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Sample records for highly diversified recombination

  1. Molecular phylogeny of the highly diversified catfish subfamily Loricariinae (Siluriformes, Loricariidae) reveals incongruences with morphological classification.

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    Covain, Raphaël; Fisch-Muller, Sonia; Oliveira, Claudio; Mol, Jan H; Montoya-Burgos, Juan I; Dray, Stéphane

    2016-01-01

    The Loricariinae belong to the Neotropical mailed catfish family Loricariidae, the most species-rich catfish family. Among loricariids, members of the Loricariinae are united by a long and flattened caudal peduncle and the absence of an adipose fin. Despite numerous studies of the Loricariidae, there is no comprehensive phylogeny of this morphologically highly diversified subfamily. To fill this gap, we present a molecular phylogeny of this group, including 350 representatives, based on the analysis of mitochondrial and nuclear genes (8426 positions). The resulting phylogeny indicates that Loricariinae are distributed into two sister tribes: Harttiini and Loricariini. The Harttiini tribe, as classically defined, constitutes a paraphyletic assemblage and is here restricted to the three genera Harttia, Cteniloricaria, and Harttiella. Two subtribes are distinguished within Loricariini: Farlowellina and Loricariina. Within Farlowellina, the nominal genus formed a paraphyletic group, as did Sturisoma and Sturisomatichthys. Within Loricariina, Loricaria, Crossoloricaria, and Apistoloricaria are also paraphyletic. To solve these issues, and given the lack of clear morphological diagnostic features, we propose here to synonymize several genera (Quiritixys with Harttia; East Andean members of Crossoloricaria, and Apistoloricaria with Rhadinoloricaria; Ixinandria, Hemiloricaria, Fonchiiichthys, and Leliella with Rineloricaria), to restrict others (Crossoloricaria, and Sturisomatichthys to the West Andean members, and Sturisoma to the East Andean species), and to revalidate the genus Proloricaria. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella.

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    Liu, Tsunglin; Li, Ching-Min; Han, Yue-Lun; Chiang, Tzen-Yuh; Chiang, Yu-Chung; Sung, Huang-Mo

    2015-03-14

    Mycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species' association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment. Using next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance. G. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.

  3. Diversifying High Schools in Racially Changing Suburban Districts: Expanding Opportunity, Creating Barriers?

    Science.gov (United States)

    Frankenberg, Erica; Ayscue, Jennifer B.; Tyler, Alison C.

    2016-01-01

    Although demographic change is happening more rapidly at the elementary school level, the intersection of these demographic trends with the changing mission of high schools may offer the opportunity to reduce some of the persistent racial gaps in educational attainment. At the same time, when schools became diverse as desegregation took place,…

  4. Analysis and design of triple-band high-impedance surface absorber with periodic diversified impedance

    Science.gov (United States)

    Rui Zhang, Guo; Heng Zhou, Pei; Bin Zhang, Hui; Bo Zhang, Lin; Liang Xie, Jian; Jiang Deng, Long

    2013-10-01

    In this paper, a triple-band planar absorber with high-impedance surface (HIS) is designed and fabricated. The absorber structure is composed of polyurethane foam sandwiched between a lossy sheet of frequency selective surfaces (FSS) and a perfect electric conductor. The lossy FSS possesses different resistances in a periodic composite unit as compared with typical HIS absorber. Losses in the FSS are introduced by printing the periodic composite square ring pattern on blank stickers using various resistive inks. Physical mechanism of the HIS absorbers is analyzed by equivalent circuit model and electric field distribution studies. The proposed absorber with periodic composite units offers superimposed triple-band absorption as compared with that of the single units having single- or dual-band absorption characteristics. The reflection loss measurements show that the 90% absorption bandwidth of the HIS absorber is increased by 42% by the proposed composite periodic units.

  5. Agnathan VIP, PACAP and their receptors: ancestral origins of today's highly diversified forms.

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    Stephanie Y L Ng

    Full Text Available VIP and PACAP are pleiotropic peptides belonging to the secretin superfamily of brain-gut peptides and interact specifically with three receptors (VPAC(1, PAC(1 and VPAC(2 from the class II B G protein-coupled receptor family. There is immense interest regarding their molecular evolution which is often described closely alongside gene and/or genome duplications. Despite the wide array of information available in various vertebrates and one invertebrate the tunicate, their evolutionary origins remain unresolved. Through searches of genome databases and molecular cloning techniques, the first lamprey VIP/PACAP ligands and VPAC receptors are identified from the Japanese lamprey. In addition, two VPAC receptors (VPACa/b are identified from inshore hagfish and ligands predicted for sea lamprey. Phylogenetic analyses group these molecules into their respective PHI/VIP, PRP/PACAP and VPAC receptor families and show they resemble ancestral forms. Japanese lamprey VIP/PACAP peptides synthesized were tested with the hagfish VPAC receptors. hfVPACa transduces signal via both adenylyl cylase and phospholipase C pathways, whilst hfVPACb was only able to transduce through the calcium pathway. In contrast to the widespread distribution of VIP/PACAP ligands and receptors in many species, the agnathan PACAP and VPAC receptors were found almost exclusively in the brain. In situ hybridisation further showed their abundance throughout the brain. The range of VIP/PACAP ligands and receptors found are highly useful, providing a glimpse into the evolutionary events both at the structural and functional levels. Though representative of ancestral forms, the VIP/PACAP ligands in particular have retained high sequence conservation indicating the importance of their functions even early in vertebrate evolution. During these nascent stages, only two VPAC receptors are likely responsible for eliciting functions before evolving later into specific subtypes post-Agnatha. We

  6. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

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    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  7. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse...... transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity...... of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment...

  8. A network approach to analyzing highly recombinant malaria parasite genes.

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    Daniel B Larremore

    Full Text Available The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs, and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences.

  9. FIRE and Diversified International Portfolio

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    Meade, Dale

    2002-11-01

    The achievement of an attractive fusion energy using magnetic confinement requires significant advances in several key areas of plasma physics and technology including: self-heated plasmas in advanced configurations, steady-state plasma confinement of high-b plasmas, plasma wall interactions at high power density and the associated plasma and nuclear technologies. A diversified international portfolio is described that would allow the early resolution of key feasibility issues for fusion. FIRE, a national design study of a next step experiment is underway to develop and assess near term opportunities for addressing the burning plasma physics issues identified above. The emphasis is on exploring and understanding the behavior of plasmas dominated by self-heating (Palpha/Pext 2) that are sustained for a duration comparable to characteristic plasma time scales (>= 10τ_E, ˜ τ_SKIN . The study has focused on a compact, high-field, cryogenic-copper-magnet, highly-shaped tokamak with the parameters: Ro = 2.14m, a= 0.595m, double-null-divertor with helium pumping, Bt(Ro) = 10 T, and Ip = 7.7 MA and flat top time 20 s ( 20 τE and 2 τ_SKIN). The results of the study (http://fire.pppl.gov) including advanced tokamak modes and areas needing additional work will be discussed. Work supported by DOE Contract # DE-AC02-76CH0 3073.

  10. High yield production of extracellular recombinant levansucrase by Bacillus megaterium.

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    Korneli, Claudia; Biedendieck, Rebekka; David, Florian; Jahn, Dieter; Wittmann, Christoph

    2013-04-01

    In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

  11. Construction of high-resolution recombination maps in Asian seabass.

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    Wang, Le; Bai, Bin; Liu, Peng; Huang, Shu Qing; Wan, Zi Yi; Chua, Elaine; Ye, Baoqing; Yue, Gen Hua

    2017-01-10

    A high-density genetic map is essential for de novo genome assembly, fine mapping QTL for important complex traits, comparative genomic studies and understanding the mechanisms of genome evolution. Although a number of genomic resources are available in Asian seabass (Lates calcarifer), a high-density linkage map is still lacking. To facilitate QTL mapping for marker-assisted selection and genome assembly, and to understand the genome-wide recombination rates, we constructed high density linkage maps using three families and genotyping by sequencing. A high-density consensus linkage map consisting of 8, 274 markers was constructed based on sex-averaged genetic maps. The genetic maps were then aligned and integrated with the current genome assembly of Asian seabass. More than 90% of the genome contig sequences were anchored onto the consensus genetic map. Evidence of assembly errors in the current genome assembly was identified. A fragment of up to 2.5 Mb belonging to LG14 was assembled into Chr15. The length of family-specific sex-averaged maps ranged from 1348.96 to 1624.65 cM. Female maps were slightly longer than male maps using common markers. Female-to-male ratios were highly variable both across chromosomes within each family and throughout three families for each chromosome. However, the distribution patterns of recombination along chromosomes were similar between sexes across the whole genome. The overall recombination rates were significantly correlated with genome-wide GC content and the correlations were revealed to be stronger in females than in males. These high-density genetic maps provide not only essential tools for facilitating de novo genome assembly and comparative genomic studies in teleosts, but also critical resources for fine mapping QTL and genome-wide association mapping for economically important traits in Asian seabass.

  12. Diversifying experiences enhance cognitive flexibility

    NARCIS (Netherlands)

    Ritter, S.M.; Damian, R.I.; Simonton, D.K.; Baaren, R.B. van; Strick, M.A.; Derks, J.G.; Dijksterhuis, A.J.

    2012-01-01

    Past research has linked creativity to unusual and unexpected experiences, such as early parental loss or living abroad. However, few studies have investigated the underlying cognitive processes. We propose that these experiences have in common a "diversifying" aspect and an active involvement,

  13. High Rydberg resonances in dielectronic recombination of pb(79+).

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    Brandau, C; Bartsch, T; Hoffknecht, A; Knopp, H; Schippers, S; Shi, W; Müller, A; Grün, N; Scheid, W; Steih, T; Bosch, F; Franzke, B; Kozhuharov, C; Mokler, P H; Nolden, F; Steck, M; Stöhlker, T; Stachura, Z

    2002-07-29

    Dielectronic recombination resonances of Pb (79+) associated with 2s(1/2)-->2p(1/2) excitations were measured at the heavy-ion storage ring ESR at GSI. The fine structure of the energetically lowest resonance manifold Pb (78+)(1s(2)2p(1/2)20l(j)) at around 18 eV could partially be resolved, and rate coefficients on an absolute scale were obtained. A comparison of the experimental data with results of a fully relativistic theoretical approach shows that high-angular-momentum components up to j=31/2 significantly contribute to the total resonance strength demonstrating the necessity to revise the widespread notion of negligible high-angular-momentum contributions at least for very highly charged ions.

  14. PRODUCTION OF RECOMBINANT HIGH pI-BARLEY α-GLUCOSIDASE

    DEFF Research Database (Denmark)

    Næsted, Henrik; Svensson, Birte

    plantlet [1]. Recently, expression and characterization of the recombinant full length, fully functional barley high pI α-glucosidase in Pichia pastoris has been achieved. To enable production of recombinant protein in mg amounts, a transformant harbouring a clone encoding the N-terminally hexa histidine...... tagged recombinant form of the enzyme was propagated using a high cell-density fermentation procedure. This system resulted in successful expression under the highly sensitive methanol utilization phase conducting the fermentation process using a BiostatB 5 L reactor. The recombinant high pI α...... and kcat for hydrolysis of maltose were 1.7 mM, 139 nM s-1 and 85 s-1 respectively. The presented data illustrate the first successful production of enzymatically active full length recombinant high pI barley α-glucosidase [2]. Further characterisation of the enzyme specificity is ongoing and positions...

  15. High-level expression of alkaline protease using recombinant ...

    African Journals Online (AJOL)

    AJL

    2012-02-16

    Feb 16, 2012 ... The apr gene was cloned into plasmid pUB110, resulting in the recombinant plasmid pUB-apr, which was then transformed into ... 2002). It is widely present in bacteria, actinomycetes and fungi. However, almost all ... the transformation method necessary for this process has not yet been developed for B.

  16. Similarity in recombination rate estimates highly correlates with genetic differentiation in humans.

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    Hafid Laayouni

    Full Text Available Recombination varies greatly among species, as illustrated by the poor conservation of the recombination landscape between humans and chimpanzees. Thus, shorter evolutionary time frames are needed to understand the evolution of recombination. Here, we analyze its recent evolution in humans. We calculated the recombination rates between adjacent pairs of 636,933 common single-nucleotide polymorphism loci in 28 worldwide human populations and analyzed them in relation to genetic distances between populations. We found a strong and highly significant correlation between similarity in the recombination rates corrected for effective population size and genetic differentiation between populations. This correlation is observed at the genome-wide level, but also for each chromosome and when genetic distances and recombination similarities are calculated independently from different parts of the genome. Moreover, and more relevant, this relationship is robustly maintained when considering presence/absence of recombination hotspots. Simulations show that this correlation cannot be explained by biases in the inference of recombination rates caused by haplotype sharing among similar populations. This result indicates a rapid pace of evolution of recombination, within the time span of differentiation of modern humans.

  17. High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway.

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    Wallace, Nicholas A; Khanal, Sujita; Robinson, Kristin L; Wendel, Sebastian O; Messer, Joshua J; Galloway, Denise A

    2017-10-15

    Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA.IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in

  18. Genetic structure and evidence of putative Darwinian diversifying selection in the Potato yellow vein virus (PYVV

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    Giovanni Chaves-Bedoya

    2013-08-01

    Full Text Available The population structure and genetic variation of Potato yellow vein virus (PYVV were estimated by analysis of the nucleotide and deduced amino acid sequence of the coat protein of 69 isolates, reported in GenBank, from Solanum tuberosum (ST and Solanum phureja (SP hosts from different regions; predominantly Cundinamarca, Antioquia and Nariño, located in central and southwestern Colombia. Bioinformatics analysis revealed that despite the wide geographic distribution of different hosts and different collecting years, PYVV maintains a genetic similarity between 97.1 to 100.0%, indicating high spatial and temporal genetic stability of the major coat protein. No recombination events were found, but evidence was seen for the first time that this protein could be undergoing Darwinian diversifying selection

  19. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

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    Senanu M Spring-Pearson

    Full Text Available The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  20. Purification and characterization of recombinant high pI Barley α-Glucosidase

    DEFF Research Database (Denmark)

    Næsted, Henrik; Bojsen, Kirsten; Svensson, Birte

    of the native enzyme purified from malt (frandsen et al.). the kinetic values km, vmax and kcat are determined to 1.7 mm, 139 nm s-1 and 85 s-1 using maltose as substrate. the presented data illustrate the first successful production of enzymatically active full length recombinant high pi barley α...... (MACGREGOR & sissons). recently expression and characterization of the recombinant full length and fully functional barley high pi α-glucosidase in pichia pastoris has been achieved. in order to facilitate protein yield in the mg range, a clone representing an n-terminal hexa histidine tagged recombinant...

  1. Characterization of recombinant high pI Barley α-Glucosidase

    DEFF Research Database (Denmark)

    Næsted, Henrik; Svensson, Birte

    (MacGregor A.W.). Here we present the recent results of the expression and characterization of the recombinant full length barley high pI α-glucosidase in Pichia Pastoris. In order to facilitate in the range of mg protein yield, a clone representing an N-terminal hexa histidine tagged recombinant form...... compared to the kinetic data of the native enzyme (Frandsen et al.). The presented data illustrates for the first time the successful production of enzymatically active full length recombinant high pI barley α-glucosidase (Tibbot et al. and Fransen et al.). Frandsen et al. 2000 Plant Physiology 123, 275...

  2. Associations of Leu72Met Polymorphism of Preproghrelin with Ratios of Plasma Lipids Are Diversified by a High-Carbohydrate Diet in Healthy Chinese Adolescents.

    Science.gov (United States)

    Su, Mi; Qiu, Li; Wang, Qian; Jiang, Zhen; Liu, Xiao Juan; Lin, Jia; Fang, Ding Zhi

    2015-01-01

    The association of preproghrelin Leu72Met polymorphism with plasma lipids profile was inconsistently reported and needs more studies to be confirmed. Our study was to investigate the changes of plasma lipids ratios after a high-carbohydrate (high-CHO) diet in healthy Chinese adolescents with different genotypes of this polymorphism. Fifty-three healthy university students were given a washout diet of 54.1% carbohydrate for 7 days, followed by a high-CHO diet of 70.1% carbohydrate for 6 days. The anthropometric and biological parameters were analyzed at baseline and before and after the high-CHO diet. When compared with those before the high-CHO diet, body mass index (BMI) decreased in the male and female Met72 allele carriers. Decreased low-/high-density lipoprotein cholesterol (LDL-C/HDL-C) was observed in all participants except the female subjects with the Leu72Leu genotype. TG/HDL-C and log (TG/HDL-C) were increased only in the female subjects with the Leu72Leu genotype. These results suggest that the Met72 allele of preproghrelin Leu72Met polymorphism may be associated with decreased BMI induced by the high-CHO diet in male and female adolescents, while the Leu72 allele with increased TG/HDL-C and log (TG/HDL-C) in the female adolescents only. Furthermore, the decreasing effect of the high-CHO diet on LDL/HDL-C may be eliminated in the female Leu72Leu homozygotes. © 2015 S. Karger AG, Basel.

  3. Recombineering: highly efficient in vivo genetic engineering using single-strand oligos.

    Science.gov (United States)

    Sawitzke, James A; Thomason, Lynn C; Bubunenko, Mikhail; Li, Xintian; Costantino, Nina; Court, Donald L

    2013-01-01

    Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations. © 2013 Elsevier Inc. All rights reserved.

  4. The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection.

    Science.gov (United States)

    Cabezas-Cruz, Alejandro; Valdés, James J; de la Fuente, José

    2014-12-10

    A new species of Ehrlichia, phylogenetically distant from E. ruminantium, was found in 2010 infecting cattle in Canada. In 2012 and 2013, we reported the in vitro propagation, molecular and ultrastructural characterization of Ehrlichia sp. UFMG-EV (E. mineirensis), a new species of Ehrlichia isolated from the haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. A new organism, named Ehrlichia sp. UFMT-BV, closely related to Ehrlichia sp. UFMG-EV, was recently described in Brazil and after experimental infection it was shown to be pathogenic for cattle. This new emerging clade of cattle Ehrlichia pathogens is closely related to E. canis. The major immunogenic Tandem Repeat Protein (TRP36; also known as gp36) is extensively used to characterize the genetic diversity of E. canis. Homologs of TRP36 were found in both Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV. Herein, we characterized the evolution of this new Ehrlichia clade using TRP36 sequences. Our working hypothesis is that Ehrlichia sp. UFMG-EV and related microorganisms evolved from a highly variable E. canis clade. In support of our hypothesis we found that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV TRP36 evolved from a highly divergent and variable clade within E. canis and this clade evolved under episodic diversifying selection with a high proportion of sites under positive selection. Our results suggest that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV evolved from a variable clade within E. canis.

  5. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

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    Neubauer Peter

    2003-04-01

    Full Text Available Abstract Background Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. Results Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. Conclusion Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

  6. A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences.

    Science.gov (United States)

    Alcantara, Luiz Carlos Junior; Cassol, Sharon; Libin, Pieter; Deforche, Koen; Pybus, Oliver G; Van Ranst, Marc; Galvão-Castro, Bernardo; Vandamme, Anne-Mieke; de Oliveira, Tulio

    2009-07-01

    Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including: http://bioafrica.mrc.ac.za/rega-genotype/html/, http://lasp.cpqgm.fiocruz.br/virus-genotype/html/, http://jose.med.kuleuven.be/genotypetool/html/.

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

    Science.gov (United States)

    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. © 2015 American Institute of Chemical Engineers.

  8. Polarization measurement of dielectronic recombination transitions in highly charged krypton ions

    CERN Document Server

    Shah, Chintan; Bernitt, Sven; Dobrodey, Stepan; Steinbrügge, René; Beilmann, Christian; Amaro, Pedro; Hu, Zhimin; Weber, Sebastian; Fritzsche, Stephan; Surzhykov, Andrey; López-Urrutia, José R Crespo; Tashenov, Stanislav

    2016-01-01

    We report linear polarization measurements of x rays emitted due to dielectronic recombination into highly charged krypton ions. The ions in the He-like through O-like charge states were populated in an electron beam ion trap with the electron beam energy adjusted to recombination resonances in order to produce $K\\alpha$ x rays. The x rays were detected with a newly developed Compton polarimeter using a beryllium scattering target and 12 silicon x-ray detector diodes sampling the azimuthal distribution of the scattered x rays. The extracted degrees of linear polarization of several dielectronic recombination transitions agree with results of relativistic distorted--wave calculations. We also demonstrate a high sensitivity of the polarization to the Breit interaction, which is remarkable for a medium-$Z$ element like krypton. The experimental results can be used for polarization diagnostics of hot astrophysical and laboratory fusion plasmas.

  9. Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping.

    Directory of Open Access Journals (Sweden)

    Timothy Billings

    Full Text Available The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.

  10. Expression and Characterisation of Recombinant Rhodocyclus tenuis High Potential Iron-Sulphur Protein

    DEFF Research Database (Denmark)

    Caspersen, Michael Bjerg; Bennet, K.; Christensen, Hans Erik Mølager

    2000-01-01

    The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein...... of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein, The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those...

  11. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    Photosynthetic microorganism like microalgae and cyanobacteria are considered as emerging biotechnology platforms for production of recombinant proteins and other high-value biomolecules with a wide range of applications. Moreover, microalgae offer significant advantages compared with other...... the potential of microalgae as a cell factory for secretion of recombinant proteins. The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further...

  12. High-level secretion of native recombinant human calreticulin in yeast

    DEFF Research Database (Denmark)

    Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

    2015-01-01

    , Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional...... recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts...

  13. A high etendue spectrometer suitable for core charge eXchange recombination spectroscopy on ITER

    NARCIS (Netherlands)

    Jaspers, R.J.E.; Scheffer, M.; Kappatou, A.; Valk, N.C.J. van der; Durkut, M.; Snijders, B.; Marchuk, O.; Biel, W.; Pokol, G.I.; Erdei, G.; Zoletnik, S.; Dunai, D.

    2012-01-01

    A feasibility study for the use of core charge exchange recombination spectroscopy on ITER has shown that accurate measurements on the helium ash require a spectrometer with a high etendue of 1mm 2sr to comply with the measurement requirements [S. Tugarinov, Rev. Sci. Instrum. 74, 2075

  14. Diversified essential properties in halogenated graphenes

    OpenAIRE

    Tran, Ngoc Thanh Thuy; Nguyen, Duy Khanh; E., Glukhova O.; Lin, Ming-Fa

    2017-01-01

    The significant halogenation effects on the essential properties of graphene are investigated by the first-principles method. The geometric structures, electronic properties, and magnetic configurations are greatly diversified under the various halogen adsorptions. Fluorination, with the strong multi-orbital chemical bondings, can create the buckled graphene structure, while the other halogenations do not change the planar {\\sigma} bonding in the presence of single-orbital hybridization. Elec...

  15. Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination.

    Science.gov (United States)

    Dutch, R E; Zemelman, B V; Lehman, I R

    1994-01-01

    The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination. Images PMID:8189511

  16. High recombination frequency creates genotypic diversity in colonies of the leaf-cutting ant Acromyrmex echinatior

    DEFF Research Database (Denmark)

    Sirviö, A.; Gadau, J.; Rueppell, O.

    2006-01-01

    Honeybees are known to have genetically diverse colonies because queens mate with many males and the recombination rate is extremely high. Genetic diversity among social insect workers has been hypothesized to improve general performance of large and complex colonies, but this idea has not been...... tested in other social insects. Here, we present a linkage map and an estimate of the recombination rate for Acromyrmex echinatior, a leaf-cutting ant that resembles the honeybee in having multiple mating of queens and colonies of approximately the same size. A map of 145 AFLP markers in 22 linkage...... groups yielded a total recombinational size of 2076 cM and an inferred recombination rate of 161 kb cM-1 (or 6.2 cM Mb-1). This estimate is lower than in the honeybee but, as far as the mapping criteria can be compared, higher than in any other insect mapped so far. Earlier studies on A. echinatior have...

  17. Recombination of atomic oxygen on sintered zirconia at high temperature in non-equilibrium air plasma

    Energy Technology Data Exchange (ETDEWEB)

    Balat-Pichelin, M., E-mail: marianne.balat@promes.cnrs.fr [Laboratoire Procedes, Materiaux et Energie Solaire, PROMES-CNRS, UPR 8521, 7 rue du four solaire, 66120 Font-Romeu Odeillo (France); Passarelli, M.; Vesel, A. [Laboratoire Procedes, Materiaux et Energie Solaire, PROMES-CNRS, UPR 8521, 7 rue du four solaire, 66120 Font-Romeu Odeillo (France)

    2010-09-01

    High temperature ceramic materials are necessary for the design of primary heat shields for future re-usable space vehicles re-entering atmospheric planet at hypersonic velocity. During the re-entry phase on earth, one of the most important phenomena occurring on the heat shield is the recombination of atomic oxygen and this phenomenon is more or less catalyzed by the material of the heat shield. This paper presents some experimental results for the recombination coefficient of atomic oxygen {gamma} based on experiments performed on the MESOX facility using optical emission spectroscopy and actinometry techniques. Experimental results on the recombination coefficient are presented for three types of sintered ZrO{sub 2} in the temperature range 900-2500 K for 200 Pa total air pressure. These three zirconia ceramics differ essentially by the chemical nature of the sintering additives (Y{sub 2}O{sub 3}, CaO or MgO). A great different behavior of the recombination coefficient versus temperature is observed according to the crystalline structure of zirconia (monoclinic and tetragonal phases) and few influence of the additive is shown.

  18. Production of low-expressing recombinant cationic biopolymers with high purity.

    Science.gov (United States)

    Chen, Xuguang; Nomani, Alireza; Patel, Niket; Hatefi, Arash

    2017-06-01

    The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications. Unfortunately, due to their highly cationic nature and complex structure, their production in E. coli expression system is marred by low expression yield which in turn complicates the possibility of obtaining pure biopolymer. SlyD and ArnA endogenous E. coli proteins are considered the major culprits that copurify with the low-expressing biopolymers during the metal affinity chromatography. Here, we compared the impact of different parameters such as the choice of expression hosts as well as metal affinity columns in order to identify the most effective approach in obtaining highly pure recombinant cationic biopolymers with acceptable yield. The results of this study showed that by using E. coli BL21(DE3) LOBSTR strain and in combination with our developed stringent expression and Ni-NTA purification protocols highly pure products in one purification step (>99% purity) can be obtained. This approach could be applied to the production of other complex and potentially toxic biopolymers with wide range of applications in biomedicine. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Atomic oxygen recombination on the ODS PM 1000 at high temperature under air plasma

    Energy Technology Data Exchange (ETDEWEB)

    Balat-Pichelin, M., E-mail: marianne.balat@promes.cnrs.fr [Laboratoire Procedes, Materiaux et Energie Solaire, PROMES-CNRS, 7 rue du four solaire, 66120 Font-Romeu Odeillo (France); Beche, E. [Laboratoire Procedes, Materiaux et Energie Solaire, PROMES-CNRS, 7 rue du four solaire, 66120 Font-Romeu Odeillo (France)

    2010-06-01

    High temperature materials are necessary for the design of primary heat shields for future reusable space vehicles re-entering atmospheric planet at hypersonic velocity. During the re-entry phase on earth, one of the most important phenomena occurring on the heat shield is the recombination of atomic oxygen and this phenomenon is more or less catalyzed by the material of the heat shield. PM 1000 is planned to be use on the EXPERT capsule to study in real conditions its catalycity. Before the flight, it is necessary to perform measurements on ground test facility. Experimental data of the recombination coefficient of atomic oxygen under air plasma flow were obtained in the diffusion reactor MESOX on pre-oxidized PM 1000, for two total pressures 300 and 1000 Pa in the temperature range (850-1650 K) using actinometry and optical emission spectroscopy. In this investigation, the evolution of the recombination coefficient is dependent of temperature, pressure level and also of the chemical composition of the surface leading to one order of magnitude for a given temperature. The recombination coefficient is increasing with temperature and also dependent on the static pressure. The surface change due to the plasma exposure is inspected with SEM, XRD and XPS. As chromium oxide is the main part of the oxide layer formed during the oxidation in air plasma conditions, a sintered Cr{sub 2}O{sub 3} sample was elaborated from powders to compare the data of the recombination coefficient obtained on PM 1000. Pre- and post-test analyses on the several materials were carried out using SEM, WDS, XRD and XPS.

  20. Un contenu riche et diversifié

    Directory of Open Access Journals (Sweden)

    Esther Cloutier

    2010-05-01

    Full Text Available Bonjour,Nous sommes très heureux de vous convier à lire ce nouveau numéro de notre revue. Son contenu est riche et diversifié. Plusieurs thèmes sont abordés : la santé et la sécurité du travail dans les petites entreprises, l’autoévaluation du stress au travail, une démarche ergonomique de transformation du contexte d’intervention, les risques routiers professionnels des conducteurs de travaux, un outil de gestion du sommeil, ainsi que les facteurs socioculturels et TMS. En premier lieu, un a...

  1. A Diversified Investment Strategy Using Autonomous Agents

    Science.gov (United States)

    Barbosa, Rui Pedro; Belo, Orlando

    In a previously published article, we presented an architecture for implementing agents with the ability to trade autonomously in the Forex market. At the core of this architecture is an ensemble of classification and regression models that is used to predict the direction of the price of a currency pair. In this paper, we will describe a diversified investment strategy consisting of five agents which were implemented using that architecture. By simulating trades with 18 months of out-of-sample data, we will demonstrate that data mining models can produce profitable predictions, and that the trading risk can be diminished through investment diversification.

  2. A diversified portfolio model of adaptability.

    Science.gov (United States)

    Chandra, Siddharth; Leong, Frederick T L

    2016-12-01

    A new model of adaptability, the diversified portfolio model (DPM) of adaptability, is introduced. In the 1950s, Markowitz developed the financial portfolio model by demonstrating that investors could optimize the ratio of risk and return on their portfolios through risk diversification. The DPM integrates attractive features of a variety of models of adaptability, including Linville's self-complexity model, the risk and resilience model, and Bandura's social cognitive theory. The DPM draws on the concept of portfolio diversification, positing that diversified investment in multiple life experiences, life roles, and relationships promotes positive adaptation to life's challenges. The DPM provides a new integrative model of adaptability across the biopsychosocial levels of functioning. More importantly, the DPM addresses a gap in the literature by illuminating the antecedents of adaptive processes studied in a broad array of psychological models. The DPM is described in relation to the biopsychosocial model and propositions are offered regarding its utility in increasing adaptiveness. Recommendations for future research are also offered. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  3. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    Science.gov (United States)

    Kilian, Oliver; Benemann, Christina S. E.; Niyogi, Krishna K.; Vick, Bertrand

    2011-01-01

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  4. Variation in Recombination Rate and Its Genetic Determinism in Sheep Populations.

