in all years except 1985. Some factor(s), such as microbial degradation, resistant M. incognita development, or environment, reduced the effect of fenamiphos. PMID:19283032

  1. Nematode numbers and crop yield in a fenamiphos-treated sweet corn-sweet potato-vetch cropping system.

    PubMed

    Johnson, A W; Dowler, C C; Glaze, N C; Chalfant, R B; Golden, A M

    1992-12-01

    Nematode population densities and yield of sweet corn and sweet potato as affected by the nematicide fenamiphos, in a sweet corn-sweet potato-vetch cropping system, were determined in a 5-year test (1981-85). Sweet potato was the best host of Meloidogyne incognita of these three crops. Fenamiphos 15G (6.7 kg a.i./ha) incorporated broadcast in the top 15 cm of the soil layer before planting of each crop increased (P yields of sweet corn in 1981 and 1982 and sweet potato number 1 grade in 1982 and 1983. Yield of sweet corn and numbers of M. incognita second-stage juveniles (J2) in the soil each month were negatively correlated from planting (r = - 0.47) to harvest (r = -0.61) in 1982. Yield of number 1 sweet potato was inversely related to numbers of J2 in the soil in July-October 1982 and July-September 1983. Yield of cracked storage roots was positively related to the numbers of J2 in the soil on one or more sampling dates in all years except 1985. Some factor(s), such as microbial degradation, resistant M. incognita development, or environment, reduced the effect of fenamiphos. PMID:19283032

  2. 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

  3. Pannexin-1 expression in developing mouse nervous system: new evidence for expression in sensory ganglia.

    PubMed

    Raslan, Abdulrahman; Hainz, Nadine; Beckmann, Anja; Tschernig, Thomas; Meier, Carola

    2016-04-01

    Pannexin1 (Panx1) is one of three members of the pannexin protein family. The expression of Panx1 mRNA has been extensively investigated from late embryonic to adult stages. In contrast, expression during early embryonic development is largely unknown. Our aim is to examine the temporal and spatial expression of Panx1 in mouse embryonic development by focusing on embryonic days (E) 9.5 to 12.5. Whole embryos are investigated in order to provide a comprehensive survey. Analyses were performed at the mRNA level by using reverse transcription plus the polymerase chain reaction and whole-mount in situ hybridization. Panx1 mRNA was detected in the heads and bodies of embryos at all developmental stages investigated (E9.5, E10.5, E11.5, E12.5). In particular, the nervous system expressed Panx1 at an early time point. Interestingly, Panx1 expression was found in afferent ganglia of the cranial nerves and spinal cord. This finding is of particular interest in the context of neuropathic pain and other Panx1-related neurological disorders. Our study shows, for the first time, that Panx1 is expressed in the central and peripheral nervous system during early developmental stages. The consequences of Panx1 deficiency or inhibition in a number of experimental paradigms might therefore be predicated on changes during early development. PMID:26453396

  4. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    PubMed Central

    2011-01-01

    Background Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests. PMID:21781331

  5. High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

    PubMed

    Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

    2016-03-01

    Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production. PMID:26555181

  6. A time- and dose-dependent STAT1 expression system

    PubMed Central

    Leitner, Nicole R; Strobl, Birgit; Bokor, Marion; Painz, Ronald; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Karaghiosoff, Marina

    2006-01-01

    Background The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. Results The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. Conclusion These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner. PMID:17184522

  7. Effects of Potato-Cotton Cropping Systems and Nematicides on Plant-Parasitic Nematodes and Crop Yields

    PubMed Central

    Crow, W. T.; Weingartner, D. P.; Dickson, D. W.

    2000-01-01

    Belonolaimus longicaudatus has been reported as damaging both potato (Solanum tuberosum) and cotton (Gossypium hirsutum). These crops are not normally grown in cropping systems together in areas where the soil is infested with B. longicaudatus. During the 1990s cotton was grown in a potato production region that was a suitable habitat for B. longicaudatus. It was not known how integrating the production of these two crops by rotation or double-cropping would affect the population densities of B. longicaudatus, other plant-parasitic nematodes common in the region, or crop yields. A 3-year field study evaluated the viability of both crops in monocropping, rotation, and double-cropping systems. Viability was evaluated using effects on population densities of plant-parasitic nematodes and yields. Rotation of cotton with potato was found to decrease population densities of B. longicaudatus and Meloidogyne incognita in comparison with continuous potato. Population densities of B. longicaudatus following double-cropping were greater than following continuous cotton. Yields of both potato and cotton in rotation were equivalent to either crop in monocropping. Yields of both crops were lower following double-cropping when nematicides were not used. PMID:19270980