    Science.gov (United States)

    Petit, Morgane; Astruc, Jean-Michel; Sarry, Julien; Drouilhet, Laurence; Fabre, Stéphane; Moreno, Carole R; Servin, Bertrand

    2017-10-01

    Recombination is a complex biological process that results from a cascade of multiple events during meiosis. Understanding the genetic determinism of recombination can help to understand if and how these events are interacting. To tackle this question, we studied the patterns of recombination in sheep, using multiple approaches and data sets. We constructed male recombination maps in a dairy breed from the south of France (the Lacaune breed) at a fine scale by combining meiotic recombination rates from a large pedigree genotyped with a 50K SNP array and historical recombination rates from a sample of unrelated individuals genotyped with a 600K SNP array. This analysis revealed recombination patterns in sheep similar to other mammals but also genome regions that have likely been affected by directional and diversifying selection. We estimated the average recombination rate of Lacaune sheep at 1.5 cM/Mb, identified ∼50,000 crossover hotspots on the genome, and found a high correlation between historical and meiotic recombination rate estimates. A genome-wide association study revealed two major loci affecting interindividual variation in recombination rate in Lacaune, including the RNF212 and HEI10 genes and possibly two other loci of smaller effects including the KCNJ15 and FSHR genes. The comparison of these new results to those obtained previously in a distantly related population of domestic sheep (the Soay) revealed that Soay and Lacaune males have a very similar distribution of recombination along the genome. The two data sets were thus combined to create more precise male meiotic recombination maps in Sheep. However, despite their similar recombination maps, Soay and Lacaune males were found to exhibit different heritabilities and QTL effects for interindividual variation in genome-wide recombination rates. This highlights the robustness of recombination patterns to underlying variation in their genetic determinism. Copyright © 2017 by the Genetics Society

  5. Detecting genetic introgression: high levels of intersubspecific recombination found in Xylella fastidiosa in Brazil.

    Science.gov (United States)

    Nunney, Leonard; Yuan, Xiaoli; Bromley, Robin E; Stouthamer, Richard

    2012-07-01

    Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee.

  6. Detecting Genetic Introgression: High Levels of Intersubspecific Recombination Found in Xylella fastidiosa in Brazil

    Science.gov (United States)

    Yuan, Xiaoli; Bromley, Robin E.; Stouthamer, Richard

    2012-01-01

    Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee. PMID:22544234

  7. Economic Factors Affecting Diversified Farming Systems

    Directory of Open Access Journals (Sweden)

    Maria S. Bowman

    2013-03-01

    Full Text Available In response to a shift toward specialization and mechanization during the 20th century, there has been momentum on the part of a vocal contingent of consumers, producers, researchers, and policy makers who call for a transition toward a new model of agriculture. This model employs fewer synthetic inputs, incorporates practices which enhance biodiversity and environmental services at local, regional, and global scales, and takes into account the social implications of production practices, market dynamics, and product mixes. Within this vision, diversified farming systems (DFS have emerged as a model that incorporates functional biodiversity at multiple temporal and spatial scales to maintain ecosystem services critical to agricultural production. Our aim is to provide an economists' perspective on the factors which make diversified farming systems (DFS economically attractive, or not-so-attractive, to farmers, and to discuss the potential for and roadblocks to widespread adoption. We focus on how a range of existing and emerging factors drive profitability and adoption of DFS. We believe that, in order for DFS to thrive, a number of structural changes are needed. These include: 1 public and private investment in the development of low-cost, practical technologies that reduce the costs of production in DFS, 2 support for and coordination of evolving markets for ecosystem services and products from DFS and 3 the elimination of subsidies and crop insurance programs that perpetuate the unsustainable production of staple crops. We suggest that subsidies and funding be directed, instead, toward points 1 and 2, as well as toward incentives for consumption of nutritious food.

  8. Signatures of diversifying selection in European pig breeds.

    Directory of Open Access Journals (Sweden)

    Samantha Wilkinson

    2013-04-01

    Full Text Available Following domestication, livestock breeds have experienced intense selection pressures for the development of desirable traits. This has resulted in a large diversity of breeds that display variation in many phenotypic traits, such as coat colour, muscle composition, early maturity, growth rate, body size, reproduction, and behaviour. To better understand the relationship between genomic composition and phenotypic diversity arising from breed development, the genomes of 13 traditional and commercial European pig breeds were scanned for signatures of diversifying selection using the Porcine60K SNP chip, applying a between-population (differentiation approach. Signatures of diversifying selection between breeds were found in genomic regions associated with traits related to breed standard criteria, such as coat colour and ear morphology. Amino acid differences in the EDNRB gene appear to be associated with one of these signatures, and variation in the KITLG gene may be associated with another. Other selection signals were found in genomic regions including QTLs and genes associated with production traits such as reproduction, growth, and fat deposition. Some selection signatures were associated with regions showing evidence of introgression from Asian breeds. When the European breeds were compared with wild boar, genomic regions with high levels of differentiation harboured genes related to bone formation, growth, and fat deposition.

  9. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

    Science.gov (United States)

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-06-26

    The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

  10. High-throughput FTIR-based bioprocess analysis of recombinant cyprosin production.

    Science.gov (United States)

    Sampaio, Pedro N; Sales, Kevin C; Rosa, Filipa O; Lopes, Marta B; Calado, Cecília R C

    2017-01-01

    To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples' second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R 2 = 0.99, RMSEP 2.8%); cyprosin activity (R 2 = 0.98, RMSEP 3.9%); glucose (R 2 = 0.93, RMSECV 7.2%); galactose (R 2 = 0.97, RMSEP 4.6%); ethanol (R 2 = 0.97, RMSEP 5.3%); and acetate (R 2 = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.

  11. A high etendue spectrometer suitable for core charge eXchange recombination spectroscopy on ITER

    Energy Technology Data Exchange (ETDEWEB)

    Jaspers, R. J. E.; Scheffer, M. [Science and Technology of Nuclear Fusion, Eindhoven University of Technology, Eindhoven (Netherlands); Kappatou, A. [FOM Institute DIFFER - Dutch Institute for Fundamental Energy Research, Association EURATOM-FOM, Nieuwegein (Netherlands); Valk, N. C. J. van der; Durkut, M.; Snijders, B. [TNO Science and Industry, P.O. Box 155, 2600 AD Delft (Netherlands); Marchuk, O.; Biel, W. [Institut fuer Energie und Klimaforschung-IEK-4 Forschungszentrum, Juelich GmbH, 52425 Juelich (Germany); Pokol, G. I. [Institute of Nuclear Techniques, Budapest University of Technology and Economics, EURATOM Association, P. O. Box 91, H-1521 Budapest (Hungary); Erdei, G. [Department of Atomic Physics, Budapest University of Technology and Economics, EURATOM Association, P. O. Box 91, H-1521 Budapest (Hungary); Zoletnik, S.; Dunai, D. [WIGNER RCP, RMKI, EURATOM Association, P. O. Box 91, H-1521 Budapest (Hungary)

    2012-10-15

    A feasibility study for the use of core charge exchange recombination spectroscopy on ITER has shown that accurate measurements on the helium ash require a spectrometer with a high etendue of 1mm{sup 2}sr to comply with the measurement requirements [S. Tugarinov et al., Rev. Sci. Instrum. 74, 2075 (2003)]. To this purpose such an instrument has been developed consisting of three separate wavelength channels (to measure simultaneously He/Be, C/Ne, and H/D/T together with the Doppler shifted direct emission of the diagnostic neutral beam, the beam emission (BES) signal), combining high dispersion (0.02 nm/pixel), sufficient resolution (0.2 nm), high efficiency (55%), and extended wavelength range (14 nm) at high etendue. The combined measurement of the BES along the same sightline within a third wavelength range provides the possibility for in situ calibration of the charge eXchange recombination spectroscopy signals. In addition, the option is included to use the same instrument for measurements of the fast fluctuations of the beam emission intensity up to 2 MHz, with the aim to study MHD activity.

  12. A high etendue spectrometer suitable for core charge eXchange recombination spectroscopy on ITERa)

    Science.gov (United States)

    Jaspers, R. J. E.; Scheffer, M.; Kappatou, A.; van der Valk, N. C. J.; Durkut, M.; Snijders, B.; Marchuk, O.; Biel, W.; Pokol, G. I.; Erdei, G.; Zoletnik, S.; Dunai, D.

    2012-10-01

    A feasibility study for the use of core charge exchange recombination spectroscopy on ITER has shown that accurate measurements on the helium ash require a spectrometer with a high etendue of 1mm2sr to comply with the measurement requirements [S. Tugarinov et al., Rev. Sci. Instrum. 74, 2075 (2003)], 10.1063/1.1537443. To this purpose such an instrument has been developed consisting of three separate wavelength channels (to measure simultaneously He/Be, C/Ne, and H/D/T together with the Doppler shifted direct emission of the diagnostic neutral beam, the beam emission (BES) signal), combining high dispersion (0.02 nm/pixel), sufficient resolution (0.2 nm), high efficiency (55%), and extended wavelength range (14 nm) at high etendue. The combined measurement of the BES along the same sightline within a third wavelength range provides the possibility for in situ calibration of the charge eXchange recombination spectroscopy signals. In addition, the option is included to use the same instrument for measurements of the fast fluctuations of the beam emission intensity up to 2 MHz, with the aim to study MHD activity.

  13. On the use of a recombination chamber for radiation measurements in CERN-EU high energy reference radiation fields

    CERN Document Server

    Golnik, N; Otto, T

    1999-01-01

    Ambient dose equivalent was determined in high energy reference radiation fields at CERN (CERF facility) using a recombination chamber and recombination methods developed in IAE. The chamber was also used for measuring the low LET background radiation which locally accompanies the fields at CERF. The measurements included determination of the absorbed dose and recombination index of radiation quality at different beam intensities. It was shown that the background might considerably influence the measurements of the absorbed dose, however, its influence on the ambient dose equivalent remains important only at low beam intensities. (16 refs).

  14. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  15. High inbreeding, limited recombination and divergent evolutionary patterns between two sympatric morel species in China.

    Science.gov (United States)

    Du, Xi-Hui; Zhao, Qi; Xu, Jianping; Yang, Zhu L

    2016-03-01

    As highly prized, popular mushrooms, morels are widely distributed in the northern hemisphere, with China as a modern centre of speciation and diversity. Overharvesting of morels has caused concern over how to effectively preserve their biological and genetic diversity. However, little is known about their population biology and life cycle. In this study, we selected two sympatric phylogenetic species, Mel-13 (124 collections from 11 geographical locations) and Morchella eohespera (156 collections from 14 geographical locations), using fragments of 4 DNA sequences, to analyse their genetic structure. Our results indicated significant differentiation among geographic locations in both species, whereas no obvious correlation between genetic and geographic distance was identified in either species. M. eohespera exhibited a predominantly clonal population structure with limited recombination detected in only 1 of the 14 geographic locations. In contrast, relatively frequent recombination was identified in 6 of the 11 geographic locations of Mel-13. Our analysis indicated that the sympatric species Mel-13 and M. eohespera might have divergent evolutionary patterns, with the former showing signatures of recent population expansion and the latter being relatively stable. Interestingly, we found no heterozygosity but strong evidence for genealogical incongruence, indicating a high level of inbreeding and hybridisation among morel species.

  16. Diverse and highly recombinant anelloviruses associated with Weddell seals in Antarctica

    Science.gov (United States)

    Fahsbender, Elizabeth; Kim, Stacy; Kraberger, Simona; Frankfurter, Greg; Eilers, Alice A.; Shero, Michelle R.; Beltran, Roxanne; Kirkham, Amy; McCorkell, Robert; Berngartt, Rachel K.; Male, Maketalena F.; Ballard, Grant; Ainley, David G.; Breitbart, Mya

    2017-01-01

    Abstract The viruses circulating among Antarctic wildlife remain largely unknown. In an effort to identify viruses associated with Weddell seals (Leptonychotes weddellii) inhabiting the Ross Sea, vaginal and nasal swabs, and faecal samples were collected between November 2014 and February 2015. In addition, a Weddell seal kidney and South Polar skua (Stercorarius maccormicki) faeces were opportunistically sampled. Using high throughput sequencing, we identified and recovered 152 anellovirus genomes that share 63–70% genome-wide identities with other pinniped anelloviruses. Genome-wide pairwise comparisons coupled with phylogenetic analysis revealed two novel anellovirus species, tentatively named torque teno Leptonychotes weddellii virus (TTLwV) -1 and -2. TTLwV-1 (n = 133, genomes encompassing 40 genotypes) is highly recombinant, whereas TTLwV-2 (n = 19, genomes encompassing three genotypes) is relatively less recombinant. This study documents ubiquitous TTLwVs among Weddell seals in Antarctica with frequent co-infection by multiple genotypes, however, the role these anelloviruses play in seal health remains unknown. PMID:28744371

  17. A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

    Science.gov (United States)

    Yang, Peng; Liu, Wenjing; Cheng, Xuelian; Wang, Jing; Qi, Qingsheng

    2016-01-01

    ABSTRACT 5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. IMPORTANCE In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future. PMID:26921424

  18. Diversified Research Methods Education in LIS: Thinking outside the Box

    Science.gov (United States)

    Luo, Lili

    2017-01-01

    A small number of LIS degree programs have adopted a diversified approach to research methods education, including offering an array of specialized research methods courses in addition to a general introductory course. The current study conducted an in-depth investigation of the diversified research methods curriculum of the LIS program at San…

  19. Ferns diversified in the shadow of angiosperms.

    Science.gov (United States)

    Schneider, Harald; Schuettpelz, Eric; Pryer, Kathleen M; Cranfill, Raymond; Magallón, Susana; Lupia, Richard

    2004-04-01

    The rise of angiosperms during the Cretaceous period is often portrayed as coincident with a dramatic drop in the diversity and abundance of many seed-free vascular plant lineages, including ferns. This has led to the widespread belief that ferns, once a principal component of terrestrial ecosystems, succumbed to the ecological predominance of angiosperms and are mostly evolutionary holdovers from the late Palaeozoic/early Mesozoic era. The first appearance of many modern fern genera in the early Tertiary fossil record implies another evolutionary scenario; that is, that the majority of living ferns resulted from a more recent diversification. But a full understanding of trends in fern diversification and evolution using only palaeobotanical evidence is hindered by the poor taxonomic resolution of the fern fossil record in the Cretaceous. Here we report divergence time estimates for ferns and angiosperms based on molecular data, with constraints from a reassessment of the fossil record. We show that polypod ferns (> 80% of living fern species) diversified in the Cretaceous, after angiosperms, suggesting perhaps an ecological opportunistic response to the diversification of angiosperms, as angiosperms came to dominate terrestrial ecosystems.

  20. The University Entrance Exam that Diversified and the Problem

    Science.gov (United States)

    Kondo, Osamu

    The percentage of students who go on to universities or junior colleges is over fifty percent which is more than half of high school students. However the nation's birthrate is in decline and the total number of university-age children shows a declining tendency. Hence we cannot expect an increase in the number of applicants in the future. On the other hand, the number of universities has been increasing year by year. The competition among universities to survive is very fierce. The diversification of entrance examination is a symbol of this competition. By diversifying entrance exams, universities aimed at the quality of excellent students in the beginning. However, they have changed their direction to pursue the quantity of students. As of Today the entrance examination is losing its original starting function which means most or all applicants can enter universities. It is time all of the universities return to its starting line.

  1. Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals.

    Science.gov (United States)

    Morandini, Francesca; Avesani, Linda; Bortesi, Luisa; Van Droogenbroeck, Bart; De Wilde, Kirsten; Arcalis, Elsa; Bazzoni, Flavia; Santi, Luca; Brozzetti, Annalisa; Falorni, Alberto; Stoger, Eva; Depicker, Ann; Pezzotti, Mario

    2011-10-01

    We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  2. Recombinant Klebsiella oxytoca strains with improved efficiency in removal of high nitrate loads

    Energy Technology Data Exchange (ETDEWEB)

    Pinar, G.; Ramos, J.L. [Estacion Experimental del Zaidin--Consejo Superior de Investigaciones Cientificas, Granada (Spain). Dept. of Biochemistry and Molecular and Cellular Biology of Plants

    1998-12-01

    Klebsiella oxytoca CECT 4460 removes high nitrate loads from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose in K. oxytoca CECT 4460 was considered. To achieve this, the authors cloned and transferred into this strain the Klebsiella pneumoniae nasB gene, which encodes assimilatory nitrite reductase. The delivery vector was either the wide-host-range plasmid pUPE2, in which the nasB gene is expressed from the Escherichia coli P{sub lac} promoter, or a mini-Tn5-Km vector, which upon random insertion in the host chromosome allowed expression of the nasB gene from an unidentified chromosomal host promoter. The effect of the increase in the dose of the nasB gene in K. oxytoca CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed that K. oxytoca CECT 4460 bearing pUPE2 accumulated 88% less nitrite than the wild-type strain, while the recombinant strain bearing the K. pneumoniae nasB gene in the host chromosome showed a 25% lower level of nitrite accumulation in the culture medium than that of the wild type.

  3. Production of highly potent recombinant siRNAs in Escherichia coli.

    Science.gov (United States)

    Huang, Linfeng; Lieberman, Judy

    2013-12-01

    We recently invented a method to produce highly potent siRNAs in Escherichia coli, based on the serendipitous discovery that ectopic expression of p19, a plant viral siRNA-binding protein, stabilizes otherwise unstable bacterial siRNAs, which we named pro-siRNAs for prokaryotic siRNAs. We present a detailed protocol describing how to produce pro-siRNAs for efficiently knocking down any gene, beginning with the design of a pro-siRNA expression plasmid and ending with siRNA purification. This protocol uses one plasmid to co-express a recombinant His-tagged p19 protein and a long hairpin RNA containing sense and antisense sequences of the target gene. pro-siRNAs are isolated and purified using nickel beads and HPLC, using methods used to produce recombinant proteins. Once a pro-siRNA plasmid is obtained, production of purified pro-siRNAs takes a few days. The pro-siRNA technique provides a reliable and renewable source of siRNAs, and it can be implemented in any laboratory whose members are skilled in routine molecular biology techniques.

  4. Highly purified HMG versus recombinant FSH for ovarian stimulation in IVF cycles

    DEFF Research Database (Denmark)

    Platteau, P.; Nyboe, Andersen A.; Loft, A.

    2008-01-01

    The objective of this study was to compare the live birth rates resulting from ovarian stimulation with highly purified human menopausal gonadotrophin (HP-HMG), which combines FSH and human chorionic gonadotrophin-driven LH activities, or recombinant FSH (rFSH) alone in women undergoing IVF cycles....... An integrated analysis was performed of the raw data from two randomized controlled trials that were highly comparable in terms of eligibility criteria and post-randomization treatment regimens with either HP-HMG or rFSH for ovarian stimulation in IVF, following a long down-regulation protocol. All randomized...... subjects who received at least one dose of gonadotrophin in an IVF cycle (HP-HMG, n = 491; rFSH, n = 495) were included in the analysis. Subjects who underwent intracytoplasmic sperm injection cycles were excluded. The superiority of one gonadotrophin preparation over the other was tested using...

  5. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

    Directory of Open Access Journals (Sweden)

    Chen Ling

    2016-01-01

    Full Text Available Although recombinant adeno-associated virus serotype 3 (AAV3 vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs from AAV3 (ITR3, as well as the trans-acting Rep proteins from AAV3 (Rep3 in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ∼10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492 were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of

  6. High-throughput 454 resequencing for allele discovery and recombination mapping in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Tan John C

    2011-02-01

    Full Text Available Abstract Background Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum genome is crucial for understanding its evolution; however the 81% (A+T genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites. Results We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs and many non crossover (NCO gene conversions throughout the genome. Conclusions By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.

  7. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins.

    Science.gov (United States)

    Leuzinger, Kahlin; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; Lai, Huafang; Zhou, Xiaohong; Chen, Qiang

    2013-07-23

    Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It

  8. Process and product monitoring of recombinant DNA-derived biopharmaceuticals with high-performance capillary electrophoresis.

    Science.gov (United States)

    Sunday, Brooks R; Sydor, Wasyl; Guariglia, Lawrence M; Obara, Julie; Mengisen, Roland

    2003-01-01

    High-performance capillary electrophoresis (HPCE) has emerged over the past 20 years as a powerful multidimensional separation tool that is orthogonal to HPLC and comparable to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gel methods. HPCE is most frequently applied in the QC release testing of recombinant DNA-derived protein and monoclonal antibody (MAb) biopharmaceuticals. HPCE is a rugged and robust separation tool that can be used like HPLC to monitor the purification process, as well as to analyze bulk drug and drug substances. Examples of the practical applications of the predominant free-solution capillary electrophoresis (FSCE) and capillary gel electrophoresis (CGE) formats of HPCE, applied for process monitoring and product monitoring of recombinant protein and MAb biotherapeutics, are presented. HPCE has been applied in FSCE mode to monitor the purification of the rDNA-derived protein, recombinant human interleukin-4 (rhIL4). FSCE is demonstrated to be a robust method that can be used to monitor multiple column chromatographic purification processes, such as immobiilized metal-ion affinity chromatography (IMAC), ion exchange chromatography (IEC), and size exclusion chromatography (SEC) columns. The FSCE data are used to pool fractions to carry forward for further purification. The FSCE method is compared to the corresponding RP-HPLC method for rhIL4. HPCE has been applied in the CGE mode to monitor the purification of an rDNA-derived IgG4 MAb. CGE is demonstrated to be a convenient and rapid method to profile the purification process, compare purification processes, and provide a fingerprint of the MAb bulk drug that is useful for determining purity and lot-to-lot consistency. The practical advantages and limitations of CGE for process monitoring and product monitoring of MAbs are presented. The CGE method is compared to the high-performance SEC separation of the MAb under nondenaturing (HP-SEC) and denaturing (HP

  9. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  10. Propargyl Recombination: Estimation of the High Temperature, Low Pressure Rate Constant from Flame Measurements

    DEFF Research Database (Denmark)

    Rasmussen, Christian Lund; Skjøth-Rasmussen, Martin Skov; Jensen, Anker

    2005-01-01

    The most important cyclization reaction in hydrocarbon flames is probably recombination of propargyl radicals. This reaction may, depending on reaction conditions, form benzene, phenyl or fulvene, as well as a range of linear products. A number of rate measurements have been reported for C3H3 + C3H......3 at temperatures below 1000 K, while data at high temperature and low pressure only can be obtained from flames. In the present work, an estimate of the rate constant for the reaction at 1400 +/- 50 K and 20 Torr is obtained from analysis of the fuel-rich acetylene flame of Westmoreland, Howard......, and Longwell. Based on an accurate modeling of the flame structure, in particular the concentration profile of propargyl, we estimate the rate constant by fitting the kinetic modeling predictions to the measured benzene and phenyl profiles. The best agreement is obtained with k = 1.3 x 10(12) cm(3)/mol...

  11. Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

    DEFF Research Database (Denmark)

    Mateu, Guaniri; Donis, Ruben O; Wakita, Takaji

    2008-01-01

    The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the cont......The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF...... into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone....... Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility...

  12. Diversifying Your Career Counseling Practice: An Overview of Options.

    Science.gov (United States)

    Rogerson, Lynda

    1997-01-01

    Career counselors should practice what they preach and develop strategies for diversifying their careers. Options include adjunct faculty, outplacement consultant, motivational speaker, mentor/advisor for special groups, job club facilitator, seminar leader, radio host, and newspaper columnist. (SK)

  13. High Efficiency Tandem Thin-Perovskite/Polymer Solar Cells with a Graded Recombination Layer.

    Science.gov (United States)

    Liu, Yao; Renna, Lawrence A; Bag, Monojit; Page, Zachariah A; Kim, Paul; Choi, Jaewon; Emrick, Todd; Venkataraman, D; Russell, Thomas P

    2016-03-23

    Perovskite-containing tandem solar cells are attracting attention for their potential to achieve high efficiencies. We demonstrate a series connection of a ∼ 90 nm thick perovskite front subcell and a ∼ 100 nm thick polymer:fullerene blend back subcell that benefits from an efficient graded recombination layer containing a zwitterionic fullerene, silver (Ag), and molybdenum trioxide (MoO3). This methodology eliminates the adverse effects of thermal annealing or chemical treatment that occurs during perovskite fabrication on polymer-based front subcells. The record tandem perovskite/polymer solar cell efficiency of 16.0%, with low hysteresis, is 75% greater than that of the corresponding ∼ 90 nm thick perovskite single-junction device and 65% greater than that of the polymer single-junction device. The high efficiency of this hybrid tandem device, achieved using only a ∼ 90 nm thick perovskite layer, provides an opportunity to substantially reduce the lead content in the device, while maintaining the high performance derived from perovskites.

  14. Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus.

    Science.gov (United States)

    Guo, Jin-Chao; Tang, Yan-Dong; Zhao, Kuan; Wang, Tong-Yun; Liu, Ji-Ting; Gao, Jia-Cong; Chang, Xiao-Bo; Cui, Hong-Yu; Tian, Zhi-Jun; Cai, Xue-Hui; An, Tong-Qing

    2016-01-01

    Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.

  15. Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system.

    Science.gov (United States)

    Sasaki, Yukari; Sone, Takefumi; Yoshida, Shouhei; Yahata, Kazuhide; Hotta, Junko; Chesnut, Jonathan D; Honda, Takeshi; Imamoto, Fumio

    2004-02-05

    Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown.

  16. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    Science.gov (United States)

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-08-20

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer.

  17. "EFFECT OF HIGH VERSUS LOW DOSES OF HUMAN RECOMBINANT ERYTHROPOIETIN ON THE ANEMIA OF PREMATURITY"

    Directory of Open Access Journals (Sweden)

    A. Mohammadzadeh

    2005-05-01

    Full Text Available Recombinant human erythropoietin (rh-EPO is known to accelerate erythropoiesis in preterm infants. The purpose of this study was to compare the effectiveness of early treatment with two doses of rh-EPO (high vs. low dose in the management of anemia of prematurity. Twenty preterm infants with hematocrit (Hct < 30% when infant’s age was between 2 to 3 weeks after birth or Hct <25% when infant’s age was more than 3 weeks after birth, were divided randomly in two groups, each group including 10 babies. Infants in high dose group received 500 u/kg rh-EPO twice per week and the low dose group received 500 u/kg rh-EPO weekly. All infants were fed human milk supplemented with enteral iron. Hematocrit and reticulocyte counts were determined for each infant at the start of the study, 3 days after start of treatment and one week after the end of treatment. The means of gestational age in high dose and low dose groups were 31.4 ± 2.2 and 31.3±2.0 weeks, respectively. Means of birth weight in high dose and low dose groups were 1366 ± 243 and 1438±249 gr, respectively. The two groups were significantly different in reticulocyte count at 3 days after treatment (P = 0.047 and in hematocrit at the end of study (P < 0.0001. We concluded the early treatment of anemia of prematurity with high dose rh-EPO with supplemental iron significantly increases hematocrit and reticulocyte in preterm infants and reduce the need for blood transfusion in these high risk neonates.

  18. Evidence for strong Breit interaction in dielectronic recombination of highly charged heavy ions.

    Science.gov (United States)

    Nakamura, Nobuyuki; Kavanagh, Anthony P; Watanabe, Hirofumi; Sakaue, Hiroyuki A; Li, Yueming; Kato, Daiji; Currell, Fred J; Ohtani, Shunsuke

    2008-02-22

    Resonant strengths have been measured for dielectronic recombination of Li-like iodine, holmium, and bismuth using an electron beam ion trap. By observing the atomic number dependence of the state-resolved resonant strength, clear experimental evidence has been obtained that the importance of the generalized Breit interaction (GBI) effect on dielectronic recombination increases as the atomic number increases. In particular, it has been shown that the GBI effect is exceptionally strong for the recombination through the resonant state [1s2s(2)2p(1/2)](1).

  19. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

    2003-01-01

    this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield......Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  20. Extensive Horizontal Transfer and Homologous Recombination Generate Highly Chimeric Mitochondrial Genomes in Yeast.

    Science.gov (United States)

    Wu, Baojun; Buljic, Adnan; Hao, Weilong

    2015-10-01

    The frequency of horizontal gene transfer (HGT) in mitochondrial DNA varies substantially. In plants, HGT is relatively common, whereas in animals it appears to be quite rare. It is of considerable importance to understand mitochondrial HGT across the major groups of eukaryotes at a genome-wide level, but so far this has been well studied only in plants. In this study, we generated ten new mitochondrial genome sequences and analyzed 40 mitochondrial genomes from the Saccharomycetaceae to assess the magnitude and nature of mitochondrial HGT in yeasts. We provide evidence for extensive, homologous-recombination-mediated, mitochondrial-to-mitochondrial HGT occurring throughout yeast mitochondrial genomes, leading to genomes that are highly chimeric evolutionarily. This HGT has led to substantial intraspecific polymorphism in both sequence content and sequence divergence, which to our knowledge has not been previously documented in any mitochondrial genome. The unexpectedly high frequency of mitochondrial HGT in yeast may be driven by frequent mitochondrial fusion, relatively low mitochondrial substitution rates and pseudohyphal fusion to produce heterokaryons. These findings suggest that mitochondrial HGT may play an important role in genome evolution of a much broader spectrum of eukaryotes than previously appreciated and that there is a critical need to systematically study the frequency, extent, and importance of mitochondrial HGT across eukaryotes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Electron Impact Excitation and Dielectronic Recombination of Highly Charged Tungsten Ions

    Directory of Open Access Journals (Sweden)

    Zhongwen Wu

    2015-11-01

    Full Text Available Electron impact excitation (EIE and dielectronic recombination (DR of tungsten ions are basic atomic processes in nuclear fusion plasmas of the International Thermonuclear Experimental Reactor (ITER tokamak. Detailed investigation of such processes is essential for modeling and diagnosing future fusion experiments performed on the ITER. In the present work, we studied total and partial electron-impact excitation (EIE and DR cross-sections of highly charged tungsten ions by using the multiconfiguration Dirac–Fock method. The degrees of linear polarization of the subsequent X-ray emissions from unequally-populated magnetic sub-levels of these ions were estimated. It is found that the degrees of linear polarization of the same transition lines, but populated respectively by the EIE and DR processes, are very different, which makes diagnosis of the formation mechanism of X-ray emissions possible. In addition, with the help of the flexible atomic code on the basis of the relativistic configuration interaction method, DR rate coefficients of highly charged W37+ to W46+ ions are also studied, because of the importance in the ionization equilibrium of tungsten plasmas under running conditions of the ITER.

  2. Design Study of the High Luminosity LHC Recombination Dipole (D2)

    Energy Technology Data Exchange (ETDEWEB)

    Sabbi, GianLuca; Wang, Xiaorong

    2014-05-26

    The interaction region design of the high-luminosity LHC requires replacing the recombination dipole magnets (D2) with new ones. The preliminary specifications include an aperture of 105 mm, with 186 mm separation between the twin-aperture axes, and an operating field in the range of 3.5 to 4.5 T. The main design challenge is to decouple the magnetic field in the two apertures and ensure good field quality. The approach adopted for the present D2 magnets, using the iron yoke as a shield between the two apertures, leads to large saturation effects. In this study, we propose an alternative approach where the iron yoke is designed primarily for low saturation, and the resulting large but current-independent cross-talk between the apertures is corrected with an asymmetric arrangement of the conductor blocks. A preliminary solution based on the LHC dipole cable is presented, and the expected harmonics for geometric, saturation and persistent current effects are provided. Finally, the feasibility of an operating field at the high end of the range considered is discussed, to minimize the D2 magnet length and facilitate the space allocation for other components.

  3. High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity

    DEFF Research Database (Denmark)

    van Vugt, Joke .J.F.A.; Storgaard, Torben; Oleksiewicz, Martin B.

    2001-01-01

    Two types of porcine reproductive and respiratory syndrome virus (PRRSV) exist, a North American type and a European type. The co-existence of both types in some countries, such as Denmark, Slovakia and Canada, creates a risk of inter-type recombination. To evaluate this risk, cell cultures were co......, but no recombination was detected between the European and North American types. Calculation of the maximum theoretical risk of European-American recombination, based on the sensitivity of the RT-PCR system, revealed that RNA recombination between the European and North American types of PRRSV is at least 10000 times...