  8. High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression

    PubMed Central

    2014-01-01

    Background Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. Results The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to

  9. Linking stomatal traits and expression of slow anion channel genes HvSLAH1 and HvSLAC1 with grain yield for increasing salinity tolerance in barley

    PubMed Central

    Liu, Xiaohui; Mak, Michelle; Babla, Mohammad; Wang, Feifei; Chen, Guang; Veljanoski, Filip; Wang, Gang; Shabala, Sergey; Zhou, Meixue; Chen, Zhong-Hua

    2014-01-01

    Soil salinity is an environmental and agricultural problem in many parts of the world. One of the keys to breeding barley for adaptation to salinity lies in a better understanding of the genetic control of stomatal regulation. We have employed a range of physiological (stomata assay, gas exchange, phylogenetic analysis, QTL analysis), and molecular techniques (RT-PCR and qPCR) to investigate stomatal behavior and genotypic variation in barley cultivars and a genetic population in four experimental trials. A set of relatively efficient and reliable methods were developed for the characterization of stomatal behavior of a large number of varieties and genetic lines. Furthermore, we found a large genetic variation of gas exchange and stomatal traits in barley in response to salinity stress. Salt-tolerant cultivar CM72 showed significantly larger stomatal aperture under 200 mM NaCl treatment than that of salt-sensitive cultivar Gairdner. Stomatal traits such as aperture width/length were found to significantly correlate with grain yield under salt treatment. Phenotypic characterization and QTL analysis of a segregating double haploid population of the CM72/Gairdner resulted in the identification of significant stomatal traits-related QTLs for salt tolerance. Moreover, expression analysis of the slow anion channel genes HvSLAH1 and HvSLAC1 demonstrated that their up-regulation is linked to higher barley grain yield in the field. PMID:25505473

  10. Crop yield summary for three wetland reservoir subirrigation systems in northwest Ohio

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wetland Reservoir Subirrigation Systems (WRSIS) are innovative agricultural water management and recycling systems comprised of three main components; a constructed wetland, a water storage reservoir, and cropland containing subsurface drainage pipe systems. Surface runoff and subsurface drainage f...

  11. Research progress in physicochemical characteristics of lactoferrin and its recombinant expression systems.

    PubMed

    Xiaonan, Pang; Xiao, Hong; Xuan, Wei; Xiwen, Chen; Jia, Liu; Defu, Chen

    2015-09-01

    Lactoferrin is an iron-binding glycoprotein with a molecular weight of about 80 kDa that belongs to the transferrin family. Due to its unique physical and chemical properties, lactoferrin has a variety of biological functions including antibacterial, antiviral, anticancer, immunomodulatory activities and regulation of iron absorption. High-yield production of recombinant lactoferrin with biological activity and its application in clinical treatment have been a hot topic for long time. With the development of genetic engineering techniques, various expression systems have been developed to produce recombinant lactoferrin. In this review, we summarize physicochemical characteristics, biological activities, clinical studies and current recombinant expression systems of lactoferrin, in order to provide references for its clinical application. PMID:26399527

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

    PubMed Central

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

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

  13. Influence of solid-liquid separation strategy on biogas yield from a stratified swine production system.

    PubMed

    Cestonaro do Amaral, André; Kunz, Airton; Radis Steinmetz, Ricardo Luis; Scussiato, Lucas Antunes; Tápparo, Deisi Cristina; Gaspareto, Taís Carla