  4. A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Karl Friehs

    2017-05-01

    Full Text Available Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS. Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector

  5. High-level secretion of native recombinant human calreticulin in yeast

    DEFF Research Database (Denmark)

    Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

    2015-01-01

    processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species...... quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric...... by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration...

  6. Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

    Science.gov (United States)

    Davis, John N.

    2013-01-01

    Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3′ end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12′GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12′GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12′GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12′GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12′GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12′GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12′GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential. PMID:23135719

  7. Highly attenuated recombinant vesicular stomatitis virus VSV-12'GFP displays immunogenic and oncolytic activity.

    Science.gov (United States)

    van den Pol, Anthony N; Davis, John N

    2013-01-01

    Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3' end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12'GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12'GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12'GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12'GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12'GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12'GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12'GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential.

  8. Diversified techniques are key to successful Venezuela operation

    Energy Technology Data Exchange (ETDEWEB)

    Acosta, J.C.; Verde, F.D.

    1975-04-01

    Texas Petroleum Co.'s Mata field is an 84,453-acre concession separated into 3 blocks that contain a total of 463 reservoirs. It is one of E. Venezuela's major fields and currently produces an average of 22,500 bpd. Production is highly diversified, i.e., natural flow, gas lift, hydraulic pump, and rod pumping. Crude gravities range from 10/sup 0/ to 50/sup 0/ API, so production is divided into 2 streams: the Mata Mesa 30/sup 0/ API and Mata Merey 17/sup 0/ to 22/sup 0/ API. At present, 78% of the production is from secondary recovery projects. The first application of secondary recovery was initiated in 1957 with a gas injection project. There are 18 gas injection projects now in operation, producing 44% of the total field production. Waterflooding, initiated in 1969 with a pilot project in a 2-well reservoir, proved highly successful, and consequently, has been extended to 19 reservoirs producing 34% of the current field production. Other subjects discussed include geology, rock properties, fluid characteristics, production history, well completion, artificial lift methods, stimulation treatments, gas injection, and waterfloods.

  9. More on the narrowing of impact broadened radio recombination lines at high principal quantum number

    Science.gov (United States)

    Bell, M. B.

    2012-07-01

    Recently Alexander and Gulyaev have suggested that the apparent decrease in impact broadening of radio recombination lines seen at high principal quantum number n may be a product of the data reduction process, possibly resulting from the presence of noise on the telescope spectra that is not present on the calculated comparison spectra. This is an interesting proposal. However, there are serious problems with their analysis that need to be pointed out. Perhaps the most important of these is the fact that for principal quantum numbers below n=200, where the widths are not in question, their processed generated profile widths do not fit the widths of the processed lines obtained at the telescope. After processing, the halfwidths of the generated and telescope profiles must agree below n=200 if we are to believe that the processed generated linewidths above n=200 are meaningful. Theirs do not. Furthermore, we find that after applying the linewidth reduction factors found by Alexander and Gulyaev for their noise added profiles to our generated profiles to simulate their noise adding effect, the processed widths we obtain still do not come close to explaining the narrowing seen in the telescope lines for n values in the range 200technique instead of simply a further manipulation of the frequency-switched data.

  10. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. A new model for volume recombination in plane-parallel chambers in pulsed fields of high dose-per-pulse

    Science.gov (United States)

    Gotz, M.; Karsch, L.; Pawelke, J.

    2017-11-01

    In order to describe the volume recombination in a pulsed radiation field of high dose-per-pulse this study presents a numerical solution of a 1D transport model of the liberated charges in a plane-parallel ionization chamber. In addition, measurements were performed on an Advanced Markus ionization chamber in a pulsed electron beam to obtain suitable data to test the calculation. The experiment used radiation pulses of 4 μs duration and variable dose-per-pulse values up to about 1 Gy, as well as pulses of variable duration up to 308 μs at constant dose-per-pulse values between 85 mGy and 400 mGy. Those experimental data were compared to the developed numerical model and existing descriptions of volume recombination. At low collection voltages the observed dose-per-pulse dependence of volume recombination can be approximated by the existing theory using effective parameters. However, at high collection voltages large discrepancies are observed. The developed numerical model shows much better agreement with the observations and is able to replicate the observed behavior over the entire range of dose-per-pulse values and collection voltages. Using the developed numerical model, the differences between observation and existing theory are shown to be the result of a large fraction of the charge being collected as free electrons and the resultant distortion of the electric field inside the chamber. Furthermore, the numerical solution is able to calculate recombination losses for arbitrary pulse durations in good agreement with the experimental data, an aspect not covered by current theory. Overall, the presented numerical solution of the charge transport model should provide a more flexible tool to describe volume recombination for high dose-per-pulse values as well as for arbitrary pulse durations and repetition rates.

  12. Relativistic, QED and nuclear effects in highly charged ions revealed by resonant electron-ion recombination in storage rings

    OpenAIRE

    Schippers, Stefan

    2008-01-01

    Dielectronic recombination (DR) of few-electron ions has evolved into a sensitive spectroscopic tool for highly charged ions. This is due to technological advances in electron-beam preparation and ion-beam cooling techniques at heavy-ion storage rings. Recent experiments prove unambiguously that DR collision spectroscopy has become sensitive to 2nd order QED and to nuclear effects. This review discusses the most recent developments in high-resolution spectroscopy of low-energy DR resonances, ...

  13. High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.

    Science.gov (United States)

    Wang, Qi; Liu, Yongdong; Zhang, Chun; Guo, Fangxia; Feng, Cui; Li, Xiunan; Shi, Hong; Su, Zhiguo

    2017-05-01

    Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. High recombination between two physically close human basement membrane collagen genes at the distal end of chromosome 13q

    Energy Technology Data Exchange (ETDEWEB)

    Bowcock, A.M.; Hebert, J.M.; Wijsman, E.; Gadi, I.; Cavalli-Sforza, L.L.; Boyd, C.D.

    1988-04-01

    Two basement membrane collagen genes coding for the pro..cap alpha..1 chain and pro..cap alpha..2 chain of type IV collagen map to 13q34 and are linked with a maximum likelihood estimate of recombination of 0.028 at a logarithm of odds (lod) score of 19.98. The single-copy sequence that identifies the locus D13S3 is also closely linked to both collagen genes. Four enzymes reveal polymorphisms with COL4A1, and 10 haplotypes have been observed in Caucasoids. Within COL4A1 a nonrandom association and alleles exists only between alleles defined by Hae III and those defined by the other three enzymes. A random association of alleles of COL4A1 and COL4A2 is observed. Between the two collagen genes were detected three meiotic recombination events that contributed to the estimate of 2.8% recombination. This is higher than expected for two genes that lie within 650 kilobases of each other. The lack of linkage disequilibrium between COL4A1 and COL4A2 is in agreement with the relatively high recombination that is observed.

  15. Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yin, Yi; Petes, Thomas D

    2013-10-01

    In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

  16. Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yi Yin

    2013-10-01

    Full Text Available In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs. Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH. In this study, LOH events induced by ultraviolet (UV light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

  17. Characterization and high expression of recombinant Ustilago maydis xylanase in Pichia pastoris.

    Science.gov (United States)

    Han, Hongjuan; You, Shuang; Zhu, Bo; Fu, Xiaoyan; Sun, Baihui; Qiu, Jin; Yu, Chengye; Chen, Lei; Peng, Rihe; Yao, Quanhong

    2015-03-01

    A recombinant xylanase gene (rxynUMB) from Ustilago maydis 521 was expressed in Pichia pastoris, and the enzyme was purified and characterized. Phylogenetic analysis demonstrated that rxynUMB belongs to glycosyl hydrolase family 11. The Trp84, Trp95, Glu93, and Glu189 residues are proposed to be present at the active site. The apparent molecular mass of the recombinant xylananse was approximately 24 kDa, and the optimum pH and temperature were 4.3 and 50 °C, respectively. Xylanase activity was enhanced by 166 and 115% with Fe(2+) and Mn(2+), respectively. The biochemical properties of this recombinant xylanase suggest that it may be a useful candidate for a variety of commercial applications.

  18. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Blanca Iglesias-Figueroa

    2016-06-01

    Full Text Available In this study, bovine lactoferrin (bLf, an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin demonstrated antibacterial activity against Escherichia coli (E. coli BL21DE3, Staphylococcus aureus (S. aureus FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

  19. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    Science.gov (United States)

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

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

    Science.gov (United States)

    Hansen, Kenneth K.; Madsen, Lars Bojer

    2017-07-01

    We present a study of nonsequential double-recombination (NSDR) high-harmonic generation (HHG) in a molecularlike system. We have calculated the HHG spectrum for a wide range of internuclear distances, and using a Coulomb-corrected three-step model we are able to analyze and predict the observed NSDR HHG cutoffs precisely for all internuclear distances. It is shown through this Coulomb-corrected three-step (CC-TSM) model that there is an intrinsic dependence on the location of the nuclei in the NSDR HHG process not seen in one-electron HHG. This dependence originates from the strong electron correlation in the NSDR HHG process, and it modifies the classically allowed return energies which in return changes the cutoffs observed in the HHG spectra. It is observed that the CC-TSM correctly predicts cutoffs at all internuclear distances with differences of more than six harmonics being observed between the CC-TSM and the normal three-step model for the laser parameters used. We also observe that the NSDR HHG process changes for internuclear distances of R ≳8 -9 a.u., which is proposed to stem from a change in the charge-transfer dynamics within the molecule. For large internuclear distances of R ≳13 a.u., we observe a clear signature of the point of emission for the first electron emitted in the NSDR HHG signal and we also see signs of molecular exchange paths contributing to the HHG spectrum for these internuclear distances.

  1. High Dose, Prolonged Epsilon Aminocaproic Acid Infusion, and Recombinant Factor VII for Massive Postoperative Retroperitoneal Hemorrhage following Splenectomy

    Directory of Open Access Journals (Sweden)

    Alex T. Lee

    2016-01-01

    Full Text Available The antifibrinolytic agent ε-aminocaproic acid is used to decrease procedural blood loss in a variety of high risk surgeries. The utility of recombinant factor VII administration in massive hemorrhage has also been reported in a variety of settings, though the impact in a surgical context remains unclear. We describe the case of a patient who underwent massive open splenectomy and developed diffuse retroperitoneal bleeding on postoperative day one. Massive transfusion was initiated, but attempts to control hemorrhage with surgical and interventional radiology approaches were unsuccessful, as was recombinant factor VII administration. Commencement of a high dose aminocaproic acid infusion was followed by a prominent rise in fibrinogen levels and stabilization of the hemorrhage. Indications, dosages, and adverse effects of ε-aminocaproic acid as described in the literature are reviewed.

  2. High Dose, Prolonged Epsilon Aminocaproic Acid Infusion, and Recombinant Factor VII for Massive Postoperative Retroperitoneal Hemorrhage following Splenectomy.

    Science.gov (United States)

    Lee, Alex T; Barnes, Christopher R; Jain, Shweta; Pauldine, Ronald

    2016-01-01

    The antifibrinolytic agent ε -aminocaproic acid is used to decrease procedural blood loss in a variety of high risk surgeries. The utility of recombinant factor VII administration in massive hemorrhage has also been reported in a variety of settings, though the impact in a surgical context remains unclear. We describe the case of a patient who underwent massive open splenectomy and developed diffuse retroperitoneal bleeding on postoperative day one. Massive transfusion was initiated, but attempts to control hemorrhage with surgical and interventional radiology approaches were unsuccessful, as was recombinant factor VII administration. Commencement of a high dose aminocaproic acid infusion was followed by a prominent rise in fibrinogen levels and stabilization of the hemorrhage. Indications, dosages, and adverse effects of ε -aminocaproic acid as described in the literature are reviewed.

  3. New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration

    DEFF Research Database (Denmark)

    Martin, L.; Ashenden, M; Bejder, Jacob

    2016-01-01

    To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity of...

  4. High density recombinant AAV particles are competent vectors for in vivo transduction

    Science.gov (United States)

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  5. Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons

    Science.gov (United States)

    Esteves, Kévin; Mosser, Thomas; Aujoulat, Fabien; Hervio-Heath, Dominique; Monfort, Patrick; Jumas-Bilak, Estelle

    2015-01-01

    Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk. PMID:26236294

  6. Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Julie Bomholt

    Full Text Available In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

  7. Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse

    OpenAIRE

    Nemoto, Masaaki; Mito, Toshiaki; Brinigar, William S; Fronticelli, Clara; Koehler, Raymond C.

    2006-01-01

    Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human α-subunit and the bovine β-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of ~3 Torr, and a cooperativity of n = 1.0. Anesthetized...

  8. Metabolic profiling of recombinant Escherichia coli cultivations based on high-throughput FT-MIR spectroscopic analysis.

    Science.gov (United States)

    Sales, Kevin C; Rosa, Filipa; Cunha, Bernardo R; Sampaio, Pedro N; Lopes, Marta B; Calado, Cecília R C

    2017-03-01

    Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:285-298, 2017. © 2016 American Institute of Chemical Engineers.

  9. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-09-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Sustainable farming: why we need to diversify.

    Science.gov (United States)

    van Dijk, Jan

    2014-06-28

    In the future, the UK farming industry will have to meet increased demands against a background of higher levels of uncertainty. argues that solely promoting the intensification of current farming systems is a high-risk strategy, whereas diversification of farming systems offers sustainability as well as opportunities for vets. British Veterinary Association.

  11. Diversifying Kinesiology: Untapped Potential of Historically Black Colleges and Universities

    Science.gov (United States)

    DiGiacinto, Kacey

    2014-01-01

    Increasing the involvement of African Americans in the field of kinesiology has been an ever present issue. While many colleges and universities are making attempts to diversify their undergraduate students, graduate students, and faculty, many are finding this a difficult task due to the lack of minority undergraduate kinesiology majors pursuing…

  12. Public Access ICT across Cultures: Diversifying Participation in the ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    2015-06-01

    Jun 1, 2015 ... Public Access ICT across Cultures: Diversifying Participation in the Network Society. Public Access ICT across ... Jordan, Rwanda). The book documents the impacts of public access, positive and negative, on individuals, society and networks, and women, and examines the policy implications of findings.

  13. Public Access ICT across Cultures : Diversifying Participation in the ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Public Access ICT across Cultures : Diversifying Participation in the Network Society. Couverture du livre Public Access ICT across Cultures. Directeur(s) : Francisco J. Proenza. Maison(s) d'édition : MIT Press, CRDI. 1 juin 2015. ISBN : 9780262527378. 472 pages. e-ISBN : 9781552505694. Téléchargez le PDF.

  14. Cooperative Learning: A Diversified Pedagogy for Diverse Classrooms

    Science.gov (United States)

    Sharan, Yael

    2010-01-01

    As a generic and diversified pedagogy, cooperative learning (CL) reaches out to the field of intercultural education with an offer to establish a reciprocal relationship. After a short description of the diversity of CL and a brief exploration of the influence that culture has on learning, this paper depicts how the partnership between CL and…

  15. Beginning the Program. Project DEEP (Diversified Educational Experiences Program).

    Science.gov (United States)

    Connett, Jane; And Others

    Project DEEP (Diversified Educational Experience Program) was developed to improve the behavior and attitudes of secondary students in schools where dropouts, absenteeism, and poor attitudes are existing problems. The open classroom with student involvement and participation in goal setting, presentation, and evaluation is the basic concept of…

  16. Cooperative Work Education in Diversified Occupations. General Related Curriculum Guide.

    Science.gov (United States)

    Brysgel, David

    This guide is intended as a working document for coordinators of cooperative work education in diversified occupations. It contains materials for use in providing students with general classroom instruction concurrent with on-the-job training. The following topics are covered in the individual units in the first-year curriculum: the cooperative…

  17. Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus.

    Science.gov (United States)

    Monazahian, M; Kippenberger, S; Müller, A; Seitz, H; Böhme, I; Grethe, S; Thomssen, R

    2000-06-01

    Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03-1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (El/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the El gene product (amino acids 222-336) and the C-terminal part of the E2 protein (amino acids 523-809). Lipoproteins did not bind to recombinant HCV core protein.

  18. Diversifying selection and color-biased dispersal in the asp viper.

    Science.gov (United States)

    Dubey, Sylvain; Zwahlen, Valérie; Mebert, Konrad; Monney, Jean-Claude; Golay, Philippe; Ott, Thomas; Durand, Thierry; Thiery, Gilles; Kaiser, Laura; Geser, Sylvia N; Ursenbacher, Sylvain

    2015-05-31

    The presence of intraspecific color polymorphism can have multiple impacts on the ecology of a species; as a consequence, particular color morphs may be strongly selected for in a given habitat type. For example, the asp viper (Vipera aspis) shows a high level of color polymorphism. A blotched morph (cryptic) is common throughout its range (central and western Europe), while a melanistic morph is frequently found in montane populations, presumably for thermoregulatory reasons. Besides, rare atypical uniformly colored individuals are known here and there. Nevertheless, we found in a restricted treeless area of the French Alps, a population containing a high proportion (>50%) of such specimens. The aim of the study is to bring insight into the presence and function of this color morph by (i) studying the genetic structure of these populations using nine microsatellite markers, and testing for (ii) a potential local diversifying selection and (iii) differences in dispersal capacity between blotched and non-blotched vipers. Our genetic analyses support the occurrence of local diversifying selection for the non-blotched phenotype. In addition, we found significant color-biased dispersal, blotched individuals dispersing more than atypical individuals. We hypothesize that, in this population, the non-blotched phenotype possess an advantage over the typical one, a phenomenon possibly due to a better background matching ability in a more open habitat. In addition, color-biased dispersal might be partly associated with the observed local diversifying selection, as it can affect the genetic structure of populations, and hence the distribution of color morphs.

  19. A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats.

    Science.gov (United States)

    Widjojoatmodjo, Myra N; Boes, Jolande; van Bers, Marleen; van Remmerden, Yvonne; Roholl, Paul J M; Luytjes, Willem

    2010-06-02

    Respiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (DeltaG) were constructed based on a clinical RSV isolate (strain 98-25147-X). Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for DeltaG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days. Collectively, the data indicate that a single dose immunization with the highly attenuated DeltaG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since DeltaG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

  20. Norovirus recombination

    National Research Council Canada - National Science Library

    Bull, Rowena A; Tanaka, Mark M; White, Peter A

    2007-01-01

    ...{at}unsw.edu.au RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV...

  1. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...... and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes....

  2. Genetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output

    Directory of Open Access Journals (Sweden)

    Michael M. Shavlovsky, PhD, ScD¹

    2012-03-01

    Full Text Available We investigated the possibility of obtaining native recombinant amyloidogenic proteins by creating genetic constructs encoding fusion proteins of target proteins with Super Folder Green Fluorescent Protein (sfGFP. In this study, we show that the structures, containing the sfGFP gene, provide a synthesis, within a bacterial system, of fusion proteins with minimal formation of inclusion bodies. Constructs containing genes of the target proteins in the 3'-terminal region of the sfGFP gene followed by a polynucleotide sequence, which allows for affinity purification fusion proteins, are optimal. Heating bacterial cultures before the induction of the expression of recombinant genes in 42°С for 30 min (heat shock was found to increase the output of the desired products, thus practically avoiding the formation of insoluble aggregates

  3. Genetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output

    OpenAIRE

    Michael M. Shavlovsky, PhD, ScD¹; Irina V. Morozova¹; Dmitry S. Polyakov, PhD¹; Tatyana D. Aleynikova, PhD¹; Anna M. Kern²; Natalya A. Grudinina, PhD¹; Kirill V. Solovyov, PhD¹

    2012-01-01

    We investigated the possibility of obtaining native recombinant amyloidogenic proteins by creating genetic constructs encoding fusion proteins of target proteins with Super Folder Green Fluorescent Protein (sfGFP). In this study, we show that the structures, containing the sfGFP gene, provide a synthesis, within a bacterial system, of fusion proteins with minimal formation of inclusion bodies. Constructs containing genes of the target proteins in the 3'-terminal region of the sfGFP gene follo...

  4. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    Science.gov (United States)

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-03

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

  5. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

    National Research Council Canada - National Science Library

    Ukkonen, Kaisa; Vasala, Antti; Ojamo, Heikki; Neubauer, Peter

    2011-01-01

    ...®) and high-aeration shake flask (Ultra Yield Flask™). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli...

  6. The genetic structure of recombinant inbred mice: high-resolution consensus maps for complex trait analysis.

    Science.gov (United States)

    Williams, R W; Gu, J; Qi, S; Lu, L

    2001-01-01

    Recombinant inbred (RI) strains of mice are an important resource used to map and analyze complex traits. They have proved particularly effective in multidisciplinary genetic studies. Widespread use of RI strains has been hampered by their modest numbers and by the difficulty of combining results derived from different RI sets. We have increased the density of typed microsatellite markers two- to five-fold in each of several major RI sets that share C57BL/6 as a parental strain (AXB, BXA, BXD, BXH and CXB). A common set of 490 markers was genotyped in just over 100 RI strains. Genotypes of around 1,100 additional microsatellites in one or more RI sets were generated, collected and checked for errors. Consensus RI maps that integrate genotypes of approximately 1,600 microsatellite loci were assembled. The genomes of individual strains typically incorporate 45-55 recombination breakpoints. The collected RI set - termed the BXN set - contains approximately 5,000 breakpoints. The distribution of recombinations approximates a Poisson distribution and distances between breakpoints average about 0.5 centimorgans (cM). Locations of most breakpoints have been defined with a precision of Hardy-Weinberg equilibrium in only a small number of intervals. Consensus maps derived from RI strains conform almost exactly to theoretical expectation and are close to the length predicted by the Haldane-Waddington equation (x3.6 for a 2-3 cM interval between markers). Non-syntenic associations between different chromosomes introduce predictable distortions in quantitative trait locus (QTL) datasets that can be partly corrected using two-locus correlation matrices.

  7. Development of Highly Functional Biomaterials by Decoupling and Recombining Material Properties.

    Science.gov (United States)

    Danoux, Charlène; Sun, Lanying; Koçer, Gülistan; Birgani, Zeinab Tahmasebi; Barata, David; Barralet, Jake; van Blitterswijk, Clemens; Truckenmüller, Roman; Habibovic, Pamela

    2016-03-02

    Development of functional biomaterials by a design-driven approach is described, whereby individual properties are first decoupled to investigate their sole effects on a biological process. Following this investigation, they are recombined in such a way that the overall performance and applicability of the biomaterial is improved. This is in contrast to classical, processing-driven biomaterials development where the properties of a material are mainly determined by the possibilities of the technique used to produce it. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. China’s diversifying demand for housing for the elderly

    OpenAIRE

    Jia, Min; Heath, Timothy

    2016-01-01

    Purpose: This paper aims to examine the trends among the elderly population in China about residential preferences and policy applications, as the elderly is a rapidly expanding demographic group that has increasing and diversifying inclinations for demanding the residential facilities for the elderly (RFEs) now and in the foreseeable future. Design/methodology/approach: Based on a review of the existing literature and policies, a model is conceptualised for understanding the demands of th...

  9. Creating mutual identification and solidarity in highly diversified ...

    African Journals Online (AJOL)

    Therefore, we can take a shared national identity as one of the building blocks of the welfare state. However, we argue that a shared cultural or civic national identity can not be a necessary condition for this sense of belonging together. The mere fact of co-operation and common participation in shared activities and projects ...

  10. Structural and economic dynamics in diversified Italian farms

    Directory of Open Access Journals (Sweden)

    Cristina Salvioni

    2013-12-01

    Full Text Available Objective of this work is to investigate the structural change and economic dynamics of farms pursuing diversification and differentiation strategies in Italy. The analysis was performed on a panel of data built on the basis of information collected by the Italian FADN between 2003-2009. For the purpose of the analysis, we divided the population of commercial Italian farms into a five-fold farm typology based on size and the extent of diversification and differentiation strategies adopted by the farms. In detail, farms are defined as differentiated when they make use of a system of quality certification, while they are defined as diversified when they take up non farming activities (agritourism, social farms etc.. The findings show that conventional farms remain by far the largest category within the population of Italian commercial farms, while only 13% of the total commercial farms are classified as differentiated and/or diversified. Farms adopting product differentiation strategies are found to have an income growth path similar to that of conventional farms. Yet the category of diversified farms is the only one showing an upward trend with regard to income per worker in the observed years, while farms relying entirely on agricultural products appear to perform poorly in terms of labour productivity.

  11. A DIVERSIFIED DEEP BELIEF NETWORK FOR HYPERSPECTRAL IMAGE CLASSIFICATION

    Directory of Open Access Journals (Sweden)

    P. Zhong

    2016-06-01

    Full Text Available In recent years, researches in remote sensing demonstrated that deep architectures with multiple layers can potentially extract abstract and invariant features for better hyperspectral image classification. Since the usual real-world hyperspectral image classification task cannot provide enough training samples for a supervised deep model, such as convolutional neural networks (CNNs, this work turns to investigate the deep belief networks (DBNs, which allow unsupervised training. The DBN trained over limited training samples usually has many “dead” (never responding or “potential over-tolerant” (always responding latent factors (neurons, which decrease the DBN’s description ability and thus finally decrease the hyperspectral image classification performance. This work proposes a new diversified DBN through introducing a diversity promoting prior over the latent factors during the DBN pre-training and fine-tuning procedures. The diversity promoting prior in the training procedures will encourage the latent factors to be uncorrelated, such that each latent factor focuses on modelling unique information, and all factors will be summed up to capture a large proportion of information and thus increase description ability and classification performance of the diversified DBNs. The proposed method was evaluated over the well-known real-world hyperspectral image dataset. The experiments demonstrate that the diversified DBNs can obtain much better results than original DBNs and comparable or even better performances compared with other recent hyperspectral image classification methods.

  12. Evolution of grain structure and recombination active dislocations in extraordinary tall conventional and high performance multi-crystalline silicon ingots

    Science.gov (United States)

    Trempa, M.; Kupka, I.; Kranert, C.; Lehmann, T.; Reimann, C.; Friedrich, J.

    2017-02-01

    In this work one high performance multi-crystalline silicon ingot and one conventional multi-crystalline silicon ingot, each with an extraordinary ingot height of 710 mm, were replicated by the successive growth of eight G1 ingots to evaluate the potential advantage of extraordinary tall HPM ingots in industrial production. By analyzing different grain structure parameters like mean grain size, grain orientation and grain boundary type distribution as well as the recombination active dislocation area over the complete ingot height, it was observed that the material properties strongly differ in the initial state of growth for the two material types. However, at ingot heights above 350 mm, the difference has vanished and the grain structure properties for both materials appear similar. It is shown that the evolution of the grain structure in both material types can be explained by the same grain selection and grain boundary generation/annihilation mechanisms whereas the current grain structure determines which mechanisms are the most dominant at a specific ingot height. Since the grain structure directly influences the dislocation content in the silicon material, also the recombination active dislocation area becomes equal in high performance and conventional multi-crystalline silicon material at ingot heights above 350 mm. From these results it is concluded that the advantage of high performance silicon material is limited to the first grown 350 mm of the ingot.

  13. Characterization of a Type-Common Human Recombinant Monoclonal Antibody to Herpes Simplex Virus with High Therapeutic Potential

    Science.gov (United States)

    De Logu, Alessandro; Williamson, R. Anthony; Rozenshteyn, Roman; Ramiro-Ibañez, Fernando; Simpson, Cindy D.; Burton, Dennis R.; Paolo Sanna, Pietro

    1998-01-01

    We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody. PMID:9774565

  14. Diversifying selection and host adaptation in two endosymbiont genomes

    Directory of Open Access Journals (Sweden)

    Slatko Barton

    2007-04-01

    Full Text Available Abstract Background The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host:symbiont coevolution. Wolbachia's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, wMel and the nematode Brugia malayi, wBm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts. Results The prevalence of selection was far greater in wMel than wBm. Genes contributing to DNA metabolism, cofactor biosynthesis, and secretion were positively selected in both lineages. In wMel there was a greater emphasis on DNA repair, cell division, protein stability, and cell envelope synthesis. Conclusion Secretion pathways and outer surface protein encoding genes are highly affected by selection in keeping with host:parasite theory. If evidence of selection on various cofactor molecules reflects possible provisioning, then both insect as

  15. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces Cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2014-01-01

    of solutes. Aquaporins constitute a family of physiologically very important integral membrane proteins that are found in all three kingdoms, eubacteria, archaea and eukaryotes. As protein channels, they facilitate passive transport of water across cell membranes. In the present study the yeast Saccharomyces...... cerevisiae was exploited as a host for heterologous expression of human aquaporins. Aquaporin cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human aquaporin was C-terminally tagged with yeast-enhanced GFP to quantify functional expression...... transcription factor and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30oC was due to in vivo mal-folding. Reduction of the expression temperature to 15oC almost completely...

  16. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Hélix-Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tag......In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...

  17. Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization.

    Science.gov (United States)

    Tomitaka, Masataka; Taguchi, Hisataka; Fukuda, Kohsai; Akamatsu, Takashi; Kida, Kenji

    2013-12-01

    A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing-Jing; Ding, Wen-Tao; Zhang, Guo-Chang; Wang, Jing-Yu [Tianjin Univ. (China). Dept. of Biochemical Engineering

    2011-08-15

    Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain's performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches. (orig.)

  19. High CO2 concentration as an inductor agent to drive production of recombinant phytotoxic antimicrobial peptides in plant biofactories.

    Science.gov (United States)

    Ruiz, Cristina; Pla, Maria; Company, Nuri; Riudavets, Jordi; Nadal, Anna

    2016-03-01

    Cationic α-helical antimicrobial peptides such as BP100 are of increasing interest for developing novel phytosanitary or therapeutic agents and products with industrial applications. Biotechnological production of these peptides in plants can be severely impaired due to the toxicity exerted on the host by high-level expression. This can be overcome by using inducible promoters with extremely low activity throughout plant development, although the yields are limited. We examined the use of modified atmospheres using the increased levels of [CO2], commonly used in the food industry, as the inductor agent to biotechnologically produce phytotoxic compounds with higher yields. Here we show that 30% [CO2] triggered a profound transcriptional response in rice leaves, including a change in the energy provision from photosynthesis to glycolysis, and the activation of stress defense mechanisms. Five genes with central roles in up-regulated pathways were initially selected and their promoters successfully used to drive the expression of phytotoxic BP100 in genetically modified (GM) rice. GM plants had a normal phenotype on development and seed production in non-induction conditions. Treatment with 30 % [CO2] led to recombinant peptide accumulation of up to 1 % total soluble protein when the Os.hb2 promoter was used. This is within the range of biotechnological production of other peptides in plants. Using BP100 as a proof-of-concept we demonstrate that very high [CO2] can be considered an economically viable strategy to drive production of recombinant phytotoxic antimicrobial peptides in plant biofactories.

  20. CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

    Science.gov (United States)

    Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

    2017-05-15

    B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  2. High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

    Directory of Open Access Journals (Sweden)

    Jahn Dieter

    2006-11-01

    Full Text Available Abstract Background During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. Results For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. Conclusion The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

  3. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

    Directory of Open Access Journals (Sweden)

    Sergio Alvarez-Pérez

    Full Text Available The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA of four core housekeeping genes (rrs, gyrB, rpoB and rpoD. A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1; P. fluorescens, P. lutea and P. syringae (NG 2; and P. rhizosphaerae (NG 3. Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

  4. Possible diversifying selection in the imprinted gene, MEDEA, in Arabidopsis.

    Science.gov (United States)

    Miyake, Takashi; Takebayashi, Naoki; Wolf, Diana E

    2009-04-01

    Coevolutionary conflict among imprinted genes that influence traits such as offspring growth may arise when maternal and paternal genomes have different evolutionary optima. This conflict is expected in outcrossing taxa with multiple paternity, but not self-fertilizing taxa. MEDEA (MEA) is an imprinted plant gene that influences seed growth. Disagreement exists regarding the type of selection acting on this gene. We present new data and analyses of sequence diversity of MEA in self-fertilizing and outcrossing Arabidopsis and its relatives, to help clarify the form of selection acting on this gene. Codon-based branch analysis among taxa (PAML) suggests that selection on the coding region is changing over time, and nonsynonymous substitution is elevated in at least one outcrossing branch. Codon-based analysis of diversity within outcrossing Arabidopsis lyrata ssp. petraea (OmegaMap) suggests that diversifying selection is acting on a portion of the gene, to cause elevated nonsynonymous polymorphism. Providing further support for balancing selection in A. lyrata, Hudson, Kreitman and Aguadé analysis indicates that diversity/divergence at silent sites in the MEA promoter and genic region is elevated relative to reference genes, and there are deviations from the neutral frequency spectrum. This combination of positive selection as well as balancing and diversifying selection in outcrossing lineages is consistent with other genes influence by evolutionary conflict, such as disease resistance genes. Consistent with predictions that conflict would be eliminated in self-fertilizing taxa, we found no evidence of positive, balancing, or diversifying selection in A. thaliana promoter or genic region.

  5. Decentralizing conservation and diversifying livelihoods within Kanchenjunga Conservation Area, Nepal.

    Science.gov (United States)

    Parker, Pete; Thapa, Brijesh; Jacob, Aerin

    2015-12-01

    To alleviate poverty and enhance conservation in resource dependent communities, managers must identify existing livelihood strategies and the associated factors that impede household access to livelihood assets. Researchers increasingly advocate reallocating management power from exclusionary central institutions to a decentralized system of management based on local and inclusive participation. However, it is yet to be shown if decentralizing conservation leads to diversified livelihoods within a protected area. The purpose of this study was to identify and assess factors affecting household livelihood diversification within Nepal's Kanchenjunga Conservation Area Project, the first protected area in Asia to decentralize conservation. We randomly surveyed 25% of Kanchenjunga households to assess household socioeconomic and demographic characteristics and access to livelihood assets. We used a cluster analysis with the ten most common income generating activities (both on- and off-farm) to group the strategies households use to diversify livelihoods, and a multinomial logistic regression to identify predictors of livelihood diversification. We found four distinct groups of household livelihood strategies with a range of diversification that directly corresponded to household income. The predictors of livelihood diversification were more related to pre-existing socioeconomic and demographic factors (e.g., more landholdings and livestock, fewer dependents, receiving remittances) than activities sponsored by decentralizing conservation (e.g., microcredit, training, education, interaction with project staff). Taken together, our findings indicate that without direct policies to target marginalized groups, decentralized conservation in Kanchenjunga will continue to exclude marginalized groups, limiting a household's ability to diversify their livelihood and perpetuating their dependence on natural resources. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Processing of recombinant Listeria monocytogenes proteins for MHC class I presentation follows a dedicated, high-efficiency pathway

    Science.gov (United States)

    Wolf, Benjamin J.; Princiotta, Michael F.

    2013-01-01

    CD8+ T lymphocytes recognize short peptides of ~8–10 amino acids bound to MHC class I molecules (pMHC) on the surface of antigen presenting cells. These peptides can be generated from either endogenous proteins synthesized by the biosynthetic machinery of the presenting cell or from exogenously sourced proteins. Because much of the research characterizing the MHC class I processing pathway has focused on endogenously synthesized proteins, it is not known whether differences exist in the processing pathway followed by endogenously synthesized versus exogenously sourced proteins. To highlight potential differences in the processing of endogenous versus exogenous proteins, we developed a model system to measure the efficiency of pMHC generation from nearly identical recombinant proteins expressed from vaccinia virus and Listeria monocytogenes. In these experiments, we uncovered a striking difference in the way recombinant Listeria antigens are processed and presented when compared to endogenously synthesized viral proteins. Specifically, we find that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived, whereas the rate of pMHC production from endogenously synthesized viral proteins is absolutely dependent on its protein half-life. Accordingly, our data demonstrate the existence of a distinct and highly efficient MHC class I presentation pathway used for the processing of at least some exogenously synthesized proteins. PMID:23396941

  7. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni2+. Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Zinc tin oxide as high-temperature stable recombination layer for mesoscopic perovskite/silicon monolithic tandem solar cells

    KAUST Repository

    Werner, Jérémie

    2016-12-05

    Perovskite/crystalline silicon tandem solar cells have the potential to reach efficiencies beyond those of silicon single-junction record devices. However, the high-temperature process of 500 °C needed for state-of-the-art mesoscopic perovskite cells has, so far, been limiting their implementation in monolithic tandem devices. Here, we demonstrate the applicability of zinc tin oxide as a recombination layer and show its electrical and optical stability at temperatures up to 500 °C. To prove the concept, we fabricate monolithic tandem cells with mesoscopic top cell with up to 16% efficiency. We then investigate the effect of zinc tin oxide layer thickness variation, showing a strong influence on the optical interference pattern within the tandem device. Finally, we discuss the perspective of mesoscopic perovskite cells for high-efficiency monolithic tandem solar cells. © 2016 Author(s)

  9. Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

    Science.gov (United States)

    Schembri, Laura; Zanese, Marion; Depierre-Plinet, Gaelle; Petit, Muriel; Elkaoukabi-Chaibi, Assia; Tauzin, Loic; Florean, Cristina; Lartigue, Lydia; Medina, Chantal; Rey, Christophe; Belloc, Francis; Reiffers, Josy; Ichas, François; De Giorgi, Francesca

    2009-12-01

    Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

  10. High quality maize centromere 10 sequence reveals evidence of frequent recombination events

    Directory of Open Access Journals (Sweden)

    Thomas Kai Wolfgruber

    2016-03-01

    Full Text Available The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR have presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 x 10-6 and 5 x 10-5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb of the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length centromeric retrotransposons from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. This repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to facilitate the repair of frequent DSBs in centromeres.

  11. Enhanced alcohol self-administration and reinstatement in a highly impulsive, inattentive recombinant inbred mouse strain

    Directory of Open Access Journals (Sweden)

    Maarten eLoos

    2013-10-01

    Full Text Available Deficits in executive control have frequently been associated with alcohol use disorder. Here we investigated to what extent pre-existing genetically encoded levels of impulsive/inattentive behavior associate with motivation to take alcohol and vulnerability to cue-induced reinstatement of alcohol seeking in an operant self-administration paradigm. We took advantage of BXD16, a recombinant inbred strain previously shown to have enhanced impulsivity and poor attentional control. We compared BXD16 with C57BL/6J mice in a simple choice reaction time task (SCRTT and confirmed its impulsive/inattentive phenotype. BXD16 mice were less active in a novel open field, and were equally active in an automated home cage environment, showing that increased impulsive responding of BXD16 mice could not be explained by enhanced general activity compared to C57BL/6J mice. After training in a sucrose/alcohol fading self-administration procedure, BXD16 showed increased motivation to earn 10% alcohol solution, both under fixed ratio (FR1 and progressive ratio (PR2 schedules of reinforcement. Responding on the active lever readily decreased during extinction training with no apparent differences between strains. However, upon re-exposure to alcohol-associated cues, alcohol seeking was reinstated to a larger extent in BXD16 than in C57BL/6J mice. Although further studies are needed to determine whether impulsivity/inattention and alcohol seeking depend on common or separate genetic loci, these data show that in mice enhanced impulsivity coincides with increased motivation to take alcohol, as well as relapse vulnerability.

  12. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    Science.gov (United States)

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    Science.gov (United States)

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Transgenes sustain epigeal insect biodiversity in diversified vegetable farm systems.

    Science.gov (United States)

    Leslie, T W; Hoheisel, G A; Biddinger, D J; Rohr, J R; Fleischer, S J

    2007-02-01

    Many ecological studies have focused on the effects of transgenes in field crops, but few have considered multiple transgenes in diversified vegetable systems. We compared the epigeal, or soil surface-dwelling, communities of Coleoptera and Formicidae between transgenic and isoline vegetable systems consisting of sweet corn, potato, and acorn squash, with transgenic cultivars expressing Cry1(A)b, Cry3, or viral coat proteins. Vegetables were grown in replicated split plots over 2 yr with integrated pest management (IPM) standards defining insecticide use patterns. More than 77.6% of 11,925 insects from 1,512 pitfall traps were identified to species, and activity density was used to compare dominance distribution, species richness, and community composition. Measures of epigeal biodiversity were always equal in transgenic vegetables, which required fewer insecticide applications than their near isolines. There were no differences in species richness between transgenic and isoline treatments at the farm system and individual crop level. Dominance distributions were also similar between transgenic and isoline farming systems. Crop type, and not genotype, had a significant influence on Carabidae and Staphylinidae community composition in the first year, but there were no treatment effects in the second year, possibly because of homogenizing effects of crop rotations. Communities were more influenced by crop type, and possibly crop rotation, than by genotype. The heterogeneity of crops and rotations in diversified vegetable farms seems to aid in preserving epigeal biodiversity, which may be supplemented by reductions in insecticide use associated with transgenic cultivars.

  15. Relationship between gene duplicability and diversifiability in the topology of biochemical networks.

    Science.gov (United States)

    Guo, Zhanyong; Jiang, Wen; Lages, Nuno; Borcherds, Wade; Wang, Degeng

    2014-07-08

    Selective gene duplicability, the extensive expansion of a small number of gene families, is universal. Quantitatively, the number of genes (P(K)) with K duplicates in a genome decreases precipitously as K increases, and often follows a power law (P(k)∝k-α). Functional diversification, either neo- or sub-functionalization, is a major evolution route for duplicate genes. Using three lines of genomic datasets, we studied the relationship between gene duplicability and diversifiability in the topology of biochemical networks. First, we explored scenario where two pathways in the biochemical networks antagonize each other. Synthetic knockout of respective genes for the two pathways rescues the phenotypic defects of each individual knockout. We identified duplicate gene pairs with sufficient divergences that represent this antagonism relationship in the yeast S. cerevisiae. Such pairs overwhelmingly belong to large gene families, thus tend to have high duplicability. Second, we used distances between proteins of duplicate genes in the protein interaction network as a metric of their diversification. The higher a gene's duplicate count, the further the proteins of this gene and its duplicates drift away from one another in the networks, which is especially true for genetically antagonizing duplicate genes. Third, we computed a sequence-homology-based clustering coefficient to quantify sequence diversifiability among duplicate genes - the lower the coefficient, the more the sequences have diverged. Duplicate count (K) of a gene is negatively correlated to the clustering coefficient of its duplicates, suggesting that gene duplicability is related to the extent of sequence divergence within the duplicate gene family. Thus, a positive correlation exists between gene diversifiability and duplicability in the context of biochemical networks - an improvement of our understanding of gene duplicability.

  16. RNA recombination in Porcine Reproductive and Respiratory Syndrome Virus is restricted to parental sequences with high similarity

    DEFF Research Database (Denmark)

    Vugt, J.J.F.A. van; Storgaard, T.; Oleksiewicz, M. B.

    2001-01-01

    Two types of porcine reproductive and respiratory syndrome virus (PRRSV) exist, a North American type and a European type. The co-existence of both types in some countries, such as Denmark, Slovakia and Canada, creates a risk of inter-type recombination. To evaluate this risk, cell cultures were co......, but no recombination was detected between the European and North American types. Calculation of the maximum theoretical risk of European–American recombination, based on the sensitivity of the RT–PCR system, revealed that RNA recombination between the European and North American types of PRRSV is at least 10000 times...

  17. Recombination monitor

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-02-03

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

  18. [Comparative multicenter study of a rabbit high-sensitivity thromboplastin and a recombinant thromboplastin with synthetic phospholipids].

    Science.gov (United States)

    Martínez-Brotóns, F; Borrell, M; Fontcuberta, J; Batlle, J; López, F; Páramo, J A; Ribera, C; Rocha, E; Vicente, V; Zuazu, I

    1994-08-01

    The purpose of the present study was to compare the results obtained with a human recombinant thromboplastin (Innovin, Baxter) (IN) and a high-sensitivity rabbit brain reagent (Thromboplastin IS, Baxter) (IS), on the performance of prothrombin time (PT) test and the functional assay of factors included in the extrinsic coagulation system, in order to establish possible differences on imprecision, diagnostic accuracy and sensitivity to the oral anticoagulant defect, between the two products. Six Spanish hospital took part in the study. Plasma samples from 221 healthy subjects, 100 patients with severe liver disease, 27 with dysfibrinogenaemia, 10 with lupus anticoagulant and from 13 individuals propositus and their relatives with congenital deficiencies of the extrinsic coagulation pathway, and their relatives were studied; 188 patients stabilized on oral anticoagulant therapy and 82 on heparin therapy were also included. The in vitro effect of heparin was tested by addition of increasing amounts of heparin (0.3 to 10.0 IU/mL) to aliquots of normal plasma. Both in the intra-assay and in the inter-assay imprecision study, a better coefficient of variation was obtained with IN when the PT was performed on abnormal samples. Prothrombin time ratio from patients with liver disease had significantly higher values with IS. On the contrary, IN had a higher sensitivity in samples from patients with dysfibrinogenaemia or from those stabilized on oral anticoagulant therapy. In showed a very low sensitivity to heparin at concentrations corresponding to the therapeutic range. The results of this field study indicate that IN, compared with a high-sensitivity rabbit brain thromboplastin, is a suitable reagent for PT determination in normal subjects, patients with liver disease or with congenital deficiencies of clotting factors. It shows a higher sensitivity in cases of dysfibrinogenaemia and in patients on oral anticoagulant therapy. In addition, the recombinant reagent had

  19. Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.

    Science.gov (United States)

    Zhang, Ningning; Dolan, Maureen; Wu, Di; Phillips, Gregory C; Xu, Jianfeng

    2016-12-01

    Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)32 tag (32 tandem repeats of "Ser-Pro" motif). The (SP)32 tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

  20. High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

    Science.gov (United States)

    Saez, Natalie J; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-07-30

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  1. High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

    Science.gov (United States)

    Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

    2016-07-30

    Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

  2. Expression of Recombinant Antibodies

    OpenAIRE

    André eFrenzel; Michael eHust; Thomas eSchirrmann

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  3. Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

    DEFF Research Database (Denmark)

    Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

    2003-01-01

    The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

  4. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    Directory of Open Access Journals (Sweden)

    Yao Nie

    successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

  5. Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

    Science.gov (United States)

    Dietzel, E; Floehr, J; Van de Leur, E; Weiskirchen, R; Jahnen-Dechent, W

    2017-01-01

    Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. The astacin

  6. Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes.

    Directory of Open Access Journals (Sweden)

    Ditte Hededam Welner

    Full Text Available Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated

  7. Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164.

    Science.gov (United States)

    Sacchi, Veronica; Mittermayr, Rainer; Hartinger, Joachim; Martino, Mikaël M; Lorentz, Kristen M; Wolbank, Susanne; Hofmann, Anna; Largo, Remo A; Marschall, Jeffrey S; Groppa, Elena; Gianni-Barrera, Roberto; Ehrbar, Martin; Hubbell, Jeffrey A; Redl, Heinz; Banfi, Andrea

    2014-05-13

    Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-PI1-8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentrations ≥25 μg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 μg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-PI1-8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

  8. Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces.

    Science.gov (United States)

    Liu, Leqian; Redden, Heidi; Alper, Hal S

    2013-12-01

    Microbial systems provide an attractive, renewable route to produce desired organic molecules such as fuels and chemicals. While attention within the field of metabolic engineering has mostly focused on Escherichia coli, yeast is a potent host and growing host for industrial products and has many outstanding, biotechnologically desirable native traits. Thus, there has been a recent shift in focus toward yeast as production hosts to replace E. coli. As such, products have diversified in yeast beyond simply ethanol. Additionally, nonconventional yeasts have been considered to move beyond Saccharomyces cerevisiae. This review highlights recent advances in metabolic engineering of yeasts for producing value-added chemical compounds including alcohols, sugar derivatives, organic acids, fats, terpenes, aromatics, and polyketides. Furthermore, we will also discuss the future direction of metabolic engineering of yeasts. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Diversified microbiota of meconium is affected by maternal diabetes status.

    Directory of Open Access Journals (Sweden)

    Jianzhong Hu

    Full Text Available This study was aimed to assess the diversity of the meconium microbiome and determine if the bacterial community is affected by maternal diabetes status.The first intestinal discharge (meconium was collected from 23 newborns stratified by maternal diabetes status: 4 mothers had pre-gestational type 2 diabetes mellitus (DM including one mother with dizygotic twins, 5 developed gestational diabetes mellitus (GDM and 13 had no diabetes. The meconium microbiome was profiled using multi-barcode 16S rRNA sequencing followed by taxonomic assignment and diversity analysis.All meconium samples were not sterile and contained diversified microbiota. Compared with adult feces, the meconium showed a lower species diversity, higher sample-to-sample variation, and enrichment of Proteobacteria and reduction of Bacteroidetes. Among the meconium samples, the taxonomy analyses suggested that the overall bacterial content significantly differed by maternal diabetes status, with the microbiome of the DM group showing higher alpha-diversity than that of no-diabetes or GDM groups. No global difference was found between babies delivered vaginally versus via Cesarean-section. Regression analysis showed that the most robust predictor for the meconium microbiota composition was the maternal diabetes status that preceded pregnancy. Specifically, Bacteroidetes (phyla and Parabacteriodes (genus were enriched in the meconium in the DM group compared to the no-diabetes group.Our study provides evidence that meconium contains diversified microbiota and is not affected by the mode of delivery. It also suggests that the meconium microbiome of infants born to mothers with DM is enriched for the same bacterial taxa as those reported in the fecal microbiome of adult DM patients.

  10. Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse.

    Science.gov (United States)

    Nemoto, Masaaki; Mito, Toshiaki; Brinigar, William S; Fronticelli, Clara; Koehler, Raymond C

    2006-05-01

    Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human alpha-subunit and the bovine beta-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of approximately 3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.

  11. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

    2010-06-29

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  12. A SIMPLE WAY OF ACHIEVING A HIGH CELL CONCENTRATION IN RECOMBINANT Escherichia coli CULTIVATION

    Directory of Open Access Journals (Sweden)

    Gombert A.K.

    1997-01-01

    Full Text Available Abstract - A cultivation strategy based on some previous knowledge of the metabolism of Escherichia coli BL21 (DE3 pLysS containing the troponin C gene cloned into plasmid pET was developed and applied through the use of simple fermentation equipment and a feed-forward control strategy in order to achieve a high cell concentration ¾ 92 g l-1 dry cell weight ¾ and a high cell productivity ¾ 3.7 g l-1 h-1.

  13. Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide.

    Science.gov (United States)

    Rothan, Hussin A; Mohamed, Zulqarnain; Suhaeb, Abdulrazzaq M; Rahman, Noorsaadah Abd; Yusof, Rohana

    2013-11-01

    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.

  14. Identification of immunoglobulin V(D)J recombinations in solid tumor specimen exome files: Evidence for high level B-cell infiltrates in breast cancer.

    Science.gov (United States)

    Tong, Wei Lue; Tu, Yaping N; Samy, Mohammad D; Sexton, Wade J; Blanck, George

    2017-03-04

    It has recently become apparent that it is possible to characterize productively recombined, T-cell receptor (TcR) gene segments in tumor exome files, which presumably include representations of the DNA of other cells in the microenvironment. Similar characterizations have been done for TcR recombinations in tumor specimen RNASeq files. While exome files have been used to characterize immunoglobulin gene segment recombinations for tumors closely related to B-cells, immunoglobulin recombinations have yet to be characterized for putative microenvironment cells for solid tumors. Here we report a novel scripted algorithm that detects productive and unproductive immunoglobulin recombinations in both B-cell related tumor exome files and in solid tumor exome files, with the most important result being the relatively high level B-cell infiltrate in breast cancer. This analysis has the potential of streamlining and dramatically augmenting the knowledge base regarding B-cell infiltrates into solid tumors; and leading to antibody reagents directed against tumor antigens and tissue resident, infectious pathogens.

  15. Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar, but geographically remote Planktothrix strains

    Directory of Open Access Journals (Sweden)

    Kristensen Tom

    2008-08-01

    Full Text Available Abstract Background Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera Microcystis, Planktothrix and Anabaena. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s is (are unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected. Results We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS cyanopeptolin gene cluster from the Japanese strain Planktothrix NIES 205 (205-oci, showed the 30 kb gene cluster to be highly similar to the oci gene cluster previously described in Planktothrix NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S and a glyceric acid loading (GA-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, ntcA and the phycocyanin cpcBA spacer were identical, supporting that these geographically separated Planktothrix strains are closely related. Conclusion A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are

  16. TP53 mutations, tetraploidy and homologous recombination repair defects in early stage high-grade serous ovarian cancer

    Science.gov (United States)

    Chien, Jeremy; Sicotte, Hugues; Fan, Jian-Bing; Humphray, Sean; Cunningham, Julie M.; Kalli, Kimberly R.; Oberg, Ann L.; Hart, Steven N.; Li, Ying; Davila, Jaime I.; Baheti, Saurabh; Wang, Chen; Dietmann, Sabine; Atkinson, Elizabeth J.; Asmann, Yan W.; Bell, Debra A.; Ota, Takayo; Tarabishy, Yaman; Kuang, Rui; Bibikova, Marina; Cheetham, R. Keira; Grocock, Russell J.; Swisher, Elizabeth M.; Peden, John; Bentley, David; Kocher, Jean-Pierre A.; Kaufmann, Scott H.; Hartmann, Lynn C.; Shridhar, Viji; Goode, Ellen L.

    2015-01-01

    To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC. PMID:25916844

  17. High-Resolution Mapping of Crossover and Non-crossover Recombination Events by Whole-Genome Re-sequencing of an Avian Pedigree.

    Directory of Open Access Journals (Sweden)

    Linnéa Smeds

    2016-05-01

    Full Text Available Recombination is an engine of genetic diversity and therefore constitutes a key process in evolutionary biology and genetics. While the outcome of crossover recombination can readily be detected as shuffled alleles by following the inheritance of markers in pedigreed families, the more precise location of both crossover and non-crossover recombination events has been difficult to pinpoint. As a consequence, we lack a detailed portrait of the recombination landscape for most organisms and knowledge on how this landscape impacts on sequence evolution at a local scale. To localize recombination events with high resolution in an avian system, we performed whole-genome re-sequencing at high coverage of a complete three-generation collared flycatcher pedigree. We identified 325 crossovers at a median resolution of 1.4 kb, with 86% of the events localized to <10 kb intervals. Observed crossover rates were in excellent agreement with data from linkage mapping, were 52% higher in male (3.56 cM/Mb than in female meiosis (2.28 cM/Mb, and increased towards chromosome ends in male but not female meiosis. Crossover events were non-randomly distributed in the genome with several distinct hot-spots and a concentration to genic regions, with the highest density in promoters and CpG islands. We further identified 267 non-crossovers, whose location was significantly associated with crossover locations. We detected a significant transmission bias (0.18 in favour of 'strong' (G, C over 'weak' (A, T alleles at non-crossover events, providing direct evidence for the process of GC-biased gene conversion in an avian system. The approach taken in this study should be applicable to any species and would thereby help to provide a more comprehensive portray of the recombination landscape across organism groups.

  18. Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

    Science.gov (United States)

    Krüger, Nadine; Sauder, Christian; Hoffmann, Markus; Örvell, Claes; Drexler, Jan Felix; Rubin, Steven; Herrler, Georg

    2016-11-01

    A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

  19. Targeted Recombinant Progeny: a design for ultra-high resolution mapping of Quantitative Trait Loci in crosses between inbred or pure lines.

    Science.gov (United States)

    Heifetz, Eliyahu M; Soller, Morris

    2015-07-07

    High-resolution mapping of the loci (QTN) responsible for genetic variation in quantitative traits is essential for positional cloning of candidate genes, and for effective marker assisted selection. The confidence interval (QTL) flanking the point estimate of QTN-location is proportional to the number of individuals in the mapping population carrying chromosomes recombinant in the given interval. Consequently, many designs for high resolution QTN mapping are based on increasing the proportion of recombinants in the mapping population. The "Targeted Recombinant Progeny" (TRP) design is a new design for high resolution mapping of a target QTN in crosses between pure, or inbred lines. It is a three-generation procedure generating a large number of recombinant individuals within a QTL previously shown to contain a QTN. This is achieved by having individuals that carry chromosomes recombinant across the target QTL interval as parents of a large mapping population; most of whom will therefore carry recombinant chromosomes targeted to the given QTL. The TRP design is particularly useful for high resolution mapping of QTN that differentiate inbred or pure lines, and hence are not amenable to high resolution mapping by genome-wide association tests. In the absence of residual polygenic variation, population sizes required for achieving given mapping resolution by the TRP-F2 design relative to a standard F2 design ranged from 0.289 for a QTN with standardized allele substitution effect = 0.2, mapped to an initial QTL of 0.2 Morgan to 0.041 for equivalent QTN mapped to an initial QTL of 0.02 M. In the presence of residual polygenic variation, the relative effectiveness of the TRP design ranges from 1.068 to 0.151 for the same initial QTL intervals and QTN effect. Thus even in the presence of polygenic variation, the TRP can still provide major savings. Simulation showed that mapping by TRP should be based on 30-50 markers spanning the initial interval; and on at least 50 or

  20. Subcutaneous absorption kinetics of two highly concentrated preparations of recombinant human growth hormone

    DEFF Research Database (Denmark)

    Laursen, Torben; Jørgensen, Jens Otto Lunde; Susgaard, Søren

    1993-01-01

    hours. Samples were taken every 30 minutes for 6 hours and then hourly. MAIN OUTCOME MEASURES: Bioavailability (F) and absorption dynamics of human GH were measured. The relative absorption fractions estimated from the areas under the individual serum concentration curves from 0 to 24 hours......Abstract OBJECTIVE: The relative bioavailability of two highly concentrated (12 IU/ml) formulations of biosynthetic human growth hormone (GH) administered subcutaneously was compared. DESIGN: A randomized, crossover study. Conventional GH therapy was withdrawn 72 hours before each study period...... of signs of endogenous GH secretion. INTERVENTIONS: At the start of each study period, GH 3 IU/m2 was injected subcutaneously. The two formulations, PenFill and PenSet, differ in the buffers used and in the relative content of mannitol and glycine. Serum profiles of GH were monitored frequently for 24...

  1. Gettering centres in high-energy ion-implanted silicon investigated by point defect recombination

    Science.gov (United States)

    Kögler, R.; Peeva, A.; Werner, P.; Skorupa, W.; Gösele, U.

    2001-04-01

    Self-interstitials were introduced by additional Si + implantation into the vacancy-dominated depth range around half of the projected ion range, RP/2, of high-energy ion-implanted Si in order to balance radiation-induced excess vacancies. The undesired gettering of Cu atoms in this region ( RP/2 effect) could be suppressed. The threshold was determined necessary to remove the Cu gettering at RP/2. It does approximately agree with the number of the calculated excess vacancies. Additional interstitial-type dislocation loops were formed during annealing at RP/2 as the Si + fluence exceeds this threshold. Interstitial clusters were not proven to be the gettering centres for Cu trapping.

  2. Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

    OpenAIRE

    Yin, Yi; Thomas D Petes

    2013-01-01

    In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells s...

  3. Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

    OpenAIRE

    Yi Yin; Thomas D Petes

    2013-01-01

    In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells s...

  4. Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry.

    Science.gov (United States)

    Okumura, Takeshi; Masuda, Kenji; Watanabe, Kazuhiko; Miyadai, Kenji; Nonaka, Koichi; Yabuta, Masayuki; Omasa, Takeshi

    2015-09-01

    To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Huang Yadong

    2010-02-01

    Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21 is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21 in Escherichia coli (E. coli is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier by polymerase chain reaction (PCR, and expressed the fused gene in E. coli BL21(DE3. Results By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC was shown to be higher than 96% with low endotoxin level (in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ injection. Conclusions This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity.

    Science.gov (United States)

    Tomasevic, Nenad; Luehrsen, Kenneth; Baer, Mark; Palath, Varghese; Martinez, David; Williams, Jason; Yi, Christina; Sujatha-Bhaskar, Swathi; Lanke, Rohini; Leung, John; Ching, Wendy; Lee, Andreia; Bai, Lu; Yarranton, Geoffrey; Bebbington, Christopher

    2014-12-01

    EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

  8. New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica.

    Science.gov (United States)

    VanDrisse, C M; Escalante-Semerena, J C

    2016-07-01

    Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.

    Science.gov (United States)

    Narasimhan Janakiraman, Vignesh; Noubhani, Abdelmajid; Venkataraman, Krishnan; Vijayalakshmi, Mookambeswaran; Santarelli, Xavier

    2016-01-01

    A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.

    Science.gov (United States)

    Cipáková, Ingrid; Hostinová, Eva; Gasperík, Juraj; Velebný, Vladimír

    2004-09-01

    In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.

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

    Science.gov (United States)

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

    2015-11-20

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

  12. Hard X-ray polarimetry with position sensitve germanium detectors. Studies of the recombination transitions into highly charged ions

    Energy Technology Data Exchange (ETDEWEB)

    Tashenov, Stanislav

    2005-07-01

    In this work a first study of the photon polarization for the process of radiative recombination has been performed. This was done at the ESR storage ring at GSI for uranium ions colliding with N2 at various collision energies. For this measurement a high purity Ge Pixel Detector with a 4 x 4 segmentation matrix was applied. The investigation was performed at the Gas-jet target of the ESR. The detector was placed at 60 and 90 observation angles. The sensitivity of the Compton scattering effect to the linear polarization of the X-Ray radiation was employed for the polarization measurement. Detailed investigations of the scattering and geometrical effects inside the detector were performed in order to develop a method to interpret the experimental data and extract the degree of the linear polarization in the hard X-Ray regime with a high precision. A special emphasis was given to the geometry of the detector and it's influence on the measured pixel-to-pixel Compton scattering intensities. The developed method enabled to achieve a precision of the order of 10% with the Pixel Detector which is dominated by the statistical uncertainties. The obtained results show a good agreement with the theoretical values derived from the exact relativistic calculations. For the case of the linear polarization of the K-REC photons, the measured data con rm the theoretical prediction that strong depolarization effects occur for high projectile charges in the forward hemisphere. The latter is in disagreement with the nonrelativistic theory which predicts a 100 % polarization regardless of the emission angle. (orig.)

  13. High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein.

    Science.gov (United States)

    Hao, Jing; Xu, Li; He, Hongde; Du, Xiaojun; Jia, Lingyun

    2013-08-01

    Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS-PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. 76 FR 51375 - Dialogues in Diversifying Clinical Trials: Successful Strategies for Engaging Women and...

    Science.gov (United States)

    2011-08-18

    ... Diversifying Clinical Trials: Successful Strategies for Engaging Women and Minorities in Clinical Trials. The... HUMAN SERVICES Food and Drug Administration Dialogues in Diversifying Clinical Trials: Successful Strategies for Engaging Women and Minorities in Clinical Trials AGENCY: Food and Drug Administration, HHS...