    2016-03-01

    As the fourth largest swine producer and exporter, Brazil has increased its participation in the global swine production market. Generally, these units concentrate a large number of animals and generate effluents that must be correctly managed to prevent environmental impacts, being anaerobic digestion is an interesting alternative for treating these effluents. The low-volatile solid concentration in the manure suggests the need for solid-liquid separation as a tool to improve the biogas generation capacity. This study aimed to determine the influence of simplified and inexpensive solid-liquid separation strategies (screening and settling) and the different manures produced during each swine production phase (gestating and farrowing sow houses, nursery houses and finishing houses) on biogas and methane yield. We collected samples in two gestating sow houses (GSH-a and GSH-b), two farrowing sow houses (FSH-a and FSH-b), a nursery house (NH) and a finishing house (FH). Biochemical methane potential (BMP) tests were performed according to international standard procedures. The settled sludge fraction comprised 20-30% of the raw manure volume, which comprises 40-60% of the total methane yield. The methane potential of the settled sludge fraction was approximately two times higher than the methane potential of the supernatant fraction. The biogas yield differed among the raw manures from different swine production phases (GSH-a 326.4 and GSH-b 577.1; FSH-a 860.1 and FSH-b 479.2; NH -970.2; FH 474.5 NmLbiogas.gVS(-1)). The differences were relative to the production phase (feed type and feeding techniques) and the management of the effluent inside the facilities (water management). Brazilian swine production has increased his participation in the global market, been the fourth producer and the fourth exporter. The segregation of swine production in multiple sites has increased its importance, due to the possibilities to have more specialized units. Generally, these units

  14. An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications

    PubMed Central

    Sochor, Matthew A.; Vasireddy, Vidyullatha; Drivas, Theodore G.; Wojno, Adam; Doung, Thu; Shpylchak, Ivan; Bennicelli, Jeannette; Chung, Daniel; Bennett, Jean; Lewis, Mitchell

    2015-01-01

    The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy. PMID:26597678

  15. Neutron Yield Study of Direct-Drive, Low-Adiabat Cryogenic D2 Implosions on OMEGA Laser System

    SciTech Connect

    Hu, S.X.; Radha, P.B.; Marozas, J.A.; Betti, R.; Collins, T.J.B.; Craxton, R.S.; Delettrez, J.A.; Edgell, D.H.; Epstein, R.; Goncharov, V.N.; Igumenshchev, I.V.; Marshall, F.J.; McCrory, R.L.; Meyerhofer, D.D.; Regan, S.P.; Sangster, T.C.; Skupsky, S.; Smalyuk, V.A.; Elbaz, Y.; Shvarts, D.

    2009-11-17

    Neutron yields of direct-drive, low-adiabat (alpha ~~ 2 to 3) cryogenic D2 target implosions on the OMEGA laser system [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)] have been systematically investigated using the two-dimensional (2D) radiation hydrodynamics code DRACO [P. B. Radha et al., Phys. Plasmas 12, 056307 (2005)]. Low-mode (ell <- 12) perturbations, including initial target offset, ice-layer roughness, and laser-beam power imbalance, were found to be the primary source of yield reduction for thin-shell (5 um), low-alpha, cryogenic targets. The 2D simulations of thin-shell implosions track experimental measurements for different target conditions and peak laser intensities ranging from 2.5 x 10^14–6 x 10^14 W/cm^2. Simulations indicate that the fusion yield is sensitive to the relative phases between the target offset and the ice-layer perturbations. The results provide a reasonable good guide to understanding the yield degradation in direct-drive, low-adiabat, cryogenic, thin-shell-target implosions. Thick-shell (10 um) implosions generally give lower yield over clean than low-ell-mode DRACO simulation predictions. Simulations including the effect of laser-beam nonuniformities indicate that high-ell-mode perturbations caused by laser imprinting further degrade the neutron yield of thick-shell implosions. To study ICF compression physics, these results suggest a target specification with a <-30 um offset and ice-roughness of sigma_rms < 3 um are required.

  16. Neutron yield study of direct-drive, low-adiabat cryogenic D{sub 2} implosions on OMEGA laser system

    SciTech Connect

    Hu, S. X.; Radha, P. B.; Marozas, J. A.; Betti, R.; Collins, T. J. B.; Craxton, R. S.; Delettrez, J. A.; Edgell, D. H.; Epstein, R.; Goncharov, V. N.; Igumenshchev, I. V.; Marshall, F. J.; McCrory, R. L.; Meyerhofer, D. D.; Regan, S. P.; Sangster, T. C.; Skupsky, S.; Smalyuk, V. A.; Elbaz, Y.; Shvarts, D.