  15. AACP Special Taskforce on Diversifying Our Investment in Human Capital Interim Update.

    Science.gov (United States)

    White, Carla; Adams, Jennifer

    2016-09-25

    The 2015-2017 AACP Special Taskforce on Diversifying our Investment in Human Capital was appointed for a two-year term, therefore the interim update from the Taskforce. A full report will be provided in 2017 in the form of a white paper for academic pharmacy on diversifying our investment in human capital.

  16. Effect of operating segments to the firm value of diversified listed companies in the Philippines

    Directory of Open Access Journals (Sweden)

    William T. Sucuahi

    2016-01-01

    Full Text Available A growing number of studies produced different result on whether diversification can create or destroy value. The objective of this study is to determine if the operating segments can predict the firm value of the publiclylisted diversified companies. Tobin’s Q was used as a proxy of firm value. This study used regression analysis to evaluate if number of operating segments and type of business engaged can affect the Tobin’s Q of diversified firms. Using 86 diversified listed companies the study shows that majority of the diversified firms have Tobin’s Q lesser than one. Diversified firms without real estate and banking in their segments have better firm value than those who have. The value that was created by diversification will depend on what type of operating segment the company engaged and not on the number of segments they operate.

  17. A novel line immunoassay based on recombinant virulence factors enables highly specific and sensitive serologic diagnosis of Helicobacter pylori infection.

    Science.gov (United States)

    Formichella, Luca; Romberg, Laura; Bolz, Christian; Vieth, Michael; Geppert, Michael; Göttner, Gereon; Nölting, Christina; Walter, Dirk; Schepp, Wolfgang; Schneider, Arne; Ulm, Kurt; Wolf, Petra; Busch, Dirk H; Soutschek, Erwin; Gerhard, Markus

    2013-11-01

    Helicobacter pylori colonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.

  18. Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.

    Science.gov (United States)

    Rengby, Olle; Johansson, Linda; Carlson, Lars A; Serini, Elena; Vlamis-Gardikas, Alexios; Kårsnäs, Per; Arnér, Elias S J

    2004-09-01

    The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.

  19. High Expression of Human Cathepsin S by Recombinant Pichia pastoris with Cod Skin as an Organic Co-Nitrogen Source.

    Science.gov (United States)

    Li, Guiying Y; Fu, Man; Qin, Mei; Xue, Liming M

    2018-02-06

    Human cathepsin S production by recombinant Pichia pastoris using cod skin as the co-nitrogen source was investigated in this study. The addition of carbon sources of glycerol in the fed-batch phase and of methanol in the induction stage was also investigated. A new approach to the highly expression of human cathepsin S was developed using 90 g/L of cod skin (wet weight). After 24 h of the initial fermentation, 4% glycerol (v/v, glycerol/culture) was added once to enhance the cell density (OD600) in the cultivation. Then, adding and maintaining methanol at 0.5% (v/v, methanol/cultivation) after about 48 h of fermentation achieved a high expression of human cathepsin S in a 5-L bioreactor. The results demonstrate that the maximum activity of human cathepsin S in the fermentation supernatant reached 7,152 U/L after 96 h of methanol induction. The methylotrophic yeast P. pastoris grown in the medium containing cod skin (90 g/L) as the co-nitrogen source provided a 21% higher cell density (OD600) and 18.3% higher human cathepsin S yield than P. pastoris grown in BMGY medium. For the first time, human cathepsin S was successfully expressed by P. pastoris with cod skin as the co-nitrogen source. The glycerol fed-batch controlling strategy and method of maintaining methanol at a constant concentration of 0.5% (v/v, methanol/cultivation) in the induction stage was efficient for P. pastoris growth and the expression of human cathepsin S. © 2018 S. Karger AG, Basel.

  20. Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens.

    Science.gov (United States)

    Park, Chang-Su; Park, Chul-Soon; Shin, Kyung-Chul; Oh, Deok-Kun

    2016-02-01

    The specific activity of recombinant Escherichia coli cells expressing the double-site variant (I33L-S213C) d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was highest at 24 h of cultivation time in Terrific Broth (TB) medium among the media tested. The contents of crude protein and DPEase in recombinant cells at 24 h were 37.0 and 8.6% (w/w), respectively, indicating that the enzyme was highly expressed. The reaction conditions for the production of d-psicose from d-fructose by whole recombinant cells with the highest specific activity were optimal at 60°C, pH 8.5, 4 g/l cells, and 700 g/l d-fructose. Under these conditions, whole recombinant cells produced 230 g/l d-psicose after 40 min, with a conversion yield of 33% (w/w), a volumetric productivity of 345 g/l/h, and a specific productivity of 86.2 g/g/h. These are the highest conversion yield and volumetric and specific productivities of d-psicose from d-fructose by cells reported thus far. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Development of an industrial medium and a novel fed-batch strategy for high-level expression of recombinant β-mananase by Pichia pastoris.

    Science.gov (United States)

    Zheng, Jia; Zhao, Wei; Guo, Ning; Lin, Fulai; Tian, Jian; Wu, Lishuang; Zhou, Hongbo

    2012-08-01

    An industrial medium, Corn Steep Liquor Powder Dextrose (CSD medium) was developed for constitutive expression of recombinant β-mananase by Pichia pastoris. The β-mananase activity (513 U/mL) with CSD medium was 1.64- and 2.5-fold higher than with YPD and BSM in shaken flasks. The β-mananase productivity with CSD medium was 61.0 U/mL h, which was 1.7- and 2.5-fold higher than with YPD and BSM in a 5-L fermenter based on a novel fed-batch strategy combining the real-time exponential feed mode with the DO-stat feed mode. The β-mananase activity, dry cell weight and the recombinant enzyme reached up to 5132 U/mL, 110.0 g/L and 4.50 g/L after 50 h cultivation in a 50-L fermenter. The high efficient expression of recombinant β-mananase by P. pastoris indicated that CSD medium and the novel fed-batch strategy have great potential for the production of recombinant β-mananase in industrial fermentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

    Science.gov (United States)

    Liu, Zhao; Wang, Dexing; Feng, Jiuhuan; Seiler, Gerald J.; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf1 is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf6, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf6 was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf6 was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes. PMID:23307903

  3. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  4. Gateway Recombinational Cloning.

    Science.gov (United States)

    Reece-Hoyes, John S; Walhout, Albertha J M

    2018-01-02

    The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel (e.g., in 96-well formats) in a standardized manner using the same enzymes. Gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the Escherichia coli genome. Because the sites of recombination (" att " sites) are much longer (25-242 bp) than restriction sites, they are extremely unlikely to occur by chance in DNA fragments. Therefore, the same recombination enzyme can be used to robustly clone many different fragments of variable size in parallel reactions. © 2018 Cold Spring Harbor Laboratory Press.

  5. A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

    Science.gov (United States)

    Seeber, Stefan; Ros, Francesca; Thorey, Irmgard; Tiefenthaler, Georg; Kaluza, Klaus; Lifke, Valeria; Fischer, Jens André Alexander; Klostermann, Stefan; Endl, Josef; Kopetzki, Erhard; Pashine, Achal; Siewe, Basile; Kaluza, Brigitte; Platzer, Josef; Offner, Sonja

    2014-01-01

    We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal. PMID:24503933

  6. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Science.gov (United States)

    Halbherr, Stefan J; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  7. The rate of nonallelic homologous recombination in males is highly variable, correlated between monozygotic twins and independent of age.

    Directory of Open Access Journals (Sweden)

    Jacqueline A L MacArthur

    2014-03-01

    Full Text Available Nonallelic homologous recombination (NAHR between highly similar duplicated sequences generates chromosomal deletions, duplications and inversions, which can cause diverse genetic disorders. Little is known about interindividual variation in NAHR rates and the factors that influence this. We estimated the rate of deletion at the CMT1A-REP NAHR hotspot in sperm DNA from 34 male donors, including 16 monozygotic (MZ co-twins (8 twin pairs aged 24 to 67 years old. The average NAHR rate was 3.5 × 10(-5 with a seven-fold variation across individuals. Despite good statistical power to detect even a subtle correlation, we observed no relationship between age of unrelated individuals and the rate of NAHR in their sperm, likely reflecting the meiotic-specific origin of these events. We then estimated the heritability of deletion rate by calculating the intraclass correlation (ICC within MZ co-twins, revealing a significant correlation between MZ co-twins (ICC = 0.784, p = 0.0039, with MZ co-twins being significantly more correlated than unrelated pairs. We showed that this heritability cannot be explained by variation in PRDM9, a known regulator of NAHR, or variation within the NAHR hotspot itself. We also did not detect any correlation between Body Mass Index (BMI, smoking status or alcohol intake and rate of NAHR. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of NAHR and are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion.

  8. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Stefan J Halbherr

    Full Text Available Highly pathogenic avian influenza viruses (HPAIV of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade. Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  9. Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

    Science.gov (United States)

    Chapat, Clément; Chettab, Kamel; Simonet, Pierre; Wang, Peng; De La Grange, Pierre; Le Romancer, Muriel; Corbo, Laura

    2017-08-21

    The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Population structure, migration, and diversifying selection in the Netherlands

    Science.gov (United States)

    Abdellaoui, Abdel; Hottenga, Jouke-Jan; Knijff, Peter de; Nivard, Michel G; Xiao, Xiangjun; Scheet, Paul; Brooks, Andrew; Ehli, Erik A; Hu, Yueshan; Davies, Gareth E; Hudziak, James J; Sullivan, Patrick F; van Beijsterveldt, Toos; Willemsen, Gonneke; de Geus, Eco J; Penninx, Brenda W J H; Boomsma, Dorret I

    2013-01-01

    Genetic variation in a population can be summarized through principal component analysis (PCA) on genome-wide data. PCs derived from such analyses are valuable for genetic association studies, where they can correct for population stratification. We investigated how to capture the genetic population structure in a well-characterized sample from the Netherlands and in a worldwide data set and examined whether (1) removing long-range linkage disequilibrium (LD) regions and LD-based SNP pruning significantly improves correlations between PCs and geography and (2) whether genetic differentiation may have been influenced by migration and/or selection. In the Netherlands, three PCs showed significant correlations with geography, distinguishing between: (1) North and South; (2) East and West; and (3) the middle-band and the rest of the country. The third PC only emerged with minimized LD, which also significantly increased correlations with geography for the other two PCs. In addition to geography, the Dutch North–South PC showed correlations with genome-wide homozygosity (r=0.245), which may reflect a serial-founder effect due to northwards migration, and also with height (♂: r=0.142, ♀: r=0.153). The divergence between subpopulations identified by PCs is partly driven by selection pressures. The first three PCs showed significant signals for diversifying selection (545 SNPs - the majority within 184 genes). The strongest signal was observed between North and South for the functional SNP in HERC2 that determines human blue/brown eye color. Thus, this study demonstrates how to increase ancestry signals in a relatively homogeneous population and how those signals can reveal evolutionary history. PMID:23531865

  11. Genome-wide signatures of population bottlenecks and diversifying selection in European wolves.

    Science.gov (United States)

    Pilot, M; Greco, C; vonHoldt, B M; Jędrzejewska, B; Randi, E; Jędrzejewski, W; Sidorovich, V E; Ostrander, E A; Wayne, R K

    2014-04-01

    Genomic resources developed for domesticated species provide powerful tools for studying the evolutionary history of their wild relatives. Here we use 61K single-nucleotide polymorphisms (SNPs) evenly spaced throughout the canine nuclear genome to analyse evolutionary relationships among the three largest European populations of grey wolves in comparison with other populations worldwide, and investigate genome-wide effects of demographic bottlenecks and signatures of selection. European wolves have a discontinuous range, with large and connected populations in Eastern Europe and relatively smaller, isolated populations in Italy and the Iberian Peninsula. Our results suggest a continuous decline in wolf numbers in Europe since the Late Pleistocene, and long-term isolation and bottlenecks in the Italian and Iberian populations following their divergence from the Eastern European population. The Italian and Iberian populations have low genetic variability and high linkage disequilibrium, but relatively few autozygous segments across the genome. This last characteristic clearly distinguishes them from populations that underwent recent drastic demographic declines or founder events, and implies long-term bottlenecks in these two populations. Although genetic drift due to spatial isolation and bottlenecks seems to be a major evolutionary force diversifying the European populations, we detected 35 loci that are putatively under diversifying selection. Two of these loci flank the canine platelet-derived growth factor gene, which affects bone growth and may influence differences in body size between wolf populations. This study demonstrates the power of population genomics for identifying genetic signals of demographic bottlenecks and detecting signatures of directional selection in bottlenecked populations, despite their low background variability.

  12. Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

    Directory of Open Access Journals (Sweden)

    Maryam Zangi

    2016-07-01

    Full Text Available Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP when compared to the absence of P19 suppressor (0.2%TSP. Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

  13. A SNP based high-density linkage map of Apis cerana reveals a high recombination rate similar to Apis mellifera.

    Science.gov (United States)

    Shi, Yuan Yuan; Sun, Liang Xian; Huang, Zachary Y; Wu, Xiao Bo; Zhu, Yong Qiang; Zheng, Hua Jun; Zeng, Zhi Jiang

    2013-01-01

    The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee. F2 workers (N = 103) were genotyped for 126,990 single nucleotide polymorphisms (SNPs). After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM) with the largest linkage group (180 loci) measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM. We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.

  14. A SNP based high-density linkage map of Apis cerana reveals a high recombination rate similar to Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Yuan Yuan Shi

    Full Text Available BACKGROUND: The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee. RESULTS: F2 workers (N = 103 were genotyped for 126,990 single nucleotide polymorphisms (SNPs. After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM with the largest linkage group (180 loci measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM. CONCLUSION: We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.

  15. Understanding Charge Transport and Recombination Losses in High Performance Polymer Solar Cells with Non-Fullerene Acceptors

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xuning; Zuo, Xiaobing; Xie, Shenkun; Yuan, Jianyu; Zhou, Huiqiong; Zhang, Yuan

    2017-09-07

    The photovoltaic performance of organic solar cells can be enhanced by achieving a fundamental understanding of the key processes that govern the device behaviour. In this work, we comprehensively investigate temperature (T)-dependent charge transport, non-geminate recombination losses and intermolecular stacking based on three representative organic bulk heterojunction (BHJ) solar cells comprising the polymeric donor of PBDB-T blended with non-fullerene small molecule ITIC and polymeric P(NDI2OD-T2) alongside PC71BM acceptors. Surprisingly, the champion solar cell based on PBDB-T: ITIC, even though exhibiting the most imbalanced transport, produces the highest PCE approaching 10%. We find that such an imbalance is in association with the decrease in the recombination reduction factor with respect to the Langevin rate constant. This beneficially leads to mitigated non-geminate recombination and gains in photoconductivity. In contrast, the all-polymer solar cell using the P(NDI2OD-T2) acceptor displays an excellent balance in mobility while suffering from a more substantial recombination, which causes severe carrier losses and reduced photocurrent. T-dependent mobility measurements indicate that the activation energy for the transport in these BHJ films is low (50-150 meV) which is rationalized by the preferential out-of-plane intermolecular pi-pi stacking mainly adopted by the donor molecules. The combined results point to an indication that the electron mobility in non-fullerene acceptors may not be a severe restraint while charge recombination losses play a critical role in ultimate photovoltaic characteristics based on these emerging materials

  16. Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

    Science.gov (United States)

    Rastawicki, Waldemar; Smietafiska, Karolina; Chrost, Anna; Wolkowicz, Tomasz; Rokosz-Chudziak, Natalia

    Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to

  17. Dielectronic recombination in He-like, Li-like, and Be-like highly charged ions in the KLL and KLM manifolds

    Science.gov (United States)

    Kavanagh, A. P.; Watanabe, H.; Li, Y. M.; O'Rourke, B. E.; Tobiyama, H.; Nakamura, N.; McMahon, S.; Yamada, C.; Ohtani, S.; Currell, F. J.

    2010-02-01

    This paper reports a systematic study of the dependence on atomic number of the dielectronic recombination resonance strengths for He-like, Li-like and Be-like ions. Recent measurements of dielectronic recombination resonance strengths for the KLL and KLM manifolds for iron, yttrium, iodine, holmium, and bismuth are also described. The resonance strengths were normalized to calculated electron impact ionization cross sections. The measured resonance strengths generally agree well with theoretical calculations using the distorted wave approximation. However, KLM resonance strength measurements on high atomic number open-shell ions gave higher values than those suggested by calculations. Using recently measured data, along with existing results, scaling laws have been generated as a function of atomic number for He-like, Li-like, and Be-like ions in the KLL and KLM manifolds.

  18. Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein.

    Science.gov (United States)

    Chowdhury, Anandã; Brinson, Robert; Wei, Beiyang; Stetler-Stevenson, William G

    2017-12-12

    Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC50 of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.

  19. Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

    Science.gov (United States)

    Odunsi, Kunle; Matsuzaki, Junko; Karbach, Julia; Neumann, Antje; Mhawech-Fauceglia, Paulette; Miller, Austin; Beck, Amy; Morrison, Carl D; Ritter, Gerd; Godoy, Heidi; Lele, Shashikant; duPont, Nefertiti; Edwards, Robert; Shrikant, Protul; Old, Lloyd J; Gnjatic, Sacha; Jäger, Elke

    2012-04-10

    Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

  20. Recombinant high-density lipoprotein nanoparticles containing gadolinium-labeled cholesterol for morphologic and functional magnetic resonance imaging of the liver.

    Science.gov (United States)

    Rui, Mengjie; Guo, Wei; Ding, Qian; Wei, Xiaohui; Xu, Jianrong; Xu, Yuhong

    2012-01-01

    Natural high-density lipoproteins (HDL) possess important physiological functions to the transport of cholesterol from the peripheral tissues to the liver for metabolic degradation and excretion in the bile. In this work, we took advantage of this pathway and prepared two different gadolinium (Gd)-DTPA-labeled cholesterol-containing recombinant HDL nanoparticles (Gd-chol-HDL) and Gd-(chol)(2)-HDL as liver-specific magnetic resonance imaging (MRI) contrast agents. The reconstituted HDL nanoparticles had structural similarity to native HDL, and could be taken up by HepG2 cells via interaction with HDL receptors in vitro. In vivo MRI studies in rats after intravenous injections of 10 μmol gadolinium per kg of recombinant HDL nanoparticles indicated that both nanoparticles could provide signal enhancement in the liver and related organs. However, different T(1)-weighted image details suggested that they participated in different cholesterol metabolism and excretion pathways in the liver. Such information could be highly useful to differentiate functional changes as well as anatomic differences in the liver. These cholesterol-derived contrast agents and their recombinant HDL preparations may warrant further development as a new class of contrast agents for MRI of the liver and related organs.

  1. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    Science.gov (United States)

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

  2. AACP Special Taskforce White Paper on Diversifying Our Investment in Human Capital

    National Research Council Canada - National Science Library

    Carla White; Jeannine M Conway; Paula K Davis; Arcelia M Johnson-Fannin; Jeffrey G Jurkas; Nanci L Murphy; W Thomas Smith; Margarita Echeverri; Sharon L Youmans; Katie C Owings; Jennifer L Adamsk

    2017-01-01

    The 2015-2017 American Association of Colleges of Pharmacy (AACP) Special Taskforce on Diversifying our Investment in Human Capital was appointed for a two-year term, due to the rigors and complexities of its charges...

  3. A high-resolution genetic map of yellow monkeyflower identifies chemical defense QTLs and recombination rate variation.

    Science.gov (United States)

    Holeski, Liza M; Monnahan, Patrick; Koseva, Boryana; McCool, Nick; Lindroth, Richard L; Kelly, John K

    2014-03-13

    Genotyping-by-sequencing methods have vastly improved the resolution and accuracy of genetic linkage maps by increasing both the number of marker loci as well as the number of individuals genotyped at these loci. Using restriction-associated DNA sequencing, we construct a dense linkage map for a panel of recombinant inbred lines derived from a cross between divergent ecotypes of Mimulus guttatus. We used this map to estimate recombination rate across the genome and to identify quantitative trait loci for the production of several secondary compounds (PPGs) of the phenylpropanoid pathway implicated in defense against herbivores. Levels of different PPGs are correlated across recombinant inbred lines suggesting joint regulation of the phenylpropanoid pathway. However, the three quantitative trait loci identified in this study each act on a distinct PPG. Finally, we map three putative genomic inversions differentiating the two parental populations, including a previously characterized inversion that contributes to life-history differences between the annual/perennial ecotypes. Copyright © 2014 Holeski et al.

  4. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

    Science.gov (United States)

    Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

    2017-08-01

    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

  5. High-throughput quantitation of Fc-containing recombinant proteins in cell culture supernatant by fluorescence polarization spectroscopy.

    Science.gov (United States)

    Thompson, Ben; Clifford, Jerry; Jenns, Mike; Smith, Andrew; Field, Ray; Nayyar, Kalpana; James, David C

    2017-10-01

    Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case. Copyright © 2017. Published by Elsevier Inc.

  6. Ecosystem Services in Biologically Diversified versus Conventional Farming Systems: Benefits, Externalities, and Trade-Offs

    Directory of Open Access Journals (Sweden)

    Claire Kremen

    2012-12-01

    Full Text Available We hypothesize that biological diversification across ecological, spatial, and temporal scales maintains and regenerates the ecosystem services that provide critical inputs - such as maintenance of soil quality, nitrogen fixation, pollination, and pest control - to agriculture. Agrobiodiversity is sustained by diversified farming practices and it also supplies multiple ecosystem services to agriculture, thus reducing environmental externalities and the need for off-farm inputs. We reviewed the literature that compares biologically diversified farming systems with conventional farming systems, and we examined 12 ecosystem services: biodiversity; soil quality; nutrient management; water-holding capacity; control of weeds, diseases, and pests; pollination services; carbon sequestration; energy efficiency and reduction of warming potential; resistance and resilience to climate change; and crop productivity. We found that compared with conventional farming systems, diversified farming systems support substantially greater biodiversity, soil quality, carbon sequestration, and water-holding capacity in surface soils, energy-use efficiency, and resistance and resilience to climate change. Relative to conventional monocultures, diversified farming systems also enhance control of weeds, diseases, and arthropod pests and they increase pollination services; however, available evidence suggests that these practices may often be insufficient to control pests and diseases or provide sufficient pollination. Significantly less public funding has been applied to agroecological research and the improvement of diversified farming systems than to conventional systems. Despite this lack of support, diversified farming systems have only somewhat reduced mean crop productivity relative to conventional farming systems, but they produce far fewer environmental and social harms. We recommend that more research and crop breeding be conducted to improve diversified farming

  7. Ultra-High Density, Transcript-Based Genetic Maps of Pepper Define Recombination in the Genome and Synteny Among Related Species.

    Science.gov (United States)

    Hill, Theresa; Ashrafi, Hamid; Chin-Wo, Sebastian Reyes; Stoffel, Kevin; Truco, Maria-Jose; Kozik, Alexander; Michelmore, Richard; Van Deynze, Allen

    2015-09-08

    Our ability to assemble complex genomes and construct ultradense genetic maps now allows the determination of recombination rates, translocations, and the extent of genomic collinearity between populations, species, and genera. We developed two ultradense genetic linkage maps for pepper from single-position polymorphisms (SPPs) identified de novo with a 30,173 unigene pepper genotyping array. The Capsicum frutescens × C. annuum interspecific and the C. annuum intraspecific genetic maps were constructed comprising 16,167 and 3,878 unigene markers in 2108 and 783 genetic bins, respectively. Accuracies of marker groupings and orders are validated by the high degree of collinearity between the two maps. Marker density was sufficient to locate the chromosomal breakpoint resulting in the P1/P8 translocation between C. frutescens and C. annuum to a single bin. The two maps aligned to the pepper genome showed varying marker density along the chromosomes. There were extensive chromosomal regions with suppressed recombination and reduced intraspecific marker density. These regions corresponded to the pronounced nonrecombining pericentromeric regions in tomato, a related Solanaceous species. Similar to tomato, the extent of reduced recombination appears to be more pronounced in pepper than in other plant species. Alignment of maps with the tomato and potato genomes shows the presence of previously known translocations and a translocation event that was not observed in previous genetic maps of pepper. Copyright © 2015 Hill et al.

  8. New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

    Science.gov (United States)

    Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

    2012-12-01

    HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

  9. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

    Science.gov (United States)

    2010-01-01

    Background Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures. Results The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 ~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l-1 cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins. Conclusions We have demonstrated that by applying the novel EnBase® Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce

  10. Assignment of resonances in dissociative recombination of HD{sup +} ions: High-resolution measurements compared with accurate computations

    Energy Technology Data Exchange (ETDEWEB)

    Waffeu Tamo, F. O. [Laboratoire Ondes et Milieux Complexes FRE-3102 CNRS and Universite du Havre, 25, rue Philippe Lebon, BP 540, F-76058 Le Havre (France); Centre for Atomic, Molecular Physics and Quantum Optics (CEPAMOQ), University of Douala, P.O. Box 8580, Douala (Cameroon); Laboratoire d' Etude du Rayonnement et de la Matiere en Astrophysique, Observatoire de Paris, F-91295 Meudon Cedex (France); Buhr, H.; Schwalm, D. [Max-Planck-Institut fuer Kernphysik, Saupfercheckweg 1, D-69117 Heidelberg (Germany); Department of Particle Physics, Weizmann Institute of Science, P.O. Box 26, 76100 Rehovot (Israel); Motapon, O. [LPF, UFD Physique et Sciences de l' Ingenieur, University of Douala, P.O. Box 24157, Douala (Cameroon); Altevogt, S.; Andrianarijaona, V. M.; Grieser, M.; Lammich, L.; Lestinsky, M.; Motsch, M.; Novotny, S.; Orlov, D. A.; Pedersen, H. B.; Sprenger, F.; Weigel, U.; Wolf, A. [Max-Planck-Institut fuer Kernphysik, Saupfercheckweg 1, D-69117 Heidelberg (Germany); Nevo, I. [Department of Particle Physics, Weizmann Institute of Science, P.O. Box 26, 76100 Rehovot (Israel); Urbain, X. [Institute of Condensed Matter and Nanosciences, Universite catholique de Louvain, chemin du cyclotron 2, B-1348 Louvain-la Neuve (Belgium); Schneider, I. F. [Laboratoire Ondes et Milieux Complexes FRE-3102 CNRS and Universite du Havre, 25, rue Philippe Lebon, BP 540, F-76058 Le Havre (France)

    2011-08-15

    The collision-energy resolved rate coefficient for dissociative recombination of HD{sup +} ions in the vibrational ground state is measured using the photocathode electron target at the heavy-ion storage ring TSR. Rydberg resonances associated with rovibrational excitation of the HD{sup +} core are scanned as a function of the electron collision energy with an instrumental broadening below 1 meV in the low-energy limit. The measurement is compared to calculations using multichannel quantum defect theory, accounting for rotational structure and interactions and considering the six lowest rotational energy levels as initial ionic states. Using thermal-equilibrium-level populations at 300 K to approximate the experimental conditions, close correspondence between calculated and measured structures is found up to the first vibrational excitation threshold of the cations near 0.24 eV. Detailed assignments, including naturally broadened and overlapping Rydberg resonances, are performed for all structures up to 0.024 eV. Resonances from purely rotational excitation of the ion core are found to have similar strengths as those involving vibrational excitation. A dominant low-energy resonance is assigned to contributions from excited rotational states only. The results indicate strong modifications in the energy dependence of the dissociative recombination rate coefficient through the rotational excitation of the parent ions, and underline the need for studies with rotationally cold species to obtain results reflecting low-temperature ionized media.

  11. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

    Directory of Open Access Journals (Sweden)

    Ramos C.R.R.

    2004-01-01

    Full Text Available We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET, expression of a short 6XHis tag at N-terminus (pET3-His and a high copy number of plasmid (pRSET. The small size of the vector (2.8 kb and the high copy number/cell (200-250 copies facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture. In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site. Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

  12. The proteomic complexity and rise of the primordial ancestor of diversified life

    Directory of Open Access Journals (Sweden)

    Kim Kyung

    2011-05-01

    Full Text Available Abstract Background The last universal common ancestor represents the primordial cellular organism from which diversified life was derived. This urancestor accumulated genetic information before the rise of organismal lineages and is considered to be either a simple 'progenote' organism with a rudimentary translational apparatus or a more complex 'cenancestor' with almost all essential biological processes. Recent comparative genomic studies support the latter model and propose that the urancestor was similar to modern organisms in terms of gene content. However, most of these studies were based on molecular sequences, which are fast evolving and of limited value for deep evolutionary explorations. Results Here we engage in a phylogenomic study of protein domain structure in the proteomes of 420 free-living fully sequenced organisms. Domains were defined at the highly conserved fold superfamily (FSF level of structural classification and an iterative phylogenomic approach was used to reconstruct max_set and min_set FSF repertoires as upper and lower bounds of the urancestral proteome. While the functional make up of the urancestral sets was complex, they represent only 5-11% of the 1,420 FSFs of extant proteomes and their make up and reuse was at least 5 and 3 times smaller than proteomes of free-living organisms, repectively. Trees of proteomes reconstructed directly from FSFs or from molecular functions, which included the max_set and min_set as articial taxa, showed that urancestors were always placed at their base and rooted the tree of life in Archaea. Finally, a molecular clock of FSFs suggests the min_set reflects urancestral genetic make up more reliably and confirms diversified life emerged about 2.9 billion years ago during the start of planet oxygenation. Conclusions The minimum urancestral FSF set reveals the urancestor had advanced metabolic capabilities, was especially rich in nucleotide metabolism enzymes, had pathways for the

  13. Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol.

    Science.gov (United States)

    Yoo, Seung Sik; Park, Sunghoon; Lee, Eun Yeol

    2008-10-01

    The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.

  14. Site directed recombination

    Science.gov (United States)

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  15. A Sequence-Anchored Linkage Map of the Plant–Parasitic Nematode Meloidogyne hapla Reveals Exceptionally High Genome-Wide Recombination

    Science.gov (United States)

    Thomas, Varghese P.; Fudali, Sylwia L.; Schaff, Jennifer E.; Liu, Qingli; Scholl, Elizabeth H.; Opperman, Charles H.; Bird, David McK; Williamson, Valerie M.

    2012-01-01

    Root-knot nematodes (Meloidogyne spp.) cause major yield losses to many of the world’s crops, but efforts to understand how these pests recognize and interact with their hosts have been hampered by a lack of genetic resources. Starting with progeny of a cross between inbred strains (VW8 and VW9) of Meloidogyne hapla that differed in host range and behavioral traits, we exploited the novel, facultative meiotic parthenogenic reproductive mode of this species to produce a genetic linkage map. Molecular markers were derived from SNPs identified between the sequenced and annotated VW9 genome and de novo sequence of VW8. Genotypes were assessed in 183 F2 lines. The colinearity of the genetic and physical maps supported the veracity of both. Analysis of local crossover intervals revealed that the average recombination rate is exceptionally high compared with that in other metazoans. In addition, F2 lines are largely homozygous for markers flanking crossover points, and thus resemble recombinant inbred lines. We suggest that the unusually high recombination rate may be an adaptation to generate within-population genetic diversity in this organism. This work presents the most comprehensive linkage map of a parasitic nematode to date and, together with genomic and transcript sequence resources, empowers M. hapla as a tractable model. Alongside the molecular map, these progeny lines can be used for analyses of genome organization and the inheritance of phenotypic traits that have key functions in modulating parasitism, behavior, and survival and for the eventual identification of the responsible genes. PMID:22870404

  16. Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    Kati, Semra; Hage, Elias; Mynarek, Martin; Ganzenmueller, Tina; Indenbirken, Daniela; Grundhoff, Adam; Schulz, Thomas F

    2015-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposi's sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 10(6) IU/ml in unconcentrated culture supernatants, representing a 10(3)-10(4)-fold improvement compared to conventional methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins

    Directory of Open Access Journals (Sweden)

    Hashimoto Yoshi

    2012-04-01

    Full Text Available Abstract After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50, entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae, is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38 cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.