    2009-11-15

    Neutron yields of direct-drive, low-adiabat ({alpha}{approx_equal}2 to 3) cryogenic D{sub 2} target implosions on the OMEGA laser system [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)] have been systematically investigated using the two-dimensional (2D) radiation hydrodynamics code DRACO[P. B. Radha et al., Phys. Plasmas 12, 056307 (2005)]. Low-mode (l{<=}12) perturbations, including initial target offset, ice-layer roughness, and laser-beam power imbalance, were found to be the primary source of yield reduction for thin-shell (5 {mu}m), low-{alpha}, cryogenic targets. The 2D simulations of thin-shell implosions track experimental measurements for different target conditions and peak laser intensities ranging from 2.5x10{sup 14}-6x10{sup 14} W/cm{sup 2}. Simulations indicate that the fusion yield is sensitive to the relative phases between the target offset and the ice-layer perturbations. The results provide a reasonable good guide to understanding the yield degradation in direct-drive, low-adiabat, cryogenic, thin-shell-target implosions. Thick-shell (10 {mu}m) implosions generally give lower yield over clean than low-l-mode DRACO simulation predictions. Simulations including the effect of laser-beam nonuniformities indicate that high-l-mode perturbations caused by laser imprinting further degrade the neutron yield of thick-shell implosions. To study ICF compression physics, these results suggest a target specification with a {<=}30 {mu}m offset and ice-roughness of {sigma}{sub rms}<3 {mu}m are required.

  17. Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

    PubMed

    Sumiya, Nobuko; Fujiwara, Takayuki; Kobayashi, Yusuke; Misumi, Osami; Miyagishima, Shin-ya

    2014-01-01

    The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage. PMID:25337786

  18. Decomposing global crop yield variability

    NASA Astrophysics Data System (ADS)

    Ben-Ari, Tamara; Makowski, David

    2014-11-01

    Recent food crises have highlighted the need to better understand the between-year variability of agricultural production. Although increasing future production seems necessary, the globalization of commodity markets suggests that the food system would also benefit from enhanced supplies stability through a reduction in the year-to-year variability. Here, we develop an analytical expression decomposing global crop yield interannual variability into three informative components that quantify how evenly are croplands distributed in the world, the proportion of cultivated areas allocated to regions of above or below average variability and the covariation between yields in distinct world regions. This decomposition is used to identify drivers of interannual yield variations for four major crops (i.e., maize, rice, soybean and wheat) over the period 1961-2012. We show that maize production is fairly spread but marked by one prominent region with high levels of crop yield interannual variability (which encompasses the North American corn belt in the USA, and Canada). In contrast, global rice yields have a small variability because, although spatially concentrated, much of the production is located in regions of below-average variability (i.e., South, Eastern and South Eastern Asia). Because of these contrasted land use allocations, an even cultivated land distribution across regions would reduce global maize yield variance, but increase the variance of global yield rice. Intermediate results are obtained for soybean and wheat for which croplands are mainly located in regions with close-to-average variability. At the scale of large world regions, we find that covariances of regional yields have a negligible contribution to global yield variance. The proposed decomposition could be applied at any spatial and time scales, including the yearly time step. By addressing global crop production stability (or lack thereof) our results contribute to the understanding of a key

  19. Regulation of gene expression in the nervous system

    SciTech Connect

    Giuffrida Stella, A.M.; Perez-Polo, J.R.; deVellis, J.

    1990-01-01

    This book offers an up-to-date account of the latest research findings concerned with the regulatory mechanisms of gene expression in neuronal and glial cells under different conditions. The book explores the cellular and neurobiological aspects of important phenomena of the nervous system and its role in health, disease and injury. Contributions form prominent scientists in the field address a variety of specific topics concerned with gene expression in the nervous system - from growth, hormonal and trophic factors to neural tissue reactions in injury or aging.

  20. A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies

    PubMed Central

    Cid-Arregui, Angel; Juárez, Victoria; Hausen, Harald zur

    2003-01-01

    A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system. PMID:12663798

  1. A photon detector system for the search for the rare muon decay {mu} {yields} e{gamma}

    SciTech Connect

    Van Ausdeln, L.A.

    1993-11-01

    An innovative and state of the art pair spectrometer system to measure the photon component of {mu}{sup +} decay to obtain an improved branching ratio limit for the decay {mu} {yields} e{gamma} is investigated. Analysis algorithms are developed and an experimental inner bremsstrahlung spectrum is obtained and agrees well with Monte Carlo simulations. Background sources are investigated and found to be highly suppressed at various stages of acquisition and analysis.

  2. Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes.

    PubMed

    Dey, Avishek; Samanta, Milan Kumar; Gayen, Srimonta; Sen, Soumitra K; Maiti, Mrinal K

    2016-01-01

    Drought is one of the major limiting factors for productivity of crops including rice (Oryza sativa L.). Understanding the role of allelic variations of key regulatory genes involved in stress-tolerance is essential for developing an effective strategy to combat drought. The bZIP transcription factors play a crucial role in abiotic-stress adaptation in plants via abscisic acid (ABA) signaling pathway. The present study aimed to search for allelic polymorphism in the OsbZIP23 gene across selected drought-tolerant and drought-sensitive rice genotypes, and to characterize the new allele through overexpression (OE) and gene-silencing (RNAi). Analyses of the coding DNA sequence (CDS) of the cloned OsbZIP23 gene revealed single nucleotide polymorphism at four places and a 15-nucleotide deletion at one place. The single-copy OsbZIP23 gene is expressed at relatively higher level in leaf tissues of drought-tolerant genotypes, and its abundance is more in reproductive stage. Cloning and sequence analyses of the OsbZIP23-promoter from drought-tolerant O. rufipogon and drought-sensitive IR20 cultivar showed variation in the number of stress-responsive cis-elements and a 35-nucleotide deletion at 5'-UTR in IR20. Analysis of the GFP reporter gene function revealed that the promoter activity of O. rufipogon is comparatively higher than that of IR20. The overexpression of any of the two polymorphic forms (1083 bp and 1068 bp CDS) of OsbZIP23 improved drought tolerance and yield-related traits significantly by retaining higher content of cellular water, soluble sugar and proline; and exhibited decrease in membrane lipid peroxidation in comparison to RNAi lines and non-transgenic plants. The OE lines showed higher expression of target genes-OsRab16B, OsRab21 and OsLEA3-1 and increased ABA sensitivity; indicating that OsbZIP23 is a positive transcriptional-regulator of the ABA-signaling pathway. Taken together, the present study concludes that the enhanced gene expression rather than

  3. Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

    PubMed Central

    Dey, Avishek; Samanta, Milan Kumar; Gayen, Srimonta; Sen, Soumitra K.; Maiti, Mrinal K.

    2016-01-01

    Drought is one of the major limiting factors for productivity of crops including rice (Oryza sativa L.). Understanding the role of allelic variations of key regulatory genes involved in stress-tolerance is essential for developing an effective strategy to combat drought. The bZIP transcription factors play a crucial role in abiotic-stress adaptation in plants via abscisic acid (ABA) signaling pathway. The present study aimed to search for allelic polymorphism in the OsbZIP23 gene across selected drought-tolerant and drought-sensitive rice genotypes, and to characterize the new allele through overexpression (OE) and gene-silencing (RNAi). Analyses of the coding DNA sequence (CDS) of the cloned OsbZIP23 gene revealed single nucleotide polymorphism at four places and a 15-nucleotide deletion at one place. The single-copy OsbZIP23 gene is expressed at relatively higher level in leaf tissues of drought-tolerant genotypes, and its abundance is more in reproductive stage. Cloning and sequence analyses of the OsbZIP23-promoter from drought-tolerant O. rufipogon and drought-sensitive IR20 cultivar showed variation in the number of stress-responsive cis-elements and a 35-nucleotide deletion at 5’-UTR in IR20. Analysis of the GFP reporter gene function revealed that the promoter activity of O. rufipogon is comparatively higher than that of IR20. The overexpression of any of the two polymorphic forms (1083 bp and 1068 bp CDS) of OsbZIP23 improved drought tolerance and yield-related traits significantly by retaining higher content of cellular water, soluble sugar and proline; and exhibited decrease in membrane lipid peroxidation in comparison to RNAi lines and non-transgenic plants. The OE lines showed higher expression of target genes-OsRab16B, OsRab21 and OsLEA3-1 and increased ABA sensitivity; indicating that OsbZIP23 is a positive transcriptional-regulator of the ABA-signaling pathway. Taken together, the present study concludes that the enhanced gene expression rather

  4. Develop a field grid system for yield mapping and machine control. Quarterly report, July 1, 1995--September 30, 1995

    SciTech Connect

    Hart, F.; Windish, J.