  18. High-yield production of recombinant virus-like particles of enterovirus 71 in Pichia pastoris and their protective efficacy against oral viral challenge in mice.

    Science.gov (United States)

    Zhang, Chao; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Chen, Tan; Ye, Xiaohua; Li, Dapeng; Jin, Xia; Huang, Zhong

    2015-05-11

    Enterovirus 71 (EV71) is one of the major causative pathogens of hand, foot and mouth disease (HFMD), which is highly prevalent in the Asia-Pacific regions. Severe HFMD cases with neurological complications and even death are often associated with EV71 infections. However, no licensed EV71 vaccine is currently available. Recombinant virus-like particles (VLPs) of EV71 have been produced and shown to be a promising vaccine candidate in preclinical studies. However, the performance of current recombinant expression systems for EV71 VLP production remains unsatisfactory with regard to VLP yield and manufacturing procedure, and thus hinders further product development. In this study, we evaluated the expression of EV71 VLPs in Pichia pastoris and determined their protective efficacy in mouse models of EV71 infections. We showed that EV71 VLPs could be produced at high levels up to 4.9% of total soluble protein in transgenic P. pastoris yeast co-expressing P1 and 3CD proteins of EV71. The resulting yeast-produced VLPs potently induced neutralizing antibodies against homologous and heterologous EV71 strains in mice. More importantly, maternal immunization with VLPs protected neonatal mice in both intraperitoneal and oral challenge experiments. Collectively, these results demonstrated the success of simple, high-yield production of EV71 VLPs in transgenic P. pastoris, thus lifting the major roadblock in commercial development of VLP-based EV71 vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. A Decision Support System for Plant Optimization in Urban Areas with Diversified Solar Radiation

    Directory of Open Access Journals (Sweden)

    Heyi Wei

    2017-02-01

    Full Text Available Sunshine is an important factor which limits the choice of urban plant species, especially in environments with high-density buildings. In practice, plant selection and configuration is a key step of landscape architecture, which has relied on an experience-based qualitative approach. However, the rationality and efficiency of this need to be improved. To maintain the diversity of plant species and to ensure their ecological adaptability (solar radiation in the context of sustainable development, we developed the Urban Plants Decision Support System (UP-DSS for assisting plant selection in urban areas with diversified solar radiation. Our methodology mainly consists of the solar radiation model and calibration, the urban plant database, and information retrieval model. The structure of UP-DSS is also presented at the end of the methodology section, which is based on the platform of Geographic Information Systems (GIS and Microsoft Excel. An application of UP-DSS is demonstrated in a residential area of Wuhan, China. The results show that UP-DSS can provide a very scientific and stable tool for the adaptive planning of shade-tolerant plants and photoperiod-sensitive plants, meanwhile, it also provides a specific plant species and the appropriate types of plant community for user decision-making according to different sunshine radiation conditions and the designer’s preferences.

  20. The Role of Rangelands in Diversified Farming Systems: Innovations, Obstacles, and Opportunities in the USA

    Directory of Open Access Journals (Sweden)

    Nathan F. Sayre

    2012-12-01

    Full Text Available Discussions of diversified farming systems (DFS rarely mention rangelands: the grasslands, shrublands, and savannas that make up roughly one-third of Earth's ice-free terrestrial area, including some 312 million ha of the United States. Although ranching has been criticized by environmentalists for decades, it is probably the most ecologically sustainable segment of the U.S. meat industry, and it exemplifies many of the defining characteristics of DFS: it relies on the functional diversity of natural ecological processes of plant and animal (reproduction at multiple scales, based on ecosystem services generated and regenerated on site rather than imported, often nonrenewable, inputs. Rangelands also provide other ecosystem services, including watershed, wildlife habitat, recreation, and tourism. Even where non-native or invasive plants have encroached on or replaced native species, rangelands retain unusually high levels of plant diversity compared with croplands or plantation forests. Innovations in management, marketing, incentives, and easement programs that augment ranch income, creative land tenure arrangements, and collaborations among ranchers all support diversification. Some obstacles include rapid landownership turnover, lack of accessible U.S. Department of Agriculture certified processing facilities, tenure uncertainty, fragmentation of rangelands, and low and variable income, especially relative to land costs. Taking advantage of rancher knowledge and stewardship, and aligning incentives with production of diverse goods and services, will support the sustainability of ranching and its associated public benefits. The creation of positive feedbacks between economic and ecological diversity should be the ultimate goal.

  1. Assignment of resonances in dissociative recombination of HD+ ions: high-resolution measurements compared with accurate computations

    CERN Document Server

    Tamo, F O Waffeu; Motapon, O; Altevogt, S; Andrianarijaona, V M; Grieser, M; Lammich, L; Lestinsky, M; Motsch, M; Nevo, I; Novotny, S; Orlov, D A; Pedersen, H B; Schwalm, D; Sprenger, F; Urbain, X; Weigel, U; Wolf, A; Schneider, I F

    2011-01-01

    The collision-energy resolved rate coefficient for dissociative recombination of HD+ ions in the vibrational ground state is measured using the photocathode electron target at the heavy-ion storage ring TSR. Rydberg resonances associated with ro-vibrational excitation of the HD+ core are scanned as a function of the electron collision energy with an instrumental broadening below 1 meV in the low-energy limit. The measurement is compared to calculations using multichannel quantum defect theory, accounting for rotational structure and interactions and considering the six lowest rotational energy levels as initial ionic states. Using thermal equilibrium level populations at 300 K to approximate the experimental conditions, close correspondence between calculated and measured structures is found up to the first vibrational excitation threshold of the cations near 0.24 eV. Detailed assignments, including naturally broadened and overlapping Rydberg resonances, are performed for all structures up to 0.024 eV. Resona...

  2. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

    Directory of Open Access Journals (Sweden)

    Marc W Gibson

    2016-05-01

    Full Text Available Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively. All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

  3. High-throughput behavioral phenotyping of drug and alcohol susceptibility traits in the expanded panel of BXD recombinant inbred strains

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Vivek M [ORNL; Ansah, T [University of Tennessee Health Science Center, Memphis; Blaha, C, [University of Tennessee Health Science Center, Memphis; Cook, Melloni N. [University of Memphis; Hamre, Kristin M. [University of Tennessee Health Science Center, Memphis; Lariviere, William R [University of Pittsburgh; Matthews, Douglas B [Baylor University; Goldowitz, Daniel [University of British Columbia, Vancouver; Chesler, Elissa J [ORNL

    2010-01-01

    Genetic reference populations, particularly the BXD recombinant inbred strains, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and co- ariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic co-regulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium have obtained behavioral phenotype data from 260 measures related to multiple behavioral assays across several domains: self-administration, response to, and withdrawal from cocaine, MDMA, morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity; and sleep/wake cycles. All traits have been measured in both sexes and the recently expanded panel of 69 additional BXD recombinant inbred strains (N=69). Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent BXD RI lines was performed. Primary data is publicly available for heritability, sex difference and genetic analyses using www.GeneNetwork.org. These analyses include QTL detection and genetic analysis of gene expression. Stored results from these analyses are available at http://ontologicaldiscovery.org for comparison to other genomic analysis results. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

  4. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

    Science.gov (United States)

    Gibson, Marc W; Dewar, Simon; Ong, Han B; Sienkiewicz, Natasha; Fairlamb, Alan H

    2016-05-01

    Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

  5. Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA, a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9 cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic and mammalian cells (CHO. While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

  6. Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries.

    Science.gov (United States)

    Hundsberger, Harald; Önder, Kamil; Schuller-Götzburg, Peter; Virok, Dezso P; Herzog, Julia; Rid, Raphaela

    2017-06-08

    Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.

  7. Controlled release from recombinant polymers.

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  9. High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines.

    Science.gov (United States)

    Wright, N; Jackson, F R; Niezgoda, M; Ellison, J A; Rupprecht, C E; Nel, L H

    2013-08-28

    Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Adaptive molecular evolution of MC1R gene reveals the evidence for positive diversifying selection in indigenous goat populations.

    Science.gov (United States)

    Ahmad, Hafiz Ishfaq; Liu, Guiqiong; Jiang, Xunping; Liu, Chenhui; Chong, Yuqing; Huarong, Huang

    2017-07-01

    Detecting signatures of selection can provide a new insight into the mechanism of contemporary breeding and artificial selection and further reveal the causal genes associated to the phenotypic variation. However, the signatures of selection on genes entailing for profitable traits between Chinese commercial and indigenous goats have been poorly interpreted. We noticed footprints of positive selection at MC1R gene containing SNPs genotyped in five Chinese native goat breeds. An experimental distribution of FST was built based on approximations of FST for each SNP across five breeds. We identified selection using the high FST outlier method and found that MC1R candidate gene show evidence of positive selection. Furthermore, adaptive selection pressure on specific codons was determined using different codon based on maximum-likelihood methods; signature of positive selection in mammalian MC1R was explored in individual codons. Evolutionary analyses were inferred under maximum likelihood models, the HyPhy package implemented in the DATAMONKEY Web Server. The results of codon selection displayed positive diversifying selection at the sites were mainly involved in development of genetic variations in coat color in various mammalian species. Positive diversifying selection inferred with recent evolutionary changes in domesticated goat MC1R provides new insights that the gene evolution may have been modulated by domestication events in goats.

  11. Distinct effects on diversifying selection by two mechanisms of immunity against Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Yuan Li

    Full Text Available Antigenic variation to evade host immunity has long been assumed to be a driving force of diversifying selection in pathogens. Colonization by Streptococcus pneumoniae, which is central to the organism's transmission and therefore evolution, is limited by two arms of the immune system: antibody- and T cell- mediated immunity. In particular, the effector activity of CD4(+ T(H17 cell mediated immunity has been shown to act in trans, clearing co-colonizing pneumococci that do not bear the relevant antigen. It is thus unclear whether T(H17 cell immunity allows benefit of antigenic variation and contributes to diversifying selection. Here we show that antigen-specific CD4(+ T(H17 cell immunity almost equally reduces colonization by both an antigen-positive strain and a co-colonized, antigen-negative strain in a mouse model of pneumococcal carriage, thus potentially minimizing the advantage of escape from this type of immunity. Using a proteomic screening approach, we identified a list of candidate human CD4(+ T(H17 cell antigens. Using this list and a previously published list of pneumococcal Antibody antigens, we bioinformatically assessed the signals of diversifying selection among the identified antigens compared to non-antigens. We found that Antibody antigen genes were significantly more likely to be under diversifying selection than the T(H17 cell antigen genes, which were indistinguishable from non-antigens. Within the Antibody antigens, epitopes recognized by human antibodies showed stronger evidence of diversifying selection. Taken together, the data suggest that T(H17 cell-mediated immunity, one form of T cell immunity that is important to limit carriage of antigen-positive pneumococcus, favors little diversifying selection in the targeted antigen. The results could provide new insight into pneumococcal vaccine design.

  12. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    Science.gov (United States)

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. A highly efficient recombinant laccase from the yeast Yarrowia lipolytica and its application in the hydrolysis of biomass.

    Directory of Open Access Journals (Sweden)

    Dayanand Kalyani

    Full Text Available A modified thermal asymmetric interlaced polymerase chain reaction was performed to obtain the first yeast laccase gene (YlLac from the isolated yeast Yarrowia lipolytica. The 1557-bp full-length cDNA of YlLac encoded a mature laccase protein containing 519 amino acids preceded by a signal peptide of 19 amino acids, and the YlLac gene was expressed in the yeast Pichia pastoris. YlLac is a monomeric glycoprotein with a molecular mass of ~55 kDa as determined by polyacrylamide-gel electrophoresis. It showed a higher catalytic efficiency towards 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate (kcat/Km = 17.5 s(-1 μM(-1 and 2,6-dimethoxyphenol (kcat/Km = 16.1 s(-1 μM(-1 than other reported laccases. The standard redox potential of the T1 site of the enzyme was found to be 772 mV. The highest catalytic efficiency of the yeast recombinant laccase, YlLac, makes it a good candidate for industrial applications: it removes phenolic compounds in acid-pretreated woody biomass (Populus balsamifera and enhanced saccharification.

  14. In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil

    Science.gov (United States)

    Pandolfi, Rafael; Ramos de Almeida, Denise; Alves Pinto, Marcelo; Kreutz, Luiz Carlos

    2017-01-01

    Hepatitis E Virus (HEV) is a zoonotic pathogen responsible for causing acute hepatitis in human, especially in developing countries. Diagnosis of HEV usually relies on the detection of antibodies mostly by enzyme-linked immunosorbent assay (ELISA). In the present study, we designed a new indirect ELISA (iELISA) based on a short recombinant peptide derived from the capsid protein (ORF2p) and demonstrated its potential for detecting human IgG against HEV genotype 3. The best polystyrene plate (Maxisorp®), optimal ORF2p coating antigen concentration (0,67μg/well) and primary antibody dilution (1:100) were determined. This iELISA showed a sensitivity of 91.4% and specificity of 95.9%. The comparison of our in house iELISA with a commercial assay (RecomWell, Mikrogen®) showed 94.25% of agreement and a kappa index of 0.88. The ORF2 recombinant ELISA was used to screen 780 blood donors for anti-HEV IgG and we found that 314 (40,25%) of these donors were IgG positive. This high prevalence of antibodies suggests, for the first time, that the Southern Brazil region might be endemic to Hepatitis E Virus genotype 3. PMID:28486512

  15. Radiative versus non-radiative recombination in high-efficiency mid-IR InSb/InAs/In(Ga,Al)As/GaAs metamorphic nanoheterostructures

    Science.gov (United States)

    Komkov, O. S.; Firsov, D. D.; Chernov, M. Yu; Solov’ev, V. A.; Sitnikova, A. A.; Kop’ev, P. S.; Ivanov, S. V.

    2018-02-01

    Nanostructures with a submonolayer InSb type-II insertion inside a InAs/InGaAs type-I quantum well (QW) have been grown by molecular beam epitaxy on GaAs (0 0 1) substrates via a convex-graded InAlAs metamorphic buffer layer (MBL). Selection of optimal growth conditions and design of the MBL-virtual substrate system enables one to increase mid-infrared photoluminescence (PL) and internal quantum efficiency (IQE) of the nanoheterostructures. The maximum low temperature IQE of about 90% has been obtained owing to the residual strain engineering which has resulted in both reduction of the extended defect density in the QW, likely responsible for Shockley–Read–Hall non-radiative recombination, and suppression of the Auger recombination channels in the InAs QW and the barriers. Temperature dependence of the integrated PL intensity was analyzed to determine an activation energy of an additional high-temperature non-radiative process (~49 meV) related presumably to hole delocalization through acceptor states in the strained InAs QW.

  16. High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

    Science.gov (United States)

    Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

    2016-08-01

    The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Effects of High Temperature and Thermal Cycling on the Performance of Perovskite Solar Cells: Acceleration of Charge Recombination and Deterioration of Charge Extraction.

    Science.gov (United States)

    Sheikh, Arif D; Munir, Rahim; Haque, Md Azimul; Bera, Ashok; Hu, Weijin; Shaikh, Parvez; Amassian, Aram; Wu, Tom

    2017-10-11

    In this work, we investigated the effects of high operating temperature and thermal cycling on the photovoltaic (PV) performance of perovskite solar cells (PSCs) with a typical mesostructured (m)-TiO2-CH3NH3PbI3-xClx-spiro-OMeTAD architecture. After temperature-dependent grazing-incidence wide-angle X-ray scattering, in situ X-ray diffraction, and optical absorption experiments were carried out, the thermal durability of PSCs was tested by subjecting the devices to repetitive heating to 70 °C and cooling to room temperature (20 °C). An unexpected regenerative effect was observed after the first thermal cycle; the average power conversion efficiency (PCE) increased by approximately 10% in reference to the as-prepared device. This increase of PCE was attributed to the heating-induced improvement of the crystallinity and p doping in the hole transporter, spiro-OMeTAD, which promotes the efficient extraction of photogenerated carriers. However, further thermal cycles produced a detrimental effect on the PV performance of PSCs, with the short-circuit current and fill factor degrading faster than the open-circuit voltage. Similarly, the PV performance of PSCs degraded at high operation temperatures; both the short-circuit current and open-circuit voltage decreased with increasing temperature, but the temperature-dependent trend of the fill factor was the opposite. Our impedance spectroscopy analysis revealed a monotonous increase of the charge-transfer resistance and a concurrent decrease of the charge-recombination resistance with increasing temperature, indicating a high recombination of charge carriers. Our results revealed that both thermal cycling and high temperatures produce irreversible detrimental effects on the PSC performance because of the deteriorated interfacial photocarrier extraction. The present findings suggest that the development of robust charge transporters and proper interface engineering are critical for the deployment of perovskite PVs in harsh

  18. Effects of High Temperature and Thermal Cycling on the Performance of Perovskite Solar Cells: Acceleration of Charge Recombination and Deterioration of Charge Extraction

    KAUST Repository

    Sheikh, Arif D.

    2017-09-18

    In this work, we investigated the effects of high operating temperature and thermal cycling on the photovoltaic performance of perovskite solar cells (PSCs) with a typical mesostructured (m)-TiO2-CH3NH3PbI3-xClx-spiro-OMeTAD architecture. After carrying out temperature-dependent grazing incidence wide-angle X-ray scattering (GIWAXS), in-situ X-ray diffraction (XRD) and optical absorption experiments, thermal durability of PSCs was tested by subjecting the devices to repetitive heating to 70 °C and cooling to room temperature (20 °C). An unexpected regenerative effect was observed after the first thermal cycle; the average power conversion efficiency (PCE) increased by approximately 10 % in reference to the as-prepared device. This increase of PCE was attributed to the heating-induced improvement of crystallinity and p-doping in the hole-transporter, Spiro-OMeTAD, which promotes the efficient extraction of photo-generated carriers. However, further thermal cycles produced a detrimental effect on the photovoltaic performance of PSCs with short-circuit current and fill factor degrading faster than the open-circuit voltage. Similarly, the photovoltaic performance of PSCs degraded at high operation temperatures; both short-circuit current and open-circuit voltage decreased with increasing temperature, but the temperature-dependent trend of fill factor was opposite. Our impedance spectroscopy analysis revealed a monotonous increase of charge transfer resistance and a concurrent decrease of charge recombination resistance with increasing temperature, indicating high recombination of charge carriers. Our results revealed that both thermal cycling and high temperatures produce irreversible detrimental effects on the PSC performance due to the deteriorated interfacial photo-carrier extraction. The present findings suggest that development of robust charge transporters and proper interface engineering are critical for the deployment of perovskite photovoltaics in harsh

  19. Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection.

    Science.gov (United States)

    Mazumder, Saumyabrata; Maji, Mithun; Ali, Nahid

    2011-12-01

    Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice. Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo. Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated

  20. Determination of recombination in Mycoplasma hominis

    DEFF Research Database (Denmark)

    Jacobsen, Iben Søgaard; Boesen, Thomas; Mygind, Tina

    2002-01-01

    indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage...... disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed...... intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species....

  1. Pedagogical Transaction in Religious Education: Diversified Society and John Dewey's Philosophy of Education

    Science.gov (United States)

    Sutinen, Ari; Kallioniemi, Arto; Pihlström, Sami

    2015-01-01

    The focus of the article is on how a new approach to religious education (RE) in diversified societies can be constructed on the basis of the theory of pedagogical transaction presented by John Dewey. Reflections of developing RE are very current in Western secularized societies. We believe that Dewey's pragmatist philosophy of education and…

  2. Diversifying Fiscal Support by Pricing Public Library Services: A Policy Impact Analysis.

    Science.gov (United States)

    Hicks, Donald A.

    1980-01-01

    Addresses the possibility of diversifying the resource base of public libraries dependent on property taxes for funding through the setting of fees for library services, and reports on a pricing policy adopted by the Dallas Public Library System. Twenty-seven references are cited. (FM)

  3. Factors Leading to Success in Diversified Occupation: A Livelihood Analysis in India

    Science.gov (United States)

    Saha, Biswarup; Bahal, Ram

    2015-01-01

    Purpose: Livelihood diversification is a sound alternative for higher economic growth and its success or failure is conditioned by the interplay of a multitude of factors. The study of the profile of the farmers in which they operate is important to highlight the factors leading to success in diversified livelihoods. Design/Methodology/Approach: A…

  4. A Guide for Implementing Project DEEP (Diversified Educational Experiences Program). Administrator's Guide.

    Science.gov (United States)

    Connett, Jane; Swanson, Monty

    The guide is designed to provide the building level administrator a step by step model for implementing Project DEEP's (Diversified Educational Experiences Program) alternative classroom management system for secondary academic classrooms with disaffected (attendance problems, discipline problems, potential dropouts), average, and gifted and…

  5. Evaluating the Context of Diversified Secondary Education in Tanzania. Preliminary Comments.

    Science.gov (United States)

    King, Kenneth

    Secondary education in Tanzania has a formal diversified curriculum in agricultural, commercial, technical, and domestic science that is reinforced outside of school hours by a set of powerful pressures on schools to engage in productive work. In many secondary schools there are two different, simultaneous traditions of diversification. The first…

  6. Divide and Recombine for Large Complex Data

    Science.gov (United States)

    2017-12-01

    SUPPLEMENTARY NOTES 14. ABSTRACT Divide and Recombine (D& R ) statistical approach was developed for analyzing ‘big data’ where the computational complexity...is very high. The analyst divides data into subsets by a D& R division technique, applying analytic methods to each subset independently, without...communication. Outputs of each analytic method are recombined by a D& R recombination procedure, which allows extensive parallel computation. DeltaRho

  7. The IT user support bookshop is moving and diversifying

    CERN Multimedia

    Jutta Megies et Roger Woolnough ( IT ), Jens Vigen ( DSU )

    2005-01-01

    The IT user support bookshop in Building 513 will officially close on 11th July before re-opening at its new location in the Central Library area on 25th July. The future choice of books will naturally continue to include a large selection of IT titles but will also include physics, engineering and other subjects to be decided according to demand. We will also continue to sell IT CDs. The initial selection of physics books for the launch will be based on input from the Library. We should like to thank you for your continued support in this move, which was stimulated by the heavy demand demonstrated at last year's highly successful book fair. Jutta Megies & Roger Woolnough ( IT ), Jens Vigen ( DSU )

  8. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

    Directory of Open Access Journals (Sweden)

    Ukkonen Kaisa

    2011-12-01

    Full Text Available Abstract This report describes the combined use of an enzyme-based glucose release system (EnBase® and high-aeration shake flask (Ultra Yield Flask™. The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

  9. Diversifying crops for food and nutrition security - a case of teff.

    Science.gov (United States)

    Cheng, Acga; Mayes, Sean; Dalle, Gemedo; Demissew, Sebsebe; Massawe, Festo

    2017-02-01

    There are more than 50000 known edible plants in the world, yet two-thirds of global plant-derived food is provided by only three major cereals - maize (Zea mays), wheat (Triticum aestivum) and rice (Oryza sativa). The dominance of this triad, now considered truly global food commodities, has led to a decline in the number of crop species contributing to global food supplies. Our dependence on only a few crop species limits our capability to deal with challenges posed by the adverse effects of climate change and the consequences of dietary imbalance. Emerging evidence suggests that climate change will cause shifts in crop production and yield loss due to more unpredictable and hostile weather patterns. One solution to this problem is through the wider use of underutilised (also called orphan or minor) crops to diversify agricultural systems and food sources. In addition to being highly nutritious, underutilised crops are resilient in natural and agricultural conditions, making them a suitable surrogate to the major crops. One such crop is teff [Eragrostis tef (Zucc.) Trotter], a warm-season annual cereal with the tiniest grain in the world. Native to Ethiopia and often the sustenance for local small farmers, teff thrives in both moisture-stressed and waterlogged soil conditions, making it a dependable staple within and beyond its current centre of origin. Today, teff is deemed a healthy wheat alternative in the West and is sought-after by health aficionados and those with coeliac disease or gluten sensitivity. The blooming market for healthy food is breathing new life into this underutilised crop, which has received relatively limited attention from mainstream research perhaps due to its 'orphan crop' status. This review presents the past, present and future of an ancient grain with a potential beyond its size. © 2015 Cambridge Philosophical Society.

  10. Efficacy of Highly Purified Urinary FSH versus Recombinant FSH in Chinese Women over 37 Years Undergoing Assisted Reproductive Techniques

    Directory of Open Access Journals (Sweden)

    Xuemei Liu

    2015-02-01

    Full Text Available Background: Urine derived follicle-stimulating hormone (uFSH contains a higher proportion of acidic isoforms, whereas recombinant FSH (rFSH contains a higher proportion of less-acidic isoforms. Less-acidic isoforms have a faster clearance, and thus a shorter half-life than the acidic FSH isoforms. The slow clearance of the acidic isoforms has a longer half-life and higher biological activity. This study was designed to determine whether uFSH or rFSH is more effective in older Chinese women undergoing assisted reproductive techniques (ART. Materials and Methods: This is a prospective, randomized, controlled cohort study. A total of 508 Chinese women over 37 years were randomized into two following study groups for their in vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI cycles: i. group A (n=254 were treated with rFSH, and ii. group B (n=254 were treated with uFSH. Both groups were suppressed with a gonadotropin-releasing hormone (GnRH analogue using a long down-regulation protocol. The main outcomes for comparison were days of stimulation, estradiol (E2 on the day of human chorionic gonadotropin (hCG administration, number of oocytes collected, amount of FSH used, quantity of FSH/oocyte, endometrial thickness at hCG day, M П oocyte rate, 2PN zygote rate, grade І embryo rate, number of embryos cryopreserved, pregnancy rate, implantation rate, abortion rate and the rate of no transferable embryos. Results: Twenty two cycles including 16 cycles with poor ovarian response and six cycles with ovarian hyperstimulation syndrome were cancelled. There were 243 cycles left in each group. The patients treated with uFSH had a significantly higher 2PN zygote rate (87.4 vs. 76.6%, p0.05. Conclusion: This study showed that uFSH produced a significantly higher proportion of grade І embryos than rFSH in older Chinese women and there was a significantly lower chance of no transferable embryos in uFSH cycles. The clinical efficacy of the two

  11. Highly recombinant VGII Cryptococcus gattii population develops clonal outbreak clusters through both sexual macroevolution and asexual microevolution.

    Science.gov (United States)

    Billmyre, R Blake; Croll, Daniel; Li, Wenjun; Mieczkowski, Piotr; Carter, Dee A; Cuomo, Christina A; Kronstad, James W; Heitman, Joseph

    2014-07-29

    An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV(+) patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors. Importance: Cryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in

  12. Molecular engineering of simple phenothiazine-based dyes to modulate dye aggregation, charge recombination, and dye regeneration in highly efficient dye-sensitized solar cells.

    Science.gov (United States)

    Hua, Yong; Chang, Shuai; He, Jian; Zhang, Caishun; Zhao, Jianzhang; Chen, Tao; Wong, Wai-Yeung; Wong, Wai-Kwok; Zhu, Xunjin

    2014-05-19

    A series of simple phenothiazine-based dyes, namely, TP, EP, TTP, ETP, and EEP have been developed, in which the thiophene (T), ethylenedioxythiophene (E), their dimers, and mixtures are present to modulate dye aggregation, charge recombination, and dye regeneration for highly efficient dye-sensitized solar cell (DSSC) applications. Devices sensitized by the dyes TP and TTP display high power conversion efficiencies (PCEs) of 8.07 (Jsc = 15.2 mA cm(-2), Voc =0.783 V, fill factor (FF) = 0.679) and 7.87 % (Jsc = 16.1 mA cm(-2), Voc = 0.717 V, FF = 0.681), respectively; these were measured under simulated AM 1.5 sunlight in conjunction with the I(-)/I3(-) redox couple. By replacing the T group with the E unit, EP-based DSSCs had a slightly lower PCE of 7.98 % with a higher short-circuit photocurrent (Jsc) of 16.7 mA cm(-2). The dye ETP, with a mixture of E and T, had an even lower PCE of 5.62 %. Specifically, the cell based on the dye EEP, with a dimer of E, had inferior Jsc and Voc values and corresponded to the lowest PCE of 2.24 %. The results indicate that the photovoltaic performance can be finely modulated through structural engineering of the dyes. The selection of T analogues as donors can not only modulate light absorption and energy levels, but also have an impact on dye aggregation and interfacial charge recombination of electrons at the interface of titania, electrolytes, and/or oxidized dye molecules; this was demonstrated through DFT calculations, electrochemical impedance analysis, and transient photovoltage studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  14. 76 FR 27738 - Order of Suspension of Trading; In the Matter of Diversified Investors Corp. (n/k/a Diverse...

    Science.gov (United States)

    2011-05-12

    ... COMMISSION Order of Suspension of Trading; In the Matter of Diversified Investors Corp. (n/k/a Diverse Holdings Corp.), Drew Resources (n/k/a Galloway Energy, Inc.), DTI Medical Corp., DTLL, Inc. (n/k/a... Diversified Investors Corp. (n/k/a Diverse Holdings Corp.) because it has not filed any periodic reports since...

  15. High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

    Science.gov (United States)

    Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

    2012-03-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

  16. Comparison of lead and cadmium contents in cruciferous vegetables grown under diversified ecological conditions: Cracow region of Poland.

    Science.gov (United States)

    Kapusta-Duch, Joanna; Leszczyńska, Teresa; Florkiewicz, Adam; Filipiak-Florkiewicz, Agnieszka

    2011-01-01

    The aim of the present study was to compare lead and cadmium contents in cruciferous vegetables grown under diversified ecological conditions for three consecutive years, independently of the climatic and agrotechnical conditions. The research was conducted in the Cracow region of Poland and tests vegetables near the Steelworks, from ecological farms, and from local markets. The heavy metal contents were determined using the validated Atomic Absorption Spectrometry method, including electrothermal atomization, with an ET-AAS graphite cuvette (Varian AA240Z, made by Varian). Cruciferous vegetables cultivated in the areas surrounding the steelworks were characterized by alarmingly high lead content versus ecological and commercially available vegetables, while the contents of this metal in vegetables from the two latter locations did not differ. It cannot be definitively stated that the origin of vegetables influenced their cadmium content.

  17. Recombination drives vertebrate genome contraction.

    Science.gov (United States)

    Nam, Kiwoong; Ellegren, Hans

    2012-01-01

    Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.

  18. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    Science.gov (United States)

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  19. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  20. Expression of Recombinant Antibodies

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  1. AACP Special Taskforce White Paper on Diversifying Our Investment in Human Capital.

    Science.gov (United States)

    White, Carla; Conway, Jeannine M; Davis, Paula K; Johnson-Fannin, Arcelia M; Jurkas, Jeffrey G; Murphy, Nanci L; Smith, W Thomas; Echeverri, Margarita; Youmans, Sharon L; Owings, Katie C; Adams, Jennifer L

    2017-10-01

    The 2015-2017 American Association of Colleges of Pharmacy (AACP) Special Taskforce on Diversifying our Investment in Human Capital was appointed for a two-year term, due to the rigors and complexities of its charges. This report serves as a white paper for academic pharmacy on diversifying our investment in human capital. The Taskforce developed and recommended a representation statement that was adapted and adopted by the AACP House of Delegates at the 2016 AACP Annual Meeting. In addition, the Taskforce developed a diversity statement for the Association that was adopted by the AACP Board of Directors in 2017. The Taskforce also provides recommendations to AACP and to academic pharmacy in this white paper.