    1995-10-01

    Build and test the Field Grid Sense system for yield mapping and machine control during harvesting. Secondly, use Field Grid Sense with chemical application equipment to demonstrate a workable in-field system. More specifically, the operation of the patented hardware/software Field Grid Sense (FGS) system will be tested in crop harvesting to demonstrate the system`s utility and to analyze the flexibility of operation under true field conditions. Additionally, FGS will again be used with chemical application equipment - equipment that needs modification to correct one or two slight shortcomings. This action will create improved systems and establish the worthiness, efficiency and necessity of chemical application equipment that is controlled and directed via the FGS package.

  5. Interactive analysis of systems biology molecular expression data

    PubMed Central

    Zhang, Mingwu; Ouyang, Qi; Stephenson, Alan; Kane, Michael D; Salt, David E; Prabhakar, Sunil; Burgner, John; Buck, Charles; Zhang, Xiang

    2008-01-01

    Background Systems biology aims to understand biological systems on a comprehensive scale, such that the components that make up the whole are connected to one another and work through dependent interactions. Molecular correlations and comparative studies of molecular expression are crucial to establishing interdependent connections in systems biology. The existing software packages provide limited data mining capability. The user must first generate visualization data with a preferred data mining algorithm and then upload the resulting data into the visualization package for graphic visualization of molecular relations. Results Presented is a novel interactive visual data mining application, SysNet that provides an interactive environment for the analysis of high data volume molecular expression information of most any type from biological systems. It integrates interactive graphic visualization and statistical data mining into a single package. SysNet interactively presents intermolecular correlation information with circular and heatmap layouts. It is also applicable to comparative analysis of molecular expression data, such as time course data. Conclusion The SysNet program has been utilized to analyze elemental profile changes in response to an increasing concentration of iron (Fe) in growth media (an ionomics dataset). This study case demonstrates that the SysNet software is an effective platform for interactive analysis of molecular expression information in systems biology. PMID:18312669

  6. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. PMID:20457256

  7. Improving potato cropping systems: longer-term effects on diseases and yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of effective cropping systems can provide the structural basis for enhanced crop production and sustainability through the conservation, maintenance, and replenishment of various soil resources. In 2004, field trials evaluating potato cropping systems focused on different specific cr...

  8. EXPRESSION SYSTEM-DEPENDENT MODULATION OF HIV-1 ENVELOPE GLYCOPROTEIN ANTIGENICITY AND IMMUNOGENICITY

    PubMed Central

    Kong, Leopold; Sheppard, Neil C.; Stewart-Jones, Guillaume B.E.; Robson, Cynthia L.; Chen, Hongying; Xu, Xiaodong; Krashias, George; Bonomelli, Camille; Scanlan, Christopher N.; Kwong, Peter D.; Jeffs, Simon A.; Jones, Ian M.; Sattentau, Quentin J.

    2010-01-01

    Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4–induced sites compared to those expressed in the system that does, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used

  9. Dryland systems yields over 24 years as impacted by potential ET site and soil landscape position.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    No-till management in the Central Great Plains has allowed for more diversified cropping systems with both spring and fall planted crops. This shift reduces the time systems are in summer fallow. The success or failure of a dryland cropping system in this semi-arid region often depends on the timing...

  10. Power system comparison for the Pluto Express mission

    SciTech Connect

    Harty, R.B.

    1995-12-31

    This paper presents a comparison of three advanced radioisotope power systems, along with a down sized RTG for the Pluto Express mission. These three advanced radioisotope power systems were the Radioisotope Alkali Metal Thermal--to-Electric Converter (RAMTEC), Radioisotope Stirling, and Radioisotope Thermophotovoltaic (RTPV). For the Pluto Express mission, the power requirement at the end of the 10-y mission is 74 We. It was found that all three advanced power systems could meet the required end of mission power with two General Purpose Heat Source (GPHS) modules. The RTG required six modules to meet the power requirement. Only the RAMTEC and RTPV met the mass goal of 9.5 kg. The AMTEC has a radiator area more than a factor of 10 lower than the Stirling and RTPV power systems, which simplifies spacecraft integration.