  2. The Red Queen theory of recombination hotspots.

    Science.gov (United States)

    Ubeda, F; Wilkins, J F

    2011-03-01

    Recombination hotspots are small chromosomal regions, where meiotic crossover events happen with high frequency. Recombination is initiated by a double-strand break (DSB) that requires the intervention of the molecular repair mechanism. The DSB repair mechanism may result in the exchange of homologous chromosomes (crossover) and the conversion of the allelic sequence that breaks into the one that does not break (biased gene conversion). Biased gene conversion results in a transmission advantage for the allele that does not break, thus preventing recombination and rendering recombination hotspots transient. How is it possible that recombination hotspots persist over evolutionary time (maintaining the average chromosomal crossover rate) when they are self-destructive? This fundamental question is known as the recombination hotspot paradox and has attracted much attention in recent years. Yet, that attention has not translated into a fully satisfactory answer. No existing model adequately explains all aspects of the recombination hotspot paradox. Here, we formulate an intragenomic conflict model resulting in Red Queen dynamics that fully accounts for all empirical observations regarding the molecular mechanisms of recombination hotspots, the nonrandom targeting of the recombination machinery to hotspots and the evolutionary dynamics of hotspot turnover. © 2010 The Authors. Journal of Evolutionary Biology © 2010 European Society For Evolutionary Biology.

  3. Level of Acceptability of Moringa oleifera Diversified Products among Rural and Urban Dwellers in Nigeria

    OpenAIRE

    Mojisola F. Oyewole; Franscisca T. Adetoro; Nkiru T. Meludu

    2015-01-01

    Moringa oleifera is a nutritious vegetable tree with varieties of potential uses, as almost every part of the Moringa oleifera tree can be used for food. This study was conducted in Oyo State, Nigeria, to find out the level of acceptability of Moringa oleifera diversified products among rural and urban dwellers. Purposive sampling was used to select two local governments' areas. Stratified sampling technique was also used to select one community each from rural and urban ...

  4. Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.

    2017-01-01

    knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test...

  5. A high-yielding, generic fed-batch process for recombinant antibody production of GS-engineered cell lines

    DEFF Research Database (Denmark)

    Fan, Li; Zhao, Liang; Sun, Yating

    2009-01-01

    . Compared to batch cultures, the fed-batch technology generated the magnitude of the increase in cell yields (5 fold) and final antibody concentrations (4-8 fold). The majority of the increase in final antibody concentration was functions of the increased cell density and the prolonged culture time....... This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines....

  6. Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164

    OpenAIRE

    Sacchi, Veronica; Mittermayr, Rainer; Hartinger, Joachim; Martino, Mikaël M.; Lorentz, Kristen M.; Wolbank, Susanne; Hofmann, Anna; Largo, Remo A.; Marschall, Jeffrey S.; Groppa, Elena; Gianni-Barrera, Roberto; Ehrbar, Martin; Hubbell, Jeffrey A.; Redl, Heinz; Banfi, Andrea

    2014-01-01

    Inducing the growth of new blood vessels by specific factors is an attractive strategy to restore blood flow in ischemic tissues. Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, yet clinical trials of VEGF gene delivery failed. Major challenges include the need to control the tissue distribution of factor dose and the duration of expression. Here, we developed a highly tunable fibrin-based platform to precisely control the dose and duration of VEGF protein d...

  7. Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange.

    Science.gov (United States)

    Baumann, Martina; Gludovacz, Elisabeth; Sealover, Natalie; Bahr, Scott; George, Henry; Lin, Nan; Kayser, Kevin; Borth, Nicole

    2017-11-01

    Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site. © 2017 Wiley Periodicals, Inc.

  8. Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.

    Science.gov (United States)

    Vallejo, Luis Felipe; Brokelmann, Maren; Marten, Sabine; Trappe, Susanne; Cabrera-Crespo, Joaquin; Hoffmann, Andrea; Gross, Gerhard; Weich, Herbert A; Rinas, Ursula

    2002-03-28

    Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.

  9. High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase.

    Science.gov (United States)

    Bordeaux, Mélanie; de Girval, Diane; Rullaud, Robin; Subileau, Maeva; Dubreucq, Eric; Drone, Jullien

    2014-01-01

    We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.

  10. Identification and Expression Patterns of Putative Diversified Carboxylesterases in the Tea Geometrid Ectropis obliqua Prout

    Directory of Open Access Journals (Sweden)

    Liang Sun

    2017-12-01

    Full Text Available Carboxylesterases (CXEs belong to a family of metabolic enzymes. Some CXEs act as odorant-degrading enzymes (ODEs, which are reportedly highly expressed in insect olfactory organs and participate in the rapid deactivation of ester pheromone components and plant volatiles. The tea geometrid Ectropis obliqua Prout produces sex pheromones consisting of non-ester functional compounds but relies heavily on acetic ester plant volatiles to search for host plants and locate oviposition sites. However, studies characterizing putative candidate ODEs in this important tea plant pest are still relatively scarce. In the present study, we identified 35 candidate EoblCXE genes from E. obliqua chemosensory organs based on previously obtained transcriptomic data. The deduced amino acid sequences possessed the typical characteristics of the insect CXE family, including oxyanion hole residues, the Ser-Glu-His catalytic triad, and the Ser active included in the conserved pentapeptide characteristic of esterases, Gly-X-Ser-X-Gly. Phylogenetic analyses revealed that the EoblCXEs were diverse, belonging to several different insect esterase clades. Tissue- and sex-related expression patterns were studied via reverse-transcription and quantitative real-time polymerase chain reaction analyses (RT- and qRT-PCR. The results showed that 35 EoblCXE genes presented a diversified expression profile; among these, 12 EoblCXEs appeared to be antenna-biased, two EoblCXEs were non-chemosensory organ-biased, 12 EoblCXEs were ubiquitous, and nine EoblCXEs showed heterogeneous expression levels among different tissues. Intriguingly, two EoblCXE genes, EoblCXE7 and EoblCXE13, were not only strongly localized to antennal sensilla tuned to odorants, such as the sensilla trichodea (Str I and II and sensilla basiconica (Sba, but were also expressed in the putative gustatory sensilla styloconica (Sst, indicating that these two CXEs might play multiple physiological roles in the E. obliqua

  11. High-throughput functional microRNAs profiling by recombinant AAV-based microRNA sensor arrays.

    Directory of Open Access Journals (Sweden)

    Wenhong Tian

    Full Text Available BACKGROUND: microRNAs (miRNAs are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking. PRINCIPAL FINDINGS: We describe an adeno-associated virus (AAV vector-based microRNA (miRNA sensor (Asensor array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc and a firefly luciferase (Fluc expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found "functional miRNA signatures" for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR. We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA. CONCLUSIONS/SIGNIFICANCE: Our approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions.

  12. Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

    Science.gov (United States)

    Thakur, Dipak; Rejtar, Tomas; Karger, Barry L; Washburn, Nathaniel J; Bosques, Carlos J; Gunay, Nur S; Shriver, Zachary; Venkataraman, Ganesh

    2009-11-01

    With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for

  13. The role of SOCS2 in recombinant human growth hormone (rhGH) regulating lipid metabolism in high-fat-diet-induced obesity mice.

    Science.gov (United States)

    Yang, Hai Li; Feng, Min; Tan, Xiao; Yan, Guo Yong; Sun, Chao

    2013-03-01

    In addition to regulate body growth and development process, growth hormone (GH) also involved in lipid metabolism, decreasing fat mass and improving lipolysis. To normal mice, GH could reduce their fat content, but events turned uncertain coming to the pattern of feeding high-fat-diet. In order to investigate the role of GH in adipogenesis of mice with high-fat-diet, the high-fat-diet feeding mice were randomly assigned into three groups and treated with recombinant human growth hormone (rhGH) and the somatostatin analogue octreotide respectively. Results demonstrated that both rhGH and octreotide could reduce the body weight but the trends diminished in the end. HDL-C level was increased in octreotide treated groups but the activity of lipase was increased significantly in both two groups. RhGH remarkable increased the expression of SOCS2, FAS (P < 0.01) and SREBP-1c (P < 0.05), decreased the expression of SOCS1, SOCS3 (P < 0.05) and HSL (P < 0.01) in subcutaneous fat mass. In visceral fat tissue, all genes were increased except SOCS2 (P < 0.01), at the same time the visceral fat mass was decreased. The protein phosphorylation of JAK2 and STAT5 which were treated with octreotide were increased in subcutaneous fat, visceral fat and liver (P < 0.01) and were increased significant in visceral fat by rhGH treated (P < 0.01). In liver, only JAK2 protein phosphorylation was raised (P < 0.01). In conclusion, rhGH and octreotide could decrease the whole body mass before 6 days; the trend was weaken in later period with high-fat-diet. RhGH could increase the subcutaneous fat mass and reduce the visceral fat mass, and SOCS2 might be involved in regulation of the mechanism through JAK2/STAT5 signaling pathway.

  14. Enzymatic hydrolysis and succinic acid fermentation from steam-exploded corn stalk at high solid concentration by recombinant Escherichia coli.

    Science.gov (United States)

    Wu, Dexi; Li, Qiang; Wang, Dan; Dong, Yugang

    2013-08-01

    Steam-exploded corn stalk biomass was used as the substrate for succinic acid production via lignocellulose enzymatic hydrolysis and fermentation. Succinic acid fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). For the washed steam-exploded corn stalk at 30 % substrate concentration, i.e., 30 % water-insoluble solids (WIS), enzymatic hydrolysis yielded 97.5 g/l glucose solution and a cellulose conversion of 73.6 %, thus a high succinic acid level up to 38.6 g/l. With the unwashed steam-exploded corn stalk, though a cellulose conversion of 71.2 % was obtained in hydrolysis at 30 % solid concentration (27.9 % WIS), its hydrolysate did not ferment at all, and the hydrolysate of 25 % solid loading containing 3.8 g/l acetic acid and 168.2 mg/l furfural exerted a strong inhibition on succinic acid production.

  15. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

  16. Mitotic illegitimate recombination is a mechanism for novel changes in high-molecular-weight glutenin subunits in wheat-rye hybrids.

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    Zhongwei Yuan

    Full Text Available Wide hybrids can have novel traits or changed expression of a quantitative trait that their parents do not have. These phenomena have long been noticed, yet the mechanisms are poorly understood. High-molecular-weight glutenin subunits (HMW-GS are seed storage proteins encoded by Glu-1 genes that only express in endosperm in wheat and its related species. Novel HMW-GS compositions have been observed in their hybrids. This research elucidated the molecular mechanisms by investigating the causative factors of novel HMW-GS changes in wheat-rye hybrids. HMW-GS compositions in the endosperm and their coding sequences in the leaves of F(1 and F(2 hybrids between wheat landrace Shinchunaga and rye landrace Qinling were investigated. Missing and/or additional novel HMW-GSs were observed in the endosperm of 0.5% of the 2078 F(1 and 22% of 36 F(2 hybrid seeds. The wildtype Glu-1Ax null allele was found to have 42 types of short repeat sequences of 3-60 bp long that appeared 2 to 100 times. It also has an in-frame stop codon in the central repetitive region. Analyzing cloned allele sequences of HMW-GS coding gene Glu-1 revealed that deletions involving the in-frame stop codon had happened, resulting in novel ∼1.8-kb Glu-1Ax alleles in some F(1 and F(2 plants. The cloned mutant Glu-1Ax alleles were expressed in Escherichia coli, and the HMW-GSs produced matched the novel HMW-GSs found in the hybrids. The differential changes between the endosperm and the plant of the same hybrids and the data of E. coli expression of the cloned deletion alleles both suggested that mitotic illegitimate recombination between two copies of a short repeat sequence had resulted in the deletions and thus the changed HMW-GS compositions. Our experiments have provided the first direct evidence to show that mitotic illegitimate recombination is a mechanism that produces novel phenotypes in wide hybrids.

  17. A Complex Recombination Pattern in the Genome of Allotetraploid Brassica napus as Revealed by a High-Density Genetic Map

    Science.gov (United States)

    Yi, Bin; Fan, Chuchuan; Edwards, David; Batley, Jacqueline; Zhou, Yongming

    2014-01-01

    Polyploidy plays a crucial role in plant evolution. Brassica napus (2n = 38, AACC), the most important oil crop in the Brassica genus, is an allotetraploid that originated through natural doubling of chromosomes after the hybridization of its progenitor species, B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC). A better understanding of the evolutionary relationship between B. napus and B. rapa, B. oleracea, as well as Arabidopsis, which has a common ancestor with these three species, will provide valuable information about the generation and evolution of allopolyploidy. Based on a high-density genetic map with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, we performed a comparative genomic analysis of B. napus with Arabidopsis and its progenitor species B. rapa and B. oleracea. Based on the collinear relationship of B. rapa and B. oleracea in the B. napus genetic map, the B. napus genome was found to consist of 70.1% of the skeleton components of the chromosomes of B. rapa and B. oleracea, with 17.7% of sequences derived from reciprocal translocation between homoeologous chromosomes between the A- and C-genome and 3.6% of sequences derived from reciprocal translocation between non-homologous chromosomes at both intra- and inter-genomic levels. The current study thus provides insights into the formation and evolution of the allotetraploid B. napus genome, which will allow for more accurate transfer of genomic information from B. rapa, B. oleracea and Arabidopsis to B. napus. PMID:25356735

  18. High level of transgene expression in primary chronic lymphocytic leukemia cells using helper-virus-free recombinant Epstein-Barr virus vectors.

    Science.gov (United States)

    Wendtner, Clemens-Martin; Kurzeder, Christian; Theiss, Hans D; Kofler, David M; Baumert, Jens; Delecluse, Henri-Jacques; Janz, Annette; Hammerschmidt, Wolfgang; Hallek, Michael

    2003-02-01

    Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells. EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009). Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.

  19. Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

    Directory of Open Access Journals (Sweden)

    Michelle Cummins

    2014-05-01

    Full Text Available Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA and a ~32 kDa subunit B (RTB. There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs, previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR array biosensor (ProteOn XPR36 indicated three distinct, non-competitive binding epitopes (“bins”. The association (ka and dissociation (kd rate constants and binding affinities (KD of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications.

  20. Hedging Long-Term Exposures of a Well-Diversified Portfolio with Short-Term Stock Index Futures Contracts

    Directory of Open Access Journals (Sweden)

    Yufang Liu

    2014-01-01

    Full Text Available It is difficult for passive portfolio strategy to manage the long-term exposure of a well-diversified portfolio because stock index futures contracts have a finite life limited by their maturity. In this paper, we investigate the problem of the rollover hedge strategy for the long-term exposure of a well-diversified portfolio. First, we consider the rollover hedge strategy for the well-diversified portfolio when the portfolio is not adjusted during the period. In order to obtain the optimal solution of the proposed model, the auxiliary models are constructed using the equivalent transformation technique. Moreover, dynamic programming is employed to derive the optimal positions of stock index futures contracts for the long-term exposure of the well-diversified portfolio. In addition, we extend the result to the case of the rollover hedge strategy with transaction costs and derive the optimal number of stock index futures contracts.

  1. In vivo Biodistribution of a Highly Attenuated Recombinant Vesicular Stomatitis Virus Expressing HIV-1 Gag Following Intramuscular, Intranasal, or Intravenous Inoculation

    Science.gov (United States)

    Johnson, J. Erik; Coleman, John W.; Kalyan, Narender K.; Calderon, Priscilla; Wright, Kevin J.; Obregon, Jennifer; Ogin-Wilson, Eleanor; Natuk, Robert J.; Clarke, David K.; Udem, Stephen A.; Cooper, David; Hendry, R. Michael

    2009-01-01

    Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSVIN- N4CT9-Gag1, and a prototypic reference virus, rVSVIN-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSVIN- N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSVIN-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host. PMID:19428903

  2. A novel continuous powder aerosolizer (CPA) for inhalative administration of highly concentrated recombinant surfactant protein-C (rSP-C) surfactant to preterm neonates.

    Science.gov (United States)

    Pohlmann, G; Iwatschenko, P; Koch, W; Windt, H; Rast, M; de Abreu, M Gama; Taut, F J H; De Muynck, C

    2013-12-01

    In pulmonary medicine, aerosolization of substances for continuous inhalation is confined to different classes of nebulizers with their inherent limitations. Among the unmet medical needs is the lack of an aerosolized surfactant preparation for inhalation by preterm neonates, to avoid the risks associated with endotracheal intubation and surfactant bolus instillation. In the present report, we describe a high-concentration continuous powder aerosolization system developed for delivery of inhalable surfactant to preterm neonates. The developed device uses a technique that allows efficient aerosolization of dry surfactant powder, generating a surfactant aerosol of high concentration. In a subsequent humidification step, the heated aerosol particles are covered with a surface layer of water. The wet surfactant aerosol is then delivered to the patient interface (e.g., nasal prongs) through a tube. The performance characteristics of the system are given as mass concentration, dose rate, and size distribution of the generated aerosol. Continuous aerosol flows of about 0.84 L/min can be generated from dry recombinant surfactant protein-C surfactant, with concentrations of up to 12 g/m(3) and median particle sizes of the humidified particles in the range of 3 to 3.5 μm at the patient interface. The system has been successfully used in preclinical studies. The device with its continuous high-concentration delivery is promising for noninvasive delivery of surfactant aerosol to neonates and has the potential for becoming a versatile disperser platform closing the gap between continuously operating nebulizers and discontinuously operating dry powder inhaler devices.

  3. Dissociative Recombination of Complex Ions

    Science.gov (United States)

    Mitchell, J. Brian A.

    1999-10-01

    The FALP-MS apparatus at the University of Rennes allows the measurement of rate coefficients for the recombination of molecular ions to be made (at 300K) even though several ions may be present in the afterglow. The recombination of a number of hydrocarbon ions derived from alkane ( Lehfaoui et al. J. Chem. Phys. 106, 5406, 1997.), alkene ( Rebrion-Rowe et al. J. Chem. Phys. 108, 7185, 1998.) and aromatic (Rebrion-Rowe et al. (Submitted to J. Chem. Phys.)) parent molecules has been studied. Despite the wide range of complexity of these compounds, the measured recombination rates are remarkably similar having values in the range of 4-10-7 cm^3.s-1. Plans are being laid for a new version of this apparatus that will allow pre-prepared ions to be injected into the inert buffer gas flow. This will allow reactive ions to be studied as well as halogen containing ions whose recombination rates would normally be masked by electron attachment to their parent gases in a conventional flowing afterglow apparatus. A high temperature modification to the CRESU supersonic flow apparatus (J.L. Le Garrec et al. J. Chem. Phys. 107, 54, 1997.) in our laboratory will allow electron attachment to radicals to be studied by means of the mass spectrometric detection of products, Langmuir probe measurement of the electron density in the flow and Laser Induced Fluorescent identification of the radical species. Such measurements are needed for the modeling of semiconductor processing plasmas.

  4. Diversified cropping systems support greater microbial cycling and retention of carbon and nitrogen

    Energy Technology Data Exchange (ETDEWEB)

    King, Alison E.; Hofmockel, Kirsten S.

    2017-03-01

    Diversifying biologically simple cropping systems often entails altering other management practices, such as tillage regime or nitrogen (N) source. We hypothesized that the interaction of crop rotation, N source, and tillage in diversified cropping systems would promote microbially-mediated soil C and N cycling while attenuating inorganic N pools. We studied a cropping systems trial in its 10th year in Iowa, USA, which tested a 2-yr cropping system of corn (Zea mays L.)/soybean [Glycine max (L.) Merr.] managed with conventional fertilizer N inputs and conservation tillage, a 3-yr cropping system of corn/soybean/small grain + red clover (Trifolium pratense L.), and a 4-yr cropping system of corn/soybean/small grain + alfalfa (Medicago sativa L.)/alfalfa. Three year and 4-yr cropping systems were managed with composted manure, reduced N fertilizer inputs, and periodic moldboard ploughing. We assayed soil microbial biomass carbon (MBC) and N (MBN), soil extractable NH4 and NO3, gross proteolytic activity of native soil, and potential activity of six hydrolytic enzymes eight times during the growing season. At the 0-20cm depth, native protease activity in the 4-yr cropping system was greater than in the 2-yr cropping system by a factor of 7.9, whereas dissolved inorganic N pools did not differ between cropping systems (P = 0.292). At the 0-20cm depth, MBC and MBN the 4-yr cropping system exceeded those in the 2-yr cropping system by factors of 1.51 and 1.57. Our findings suggest that diversified crop cropping systems, even when periodically moldboard ploughed, support higher levels of microbial biomass, greater production of bioavailable N from SOM, and a deeper microbially active layer than less diverse cropping systems.

  5. The Social Dimensions of Sustainability and Change in Diversified Farming Systems

    Directory of Open Access Journals (Sweden)

    Christopher M. Bacon

    2012-12-01

    Full Text Available Agricultural systems are embedded in wider social-ecological processes that must be considered in any complete discussion of sustainable agriculture. Just as climatic profiles will influence the future viability of crops, institutions, i.e., governance agreements, rural household and community norms, local associations, markets, and agricultural ministries, to name but a few, create the conditions that foster sustainable food systems. Because discussions of agricultural sustainability often overlook the full range of social dimensions, we propose a dual focus on a broad set of criteria, i.e., human health, labor, democratic participation, resiliency, biological and cultural diversity, equity, and ethics, to assess social outcomes, and on institutions that could support diversified farming systems (DFS. A comparative analysis of case studies from California's Central Valley, Mesoamerican coffee agroforestry systems, and European Union agricultural parks finds that DFS practices are unevenly adopted within and among these systems and interdependent with institutional environments that specifically promote diversified farming practices. Influential institutions in these cases include state policies, farmers' cooperatives/associations, and organized civic efforts to influence agroenvironmental policy, share knowledge, and shape markets for more 'sustainable' products. The Californian and Mesoamerican cases considers organic and fair trade certifications, finding that although they promote several DFS practices and generate social benefits, they are inadequate as a single strategy to promote agricultural sustainability. The complex governance and multifunctional management of Europe's peri-urban agricultural parks show unexpected potential for promoting DFS. Unless DFS are anchored in supportive institutions and evaluated against an inclusive set of social and environmental criteria, short-term investments to advance diversified agriculture could

  6. Diversified Investments, Market Returns and Low Transaction Costs: Which is the Best Combination of these Factors?

    Directory of Open Access Journals (Sweden)

    Jose M. Ventura

    2000-08-01

    Full Text Available The article presents a way to build a diversified portfolio at a cost lower than what most investment service firms and advisors can provide through active fund management. The investment strategy presented consists of selecting the Index that is appropriate for the investment aims and investing the portfolio based on the mix of different indexes that reflect the investor's goal. Since index investing by definition does not select any other security than the one in the index and has to be purchased in the proportion reflected in the index, stock selection is straightforward and management fees are at a low level or even beat the minimum, if competition works.

  7. A trial of production of the plant-derived high-value protein in a plant factory: photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits.

    Science.gov (United States)

    Kato, Kazuhisa; Maruyama, Shinichiro; Hirai, Tadayoshi; Hiwasa-Tanase, Kyoko; Mizoguchi, Tsuyoshi; Goto, Eiji; Ezura, Hiroshi

    2011-08-01

    One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories. To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m(-2) s(-)1) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.

  8. The recombinational anatomy of a mouse chromosome.

    Directory of Open Access Journals (Sweden)

    Kenneth Paigen

    2008-07-01

    Full Text Available Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1-2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2x higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots.

  9. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Borup, Rehannah; Lee, Young Bae

    2009-01-01

    randomized study. SETTING: University-based facilities for clinical services and research. PATIENT(S): Thirty women undergoing treatment with vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). INTERVENTION(S): Patients were randomly allocated to receive recombinant FSH or human (hMG) COH......-binding-protein-P (anti-apoptosis protein) were expressed at higher levels in hMG than in recombinant FSH. CONCLUSION(S): The different hormone compositions of the two drugs used for COH had a statistically significant impact on the gene expression profile of preovulatory granulosa cells. Some of these genes may...

  10. Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice

    Directory of Open Access Journals (Sweden)

    Margarita Vida

    2015-07-01

    Full Text Available Interleukin-6 (IL-6 has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD in wild-type (WT and IL-6-deficient (IL-6−/− mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6. Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1 and signal transducer and activator of transcription-3 (STAT3, increased AMP kinase phosphorylation (p-AMPK, and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS and stearoyl-CoA desaturase 1 (SCD1. The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β, FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis.

  11. Homologous Recombination DNA Repair Pathway Disruption and Retinoblastoma Protein Loss Are Associated with Exceptional Survival in High-Grade Serous Ovarian Cancer.

    Science.gov (United States)

    Garsed, Dale W; Alsop, Kathryn; Fereday, Sian; Emmanuel, Catherine; Kennedy, Catherine J; Etemadmoghadam, Dariush; Gao, Bo; Gebski, Val; Garès, Valérie; Christie, Elizabeth L; Wouters, Maartje C A; Milne, Katy; George, Joshy; Patch, Ann-Marie; Li, Jason; Arnau, Gisela Mir; Semple, Timothy; Gadipally, Sreeja R; Chiew, Yoke-Eng; Hendley, Joy; Mikeska, Thomas; Zapparoli, Giada V; Amarasinghe, Kaushalya; Grimmond, Sean M; Pearson, John V; Waddell, Nicola; Hung, Jillian; Stewart, Colin J R; Sharma, Raghwa; Allan, Prue E; Rambau, Peter F; Traficante, Nadia; McNally, Orla; Mileshkin, Linda; Hamilton, Anne; Ananda, Sumitra; Grossi, Marisa; Cohen, Paul A; Leung, Yee C; Rome, Robert M; Beale, Philip; Blomfield, Penny; Friedlander, Michael; Brand, Alison; Dobrovic, Alexander; Köbel, Martin; Harnett, Paul; Nelson, Brad H; Bowtell, David D L; deFazio, Anna

    2017-10-23

    Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 1-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  12. In vitro fertilisation with recombinant follicle stimulating hormone requires less IU usage compared with highly purified human menopausal gonadotrophin: results from a European retrospective observational chart review

    Directory of Open Access Journals (Sweden)

    Blackmore Stuart

    2010-11-01

    Full Text Available Abstract Background Previous studies have reported conflicting results for the comparative doses of recombinant follicle stimulating hormone (rFSH and highly purified human menopausal gonadotrophin (hMG-HP required per cycle of in vitro fertilisation (IVF; the aim of this study was to determine the average total usage of rFSH versus hMG-HP in a 'real-world' setting using routine clinical practice. Methods This retrospective chart review of databases from four European countries investigated gonadotrophin usage, oocyte and embryo yield, and pregnancy outcomes in IVF cycles (± intra-cytoplasmic sperm injection using rFSH or hMG-HP alone. Included patients met the National Institute for Health and Clinical Excellence (NICE guideline criteria for IVF and received either rFSH or hMG-HP. Statistical tests were conducted at 5% significance using Chi-square or t-tests. Results Of 30,630 IVF cycles included in this review, 74% used rFSH and 26% used hMG-HP. A significantly lower drug usage per cycle for rFSH than hMG-HP (2072.53 +/- 76.73 IU vs. 2540.14 +/- 883.08 IU, 22.6% higher for hMG-HP; p Conclusions Based on these results, IVF treatment cycles with rFSH yield statistically more oocytes (and more mature oocytes, using significantly less IU per cycle, versus hMG-HP. The incidence of all OHSS and hospitalisations due to OHSS was significantly higher in the rFSH cycles compared to the hMG-HP cycles. However, the absolute incidence of hospitalisations due to OHSS was similar to that reported previously. These results suggest that the perceived required dosage with rFSH is currently over-estimated, and the higher unit cost of rFSH may be offset by a lower required dosage compared with hMG-HP.

  13. Nurturing Diversified Farming Systems in Industrialized Countries: How Public Policy Can Contribute

    Directory of Open Access Journals (Sweden)

    Alastair Iles

    2012-12-01

    Full Text Available If diversified farming systems (DFS are to thrive again in the United States, policies and preferences must evolve to reward the environmental and social benefits of sustainable farming and landscape management. Compared with conventional agricultural policies, policies aiding ecological diversification are underdeveloped and fragmented. We consider several examples of obstacles to the adoption and spread of diversified farming practices in the U.S. industrialized agricultural system. These include the broader political economic context of industrialized agriculture, the erosion of farmer knowledge and capacity, and supply chain and marketing conditions that limit the ability of farmers to adopt sustainable practices. To overcome these obstacles and nurture DFS, policy makers, researchers, industry, farmers, consumers, and local communities can play pivotal roles to transform agricultural research, develop peer-to-peer learning processes, support the recruitment and retention of new farmers through access to credit and land, invest in improved agricultural conservation programs, provide compensation for provision of ecological services in working landscapes, and develop links to consumer and institutional markets.

  14. The diversified function and potential therapy of ectopic olfactory receptors in non-olfactory tissues.

    Science.gov (United States)

    Chen, Zhe; Zhao, Hong; Fu, Nian; Chen, Linxi

    2017-03-24

    Olfactory receptors (ORs) are mainly distributed in olfactory neurons and play a key role in detecting volatile odorants, eventually resulting in the production of smell perception. Recently, it is also reported that ORs are expressed in non-olfactory tissues including heart, lung, sperm, skin, and cancerous tissues. Interestingly, ectopic ORs are associated with the development of diseases in non-olfactory tissues. For instance, ectopic ORs initiate the hypoxic ventilatory responses and maintain the oxygen homeostasis of breathing in the carotid body when oxygen levels decline. Ectopic ORs induce glucose homeostasis in diabetes. Ectopic ORs regulate systemic blood pressure by increasing renin secretion and vasodilation. Ectopic ORs participate in the process of tumor cell proliferation, apoptosis, metastasis, and invasiveness. Ectopic ORs accelerate the occurrence of obesity, angiogenesis and wound-healing processes. Ectopic ORs affect fetal hemoglobin levels in sickle cell anemia and thalassemia. Finally, we also elaborate some ligands targeting for ORs. Obviously, the diversified function and related signal pathway of ectopic ORs may play a potential therapeutic target in non-olfactory tissues. Thus, this review focuses on the latest research results about the diversified function and therapeutic potential of ectopic ORs in non-olfactory tissues. © 2017 Wiley Periodicals, Inc.

  15. Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution

    Directory of Open Access Journals (Sweden)

    Monjane Adérito L

    2011-12-01

    Full Text Available Abstract Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae, the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots

  16. Charge transfer and recombination at the metal oxide/CH3NH3PbClI2/spiro-OMeTAD interfaces: uncovering the detailed mechanism behind high efficiency solar cells.

    Science.gov (United States)

    Shen, Qing; Ogomi, Yuhei; Chang, Jin; Tsukamoto, Syota; Kukihara, Kenji; Oshima, Takuya; Osada, Naoya; Yoshino, Kenji; Katayama, Kenji; Toyoda, Taro; Hayase, Shuzi

    2014-10-07

    In recent years, organometal halide perovskite-based solid-state hybrid solar cells have attracted unexpected increasing interest because of their high efficiency (the record power conversion efficiency has been reported to be over 15%) and low fabrication cost. It has been accepted that the high efficiency was mainly attributed to the strong optical absorption (absorption coefficient: 15,000 cm(-1) at 550 nm) over a broader range (up to 800 nm) and the long lifetimes of photoexcited charge carriers (in the order of 10 ns - a few 100 ns) of the perovskite absorbers. However, much of the fundamental photophysical properties of perovskite relating to the high photovoltaic performance are remained to be investigated. The charge separation and recombination processes at the material interfaces are particularly important for solar cell performances. To better understand the high efficiency of perovskite solar cells, we systematically investigated the charge separation (electron and hole injection) and charge recombination dynamics of CH3NH3PbClI2 hybrid solar cells employing TiO2 nanostructures as the electron transfer material (ETM) and spiro-OMeTAD as the hole transfer material (HTM). The measurements were carried out using transient absorption (TA) techniques on a time scale from sub-picoseconds to milliseconds. We clarified the timescales of electron injection, hole injection, and recombination processes in TiO2/CH3NH3PbClI2/spiro-OMeTAD solar cells. Charge separation and collection efficiency of the perovskite-based solar cells were discussed. In addition, the effect of TiO2 size on the charge separation and recombination dynamics was also investigated. It was found that all TiO2-based perovskite solar cells possessed similar charge separation processes, but quite different recombination dynamics. Our results indicate that charge recombination was crucial to the performance of the perovskite solar cells, which could be effectively suppressed through optimising

  17. The amphioxus (Branchiostoma floridae genome contains a highly diversified set of G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Schiöth Helgi B

    2008-01-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs are one of the largest families of genes in mammals. Branchiostoma floridae (amphioxus is one of the species most closely related species to vertebrates. Results Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18, Rhodopsin (570, Adhesion (37, Frizzled (6 and Secretin (16. Surprisingly, the Adhesion GPCR repertoire in amphioxus includes receptors with many new domains not previously observed in this family. We found many Rhodopsin GPCRs from all main groups including many amine and peptide binding receptors and several previously uncharacterized expansions were also identified. This genome has however no genes coding for bitter taste receptors (TAS2, the sweet and umami (TAS1, pheromone (VR1 or VR2 or mammalian olfactory receptors. Conclusion The amphioxus genome is remarkably rich in various GPCR subtypes while the main GPCR groups known to sense exogenous substances (such as Taste 2, mammalian olfactory, nematode chemosensory, gustatory, vomeronasal and odorant receptors in other bilateral species are absent.

  18. Gr39a, a highly diversified gustatory receptor in Drosophila, has a role in sexual behavior.

    Science.gov (United States)

    Watanabe, Kanako; Toba, Gakuta; Koganezawa, Masayuki; Yamamoto, Daisuke

    2011-09-01

    Sexual recognition among individuals is crucial for the reproduction of animals. In Drosophila, like in many other animals, pheromones are suggested to play an important role in conveying information about an individual, such as sex, maturity and mating status. Sex-specific cuticular hydrocarbon components are thought to be major sex pheromones in Drosophila, and are postulated to act through the gustatory system, since they are mostly non-volatile chemicals. However, very little is known about the molecular and neural bases of gustatory pheromone reception. So far, a few putative gustatory receptors, including Gr32a and Gr68a, have been implicated in courtship behavior. Here, we examine another putative gustatory receptor, Gr39a, which shares a cluster with both Gr32a and Gr68a in a molecular phylogeny of the gustatory receptor family, for its potential role in courtship behavior. The Gr39a gene produces four isoforms through alternative splicing of different 5'-most exons. A quantitative real-time PCR analysis showed that the expression levels of all four splice variants of Gr39a were reduced in a fly line in which a P element was inserted into the Gr39a locus. Homozygous and hemizygous males for the P-element insertion, as well as males in which Gr39a was knocked down by RNAi, all showed reduced courtship levels toward females. The courtship levels returned to normal when the P element was excised out. A close analysis of courtship behavior of the mutant males revealed that the average duration of a continuous courtship bout was significantly shorter in the mutants than in the wild type. The results suggest that Gr39a has a role in sustaining courtship behavior in males, possibly through the reception of a stimulating arrestant pheromone.

  19. Efficient electron transfer and reduced recombination with Nd:YAG laser scribing for high-efficiency quantum dot-sensitized solar cells

    Science.gov (United States)

    Zheng, Tao; Kim, Hee-Je; Gopi, Chandu V. V. M.; Venkata-Haritha, Mallineni; Son, Min-Kyu; Seo, Hyunwoong

    2017-09-01

    Inefficient charge transfer and charge recombination are critical but challenging issues that restrict the power conversion efficiency (PCE) of quantum-dot-sensitized solar cells (QDSSCs). These issues must be addressed to boost the performance of QDSSCs. We present a novel Nd:YAG laser scribing treatment for fluorine doped tin oxide (FTO) substrate that reduces electron loss by reducing the moving distance of electrons and strongly inhibiting interfacial recombination processes in QDSSCs. Consequently, TiO2/CdS/CdSe/Mn-ZnSe QDSSCs on the Nd:YAG laser scribed FTO exhibited a PCE of 6.26% under 1 sun (100 mW cm-2) irradiation, while TiO2/CdS/CdSe/Mn-ZnSe QDSSCs on the FTO without Nd:YAG laser scribing exhibited a PCE of 5.51%. The short circuit current density and fill factor are also increased after laser scribing, which arises from increased electron transfer with reduced recombination. Electrochemical impedance spectroscopy modeling reveals that the Nd:YAG laser scribed QDSSC has increased charge collection efficiency and reduced interfacial recombination compared with normal QDSSC.

  20. Creation of a high yielding recombinant maize hybrid for the production of a microbicide for the prevention of HIV-1 transmission

    CSIR Research Space (South Africa)

    Barros, E

    2010-06-01

    Full Text Available The aim of this study was to use conventional breeding to increase the production in maize of the human monoclonal antibody 2G12, known to have potential therapeutic properties in the prevention of HIV-1 transmission. The recombinant antibody...

  1. Dispersive and steady-state recombination in organic disordered semiconductors

    Science.gov (United States)

    Hofacker, Andreas; Neher, Dieter

    2017-12-01

    Charge carrier recombination in organic disordered semiconductors is strongly influenced by the thermalization of charge carriers in the density of states (DOS). Measurements of recombination dynamics, conducted under transient or steady-state conditions, can easily be misinterpreted when a detailed understanding of the interplay of thermalization and recombination is missing. To enable adequate measurement analysis, we solve the multiple-trapping problem for recombining charge carriers and analyze it in the transient and steady excitation paradigm for different DOS distributions. We show that recombination rates measured after pulsed excitation are inherently time dependent since recombination gradually slows down as carriers relax in the DOS. When measuring the recombination order after pulsed excitation, this leads to an apparent high-order recombination at short times. As times goes on, the recombination order approaches an asymptotic value. For the Gaussian and the exponential DOS distributions, this asymptotic value equals the recombination order of the equilibrated system under steady excitation. For a more general DOS distribution, the recombination order can also depend on the carrier density, under both transient and steady-state conditions. We conclude that transient experiments can provide rich information about recombination in and out of equilibrium and the underlying DOS occupation provided that consistent modeling of the system is performed.

  2. Initiation of meiotic recombination in Ustilago maydis.

    Science.gov (United States)

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

    2013-12-01

    A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

  3. DNA barcoding of Rhodiola (crassulaceae: a case study on a group of recently diversified medicinal plants from the Qinghai-Tibetan Plateau.

    Directory of Open Access Journals (Sweden)

    Jian-Qiang Zhang

    Full Text Available DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences - rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS - for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau.

  4. Amphioxus encodes the largest known family of green fluorescent proteins, which have diversified into distinct functional classes

    Directory of Open Access Journals (Sweden)

    Deheyn Dimitri D

    2009-04-01

    Full Text Available Abstract Background Green fluorescent protein (GFP has been found in a wide range of Cnidaria, a basal group of metazoans in which it is associated with pigmentation, fluorescence, and light absorbance. A GFP has been recently discovered in the pigmentless chordate Branchiostoma floridae (amphioxus that shows intense fluorescence mainly in the head region. Results The amphioxus genome encodes 16 closely-related GFP-like proteins, all of which appear to be under purifying selection. We divide them into 6 clades based on protein sequence identity and show that representatives of each clade have significant differences in fluorescence intensity, extinction coefficients, and absorption profiles. Furthermore, GFPs from two clades exhibit antioxidant capacity. We therefore propose that amphioxus GFPs have diversified their functions into fluorescence, redox, and perhaps just light absorption in relation to pigmentation and/or photoprotection. Conclusion The rapid radiation of amphioxus GFP into clades with distinct functions and spectral properties reveals functional plasticity of the GFP core. The high sequence similarities between different clades provide a model system to map sequence variation to functional changes, to better understand and engineer GFP.

  5. Signatures of diversifying selection at EST-SSR loci and association with climate in natural Eucalyptus populations.

    Science.gov (United States)

    Bradbury, Donna; Smithson, Ann; Krauss, Siegfried L

    2013-10-01

    Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian FST outlier tests and allele-climate association analyses to reveal two Eucalyptus EST-SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS-like gene was an FST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second FST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene-homologous EST-SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity. © 2013 John Wiley & Sons Ltd.

  6. Hospital and community ampicillin-resistant Enterococcus faecium are evolutionarily closely linked but have diversified through niche adaptation.

    Directory of Open Access Journals (Sweden)

    Marieke J A de Regt

    Full Text Available BACKGROUND: Ampicillin-resistant Enterococcus faecium (ARE has emerged as a nosocomial pathogen. Here, we quantified ARE carriage in different community sources and determined genetic relatedness with hospital ARE. METHODS AND RESULTS: ARE was recovered from rectal swabs of 24 of 79 (30% dogs, 11 of 85 (13% cats and 0 of 42 horses and from 3 of 40 (8% faecal samples of non-hospitalized humans receiving amoxicillin. Multi-locus Sequence Typing revealed 21 sequence types (STs, including 5 STs frequently associated with hospital-acquired infections. Genes previously found to be enriched in hospital ARE, such as IS16, orf903, orf905, orf907, were highly prevalent in community ARE (≥79%, while genes with a proposed role in pathogenesis, such as esp, hyl and ecbA, were found rarely (≤5% in community isolates. Comparative genome analysis of 2 representative dog isolates revealed that the dog strain of ST192 was evolutionarily closely linked to two previously sequenced hospital ARE, but had, based on gene content, more genes in common with the other, evolutionarily more distantly related, dog strain (ST266. CONCLUSION: ARE were detected in dogs, cats and sporadically in healthy humans, with evolutionary linkage to hospital ARE. Yet, their accessory genome has diversified, probably as a result of niche adaptation.

  7. Differential Evolution Algorithm with Diversified Vicinity Operator for Optimal Routing and Clustering of Energy Efficient Wireless Sensor Networks.

    Science.gov (United States)

    Sumithra, Subramaniam; Victoire, T Aruldoss Albert

    2015-01-01

    Due to large dimension of clusters and increasing size of sensor nodes, finding the optimal route and cluster for large wireless sensor networks (WSN) seems to be highly complex and cumbersome. This paper proposes a new method to determine a reasonably better solution of the clustering and routing problem with the highest concern of efficient energy consumption of the sensor nodes for extending network life time. The proposed method is based on the Differential Evolution (DE) algorithm with an improvised search operator called Diversified Vicinity Procedure (DVP), which models a trade-off between energy consumption of the cluster heads and delay in forwarding the data packets. The obtained route using the proposed method from all the gateways to the base station is comparatively lesser in overall distance with less number of data forwards. Extensive numerical experiments demonstrate the superiority of the proposed method in managing energy consumption of the WSN and the results are compared with the other algorithms reported in the literature.

  8. Dissociation of recombinant prion autocatalysis from infectivity.

    Science.gov (United States)

    Noble, Geoffrey P; Supattapone, Surachai

    2015-01-01

    Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

  9. Study on diversified cultivation orientation and pattern of optoelectronic major undergraduates

    Science.gov (United States)

    Liu, Zhiying

    2017-08-01

    To improve the research quality preparation for graduate study and looking for job competition ability of undergraduates students, the education orientation objective need to be explicit. Universities need develop undergraduates' cultivation plan according to students' classification. Based on analysis of students export characteristic, there will be corresponding cultivation plan. Keep tracking study during the cultivation plan implantation process, the Curriculum system and related manage documents are revised corresponding to exist problems. There are mainly three kinds of undergraduates' career direction plan for opto-electronic major undergraduates. In addition to the vast majority university graduates opting for direct employment, nearly one third of university students choose to take part in the postgraduate entrance exams and other further education abroad, and also one-tenth choose their own businesses, university chooses are diversified. The exports are further studying as graduates, working and study abroad. Because national defense students are also recruited, the cultivation plan will be diversified to four types. For students, who go to work directly after graduation, the "Excellence engineers plan" is implemented to enhance their practice ability. For students, who will study further as graduate student, the scientific innovation research ability cultivation is paid more attention to make good foundation for their subsequent development. For students, who want to study abroad after graduation, the bilingual teaching method is introduced, and the English environment is built. We asked foreign professionals to give lectures for students. The knowledge range is extending, and the exchange and cooperation chance is provided at the same time. And the cultivation plan is revised during docking with Universities abroad. For national defense students, combat training and other defense theory courses are added to make them familiar with force knowledge. And

  10. Epigenetic control of meiotic recombination in plants.

    Science.gov (United States)

    Yelina, Natasha; Diaz, Patrick; Lambing, Christophe; Henderson, Ian R

    2015-03-01

    Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination. Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.

  11. High-Efficiency Aqueous-Processed Polymer/CdTe Nanocrystals Planar Heterojunction Solar Cells with Optimized Band Alignment and Reduced Interfacial Charge Recombination.

    Science.gov (United States)

    Zeng, Qingsen; Hu, Lu; Cui, Jian; Feng, Tanglue; Du, Xiaohang; Jin, Gan; Liu, Fangyuan; Ji, Tianjiao; Li, Fenghong; Zhang, Hao; Yang, Bai

    2017-09-20

    Aqueous-processed nanocrystal solar cells have attracted increasing attention due to the advantage of its environmentally friendly nature, which provides a promising approach for large-scale production. The urgent affair is boosting the power conversion efficiency (PCE) for further commercial applications. The low PCE is mainly attributed to the imperfect device structure, which leads to abundant nonradiative recombination at the interfaces. In this work, an environmentally friendly and efficient method is developed to improve the performance of aqueous-processed CdTe nanocrystal solar cells. Polymer/CdTe planar heterojunction solar cells (PHSCs) with optimized band alignment are constructed, which results in reduced interfacial charge recombination, enhanced carrier collection efficiency and built-in field. Finally, a champion PCE of 5.9%, which is a record for aqueous-processed solar cells based on CdTe nanocrystals, is achieved after optimizing the photovoltaic device.

  12. Teamwork Seminar Practice to Foster Diversified Thinking and Leadership Among Students

    Science.gov (United States)

    Maruyama, Naoki; Yoshida, Kazumi; Yamao, Hidenori

    A new course entitled “Mechanical Engineering Seminar” has begun in the Department of Mechanical Engineering, Mie University. This course consists of three parts, a teamwork seminar, a creative design seminar and a comprehensive achievement examination. Its aim is to foster a broad social and international outlook, ethical thinking, autonomy, partnership, leadership, presentation ability, originality, overall creativity in students, and to help them become aware of their real ability. The teaching method used in this seminar is based on problem-based learning (PBL) , and pro-active student participation is required. The purpose of this paper is to report the features, teaching method and educational effectiveness of the teamwork seminar, which seeks to educate students with a broad, diversified outlook. The results of a student questionnaire show that these new fields of study stimulate students' will to learn, and they express general satisfaction with the seminar.

  13. Diversifying Food Systems in the Pursuit of Sustainable Food Production and Healthy Diets.

    Science.gov (United States)

    Dwivedi, Sangam L; Lammerts van Bueren, Edith T; Ceccarelli, Salvatore; Grando, Stefania; Upadhyaya, Hari D; Ortiz, Rodomiro

    2017-10-01

    Increasing demand for nutritious, safe, and healthy food because of a growing population, and the pledge to maintain biodiversity and other resources, pose a major challenge to agriculture that is already threatened by a changing climate. Diverse and healthy diets, largely based on plant-derived food, may reduce diet-related illnesses. Investments in plant sciences will be necessary to design diverse cropping systems balancing productivity, sustainability, and nutritional quality. Cultivar diversity and nutritional quality are crucial. We call for better cooperation between food and medical scientists, food sector industries, breeders, and farmers to develop diversified and nutritious cultivars that reduce soil degradation and dependence on external inputs, such as fertilizers and pesticides, and to increase adaptation to climate change and resistance to emerging pests. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Smart Morning in an African Village: Diversifying Technologies within a Tanzanian Context

    Directory of Open Access Journals (Sweden)

    Mikko Vesisenaho

    2010-11-01

    Full Text Available Information technology (IT can make a difference in a developing country only if it is designed in close collaboration with its users. The experiences from an ethnocomputing-based IT education initiative at Tumaini University, located in the rural area of Southern Tanzania, indicates promising opportunities for engaging children and students as creative co-designers and users for diversifying, novel information technologies. The local context with its needs was taken into account when using robotics (I-Blocks and culture-based learning materials and implementing students’ village outreach projects in local schools and hospitals. The CATI model suggests the steps Contextualize, Apply, Transfer, and Import for sustainable, inductive IT design. Together with the concept of ethnocomputing, the CATI model proved useful for building and starting a new needs-based, contextualized IT undergraduate program at Tumaini University in Tanzania in 2007. 

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

    OpenAIRE

    Lee, Meng-Shiou; Hseu, You-Cheng; Lai, Guan-Hua; Chang, Wen-Te; Chen, Hsi-Jien; Huang, Chi-Hung; Lee, Meng-Shiunn; Wang, Min-Ying; Kao, Jung-Yie; You, Bang-Jau; Lin, Wen- Hsin; Lien, Yi-Yang; Lin, Ming-Kuem

    2011-01-01

    Abstract 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 V...

  16. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

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

  17. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  18. Phytoplankton can actively diversify their migration strategy in response to turbulent cues

    Science.gov (United States)

    Sengupta, Anupam; Carrara, Francesco; Stocker, Roman

    2017-03-01

    Marine phytoplankton inhabit a dynamic environment where turbulence, together with nutrient and light availability, shapes species fitness, succession and selection. Many species of phytoplankton are motile and undertake diel vertical migrations to gain access to nutrient-rich deeper layers at night and well-lit surface waters during the day. Disruption of this migratory strategy by turbulence is considered to be an important cause of the succession between motile and non-motile species when conditions turn turbulent. However, this classical view neglects the possibility that motile species may actively respond to turbulent cues to avoid layers of strong turbulence. Here we report that phytoplankton, including raphidophytes and dinoflagellates, can actively diversify their migratory strategy in response to hydrodynamic cues characteristic of overturning by Kolmogorov-scale eddies. Upon experiencing repeated overturning with timescales and statistics representative of ocean turbulence, an upward-swimming population rapidly (5-60 min) splits into two subpopulations, one swimming upward and one swimming downward. Quantitative morphological analysis of the harmful-algal-bloom-forming raphidophyte Heterosigma akashiwo together with a model of cell mechanics revealed that this behaviour was accompanied by a modulation of the cells’ fore-aft asymmetry. The minute magnitude of the required modulation, sufficient to invert the preferential swimming direction of the cells, highlights the advanced level of control that phytoplankton can exert on their migratory behaviour. Together with observations of enhanced cellular stress after overturning and the typically deleterious effects of strong turbulence on motile phytoplankton, these results point to an active adaptation of H. akashiwo to increase the chance of evading turbulent layers by diversifying the direction of migration within the population, in a manner suggestive of evolutionary bet-hedging. This migratory

  19. Diversified Farming Systems: An Agroecological, Systems-based Alternative to Modern Industrial Agriculture

    Directory of Open Access Journals (Sweden)

    Claire Kremen

    2012-12-01

    Full Text Available This Special Issue on Diversified Farming Systems is motivated by a desire to understand how agriculture designed according to whole systems, agroecological principles can contribute to creating a more sustainable, socially just, and secure global food system. We first define Diversified Farming Systems (DFS as farming practices and landscapes that intentionally include functional biodiversity at multiple spatial and/or temporal scales in order to maintain ecosystem services that provide critical inputs to agriculture, such as soil fertility, pest and disease control, water use efficiency, and pollination. We explore to what extent DFS overlap or are differentiated from existing concepts such as sustainable, multifunctional, organic or ecoagriculture. DFS are components of social-ecological systems that depend on certain combinations of traditional and contemporary knowledge, cultures, practices, and governance structures. Further, as ecosystem services are generated and regenerated within a DFS, the resulting social benefits in turn support the maintenance of the DFS, enhancing its ability to provision these services sustainably. We explore how social institutions, particularly alternative agri-food networks and agrarian movements, may serve to promote DFS approaches, but note that such networks and movements have other primary goals and are not always explicitly connected to the environmental and agroecological concerns embodied within the DFS concept. We examine global trends in agriculture to investigate to what extent industrialized forms of agriculture are replacing former DFS, assess the current and potential contributions of DFS to food security, food sovereignty and the global food supply, and determine where and under what circumstances DFS are expanding rather than contracting.

  20. Phytoplankton can actively diversify their migration strategy in response to turbulent cues.

    Science.gov (United States)

    Sengupta, Anupam; Carrara, Francesco; Stocker, Roman

    2017-03-23

    Marine phytoplankton inhabit a dynamic environment where turbulence, together with nutrient and light availability, shapes species fitness, succession and selection. Many species of phytoplankton are motile and undertake diel vertical migrations to gain access to nutrient-rich deeper layers at night and well-lit surface waters during the day. Disruption of this migratory strategy by turbulence is considered to be an important cause of the succession between motile and non-motile species when conditions turn turbulent. However, this classical view neglects the possibility that motile species may actively respond to turbulent cues to avoid layers of strong turbulence. Here we report that phytoplankton, including raphidophytes and dinoflagellates, can actively diversify their migratory strategy in response to hydrodynamic cues characteristic of overturning by Kolmogorov-scale eddies. Upon experiencing repeated overturning with timescales and statistics representative of ocean turbulence, an upward-swimming population rapidly (5-60 min) splits into two subpopulations, one swimming upward and one swimming downward. Quantitative morphological analysis of the harmful-algal-bloom-forming raphidophyte Heterosigma akashiwo together with a model of cell mechanics revealed that this behaviour was accompanied by a modulation of the cells' fore-aft asymmetry. The minute magnitude of the required modulation, sufficient to invert the preferential swimming direction of the cells, highlights the advanced level of control that phytoplankton can exert on their migratory behaviour. Together with observations of enhanced cellular stress after overturning and the typically deleterious effects of strong turbulence on motile phytoplankton, these results point to an active adaptation of H. akashiwo to increase the chance of evading turbulent layers by diversifying the direction of migration within the population, in a manner suggestive of evolutionary bet-hedging. This migratory behaviour

  1. Insights into the molecular mechanisms underlying diversified wing venation among insects.

    Science.gov (United States)

    Shimmi, Osamu; Matsuda, Shinya; Hatakeyama, Masatsugu

    2014-08-22

    Insect wings are great resources for studying morphological diversities in nature as well as in fossil records. Among them, variation in wing venation is one of the most characteristic features of insect species. Venation is therefore, undeniably a key factor of species-specific functional traits of the wings; however, the mechanism underlying wing vein formation among insects largely remains unexplored. Our knowledge of the genetic basis of wing development is solely restricted to Drosophila melanogaster. A critical step in wing vein development in Drosophila is the activation of the decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signalling pathway during pupal stages. A key mechanism is the directional transport of Dpp from the longitudinal veins into the posterior crossvein by BMP-binding proteins, resulting in redistribution of Dpp that reflects wing vein patterns. Recent works on the sawfly Athalia rosae, of the order Hymenoptera, also suggested that the Dpp transport system is required to specify fore- and hindwing vein patterns. Given that Dpp redistribution via transport is likely to be a key mechanism for establishing wing vein patterns, this raises the interesting possibility that distinct wing vein patterns are generated, based on where Dpp is transported. Experimental evidence in Drosophila suggests that the direction of Dpp transport is regulated by prepatterned positional information. These observations lead to the postulation that Dpp generates diversified insect wing vein patterns through species-specific positional information of its directional transport. Extension of these observations in some winged insects will provide further insights into the mechanisms underlying diversified wing venation among insects. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  2. Does Size Matter? Atmospheric CO2 May Be a Stronger Driver of Stomatal Closing Rate Than Stomatal Size in Taxa That Diversified under Low CO2

    Science.gov (United States)

    Elliott-Kingston, Caroline; Haworth, Matthew; Yearsley, Jon M.; Batke, Sven P.; Lawson, Tracy; McElwain, Jennifer C.

    2016-01-01

    One strategy for plants to optimize stomatal function is to open and close their stomata quickly in response to environmental signals. It is generally assumed that small stomata can alter aperture faster than large stomata. We tested the hypothesis that species with small stomata close faster than species with larger stomata in response to darkness by comparing rate of stomatal closure across an evolutionary range of species including ferns, cycads, conifers, and angiosperms under controlled ambient conditions (380 ppm CO2; 20.9% O2). The two species with fastest half-closure time and the two species with slowest half-closure time had large stomata while the remaining three species had small stomata, implying that closing rate was not correlated with stomatal size in these species. Neither was response time correlated with stomatal density, phylogeny, functional group, or life strategy. Our results suggest that past atmospheric CO2 concentration during time of taxa diversification may influence stomatal response time. We show that species which last diversified under low or declining atmospheric CO2 concentration close stomata faster than species that last diversified in a high CO2 world. Low atmospheric [CO2] during taxa diversification may have placed a selection pressure on plants to accelerate stomatal closing to maintain adequate internal CO2 and optimize water use efficiency. PMID:27605929

  3. Does size matter? Atmospheric CO2 may be a stronger driver of stomatal closing rate than stomatal size in taxa that diversified under low CO2.

    Directory of Open Access Journals (Sweden)

    Caroline Elliott-Kingston

    2016-08-01

    Full Text Available (1 One strategy for plants to optimise stomatal function is to open and close their stomata quickly in response to environmental signals. It is generally assumed that small stomata can alter aperture faster than large stomata. (2 We tested the hypothesis that species with small stomata close faster than species with larger stomata in response to darkness by comparing rate of stomatal closure across an evolutionary range of species including ferns, cycads, conifers and angiosperms under controlled ambient conditions (380ppm CO2; 20.9% O2. (3 The two species with fastest half-closure time and the two species with slowest half-closure time had large stomata while the remaining three species had small stomata, implying that closing rate was not correlated with stomatal size in these species. Neither was response time correlated with stomatal density, phylogeny, functional group or life strategy. (4 Our results suggest that past atmospheric CO2 concentration during time of taxa diversification may influence stomatal response time. We show that species which last diversified under low or declining atmospheric CO2 concentration close stomata faster than species that last diversified in a high CO2 world. Low atmospheric [CO2] during taxa diversification may have placed a selection pressure on plants to accelerate stomatal closing to maintain adequate internal CO2 and optimise water use efficiency.

  4. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    Science.gov (United States)

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  5. Mobility dependent recombination models for organic solar cells

    Science.gov (United States)

    Wagenpfahl, Alexander

    2017-09-01

    Modern solar cell technologies are driven by the effort to enhance power conversion efficiencies. A main mechanism limiting power conversion efficiencies is charge carrier recombination which is a direct function of the encounter probability of both recombination partners. In inorganic solar cells with rather high charge carrier mobilities, charge carrier recombination is often dominated by energetic states which subsequently trap both recombination partners for recombination. Free charge carriers move fast enough for Coulomb attraction to be irrelevant for the encounter probability. Thus, charge carrier recombination is independent of charge carrier mobilities. In organic semiconductors charge carrier mobilities are much lower. Therefore, electrons and holes have more time react to mutual Coulomb-forces. This results in the strong charge carrier mobility dependencies of the observed charge carrier recombination rates. In 1903 Paul Langevin published a fundamental model to describe the recombination of ions in gas-phase or aqueous solutions, known today as Langevin recombination. During the last decades this model was used to interpret and model recombination in organic semiconductors. However, certain experiments especially with bulk-heterojunction solar cells reveal much lower recombination rates than predicted by Langevin. In search of an explanation, many material and device properties such as morphology and energetic properties have been examined in order to extend the validity of the Langevin model. A key argument for most of these extended models is, that electron and hole must find each other at a mutual spatial location. This encounter may be limited for instance by trapping of charges in trap states, by selective electrodes separating electrons and holes, or simply by the morphology of the involved semiconductors, making it impossible for electrons and holes to recombine at high rates. In this review, we discuss the development of mobility limited

  6. Evidence of recombination and positive selection in cetacean papillomaviruses

    Energy Technology Data Exchange (ETDEWEB)

    Robles-Sikisaka, Refugio, E-mail: refugio.robles1@gmail.com [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Rivera, Rebecca, E-mail: RRivera@hswri.org [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Nollens, Hendrik H., E-mail: Hendrik.Nollens@SeaWorld.com [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); College of Veterinary Medicine, University of Florida, PO Box 110885, Gainesville, FL 32611 (United States); SeaWorld San Diego, 500 SeaWorld Drive, San Diego, CA 92109 (United States); St Leger, Judy, E-mail: Judy.St.Leger@SeaWorld.com [SeaWorld San Diego, 500 SeaWorld Drive, San Diego, CA 92109 (United States); Durden, Wendy N., E-mail: WNoke@hswri.org [Hubbs-SeaWorld Research Institute, 3830 South Highway A1A 4-181, Melbourne Beach, FL 32951 (United States); Stolen, Megan, E-mail: MStolen@hswri.org [Hubbs-SeaWorld Research Institute, 3830 South Highway A1A 4-181, Melbourne Beach, FL 32951 (United States); Burchell, Jennifer, E-mail: JBurchell@hswri.org [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Wellehan, James F.X., E-mail: WellehanJ@ufl.edu [College of Veterinary Medicine, University of Florida, PO Box 110885, Gainesville, FL 32611 (United States)

    2012-06-05

    Papillomaviruses (PVs) are small DNA viruses that have been associated with increased epithelial proliferation. Over one hundred PV types have been identified in humans; however, only three have been identified in bottlenose dolphins (Tursiops truncatus) to date. Using rolling circle amplification and degenerate PCR, we identified four novel PV genomes of bottlenose dolphins. TtPV4, TtPV5 and TtPV6 were identified in genital lesions while TtPV7 was identified in normal genital mucosa. Bayesian analysis of the full-length L1 genes found that TtPV4 and TtPV7 group within the Upsilonpapillomavirus genus while TtPV5 and TtPV6 group with Omikronpapillomavirus. However, analysis of the E1 gene did not distinguish these genera, implying that these genes may not share a common history, consistent with recombination. Recombination analyses identified several probable events. Signals of positive selection were found mostly in the E1 and E2 genes. Recombination and diversifying selection pressures constitute important driving forces of cetacean PV evolution.

  7. Experimental estimation of effective recombination coefficients in the D-region ionosphere at high latitudes during solar eclipses by the method of partial reflections

    Directory of Open Access Journals (Sweden)

    Chernyakov S. M.

    2017-03-01

    Full Text Available The photochemical theory of processes in the lower ionosphere is very complicated and up to now it is not completely developed. Therefore introduction of the effective coefficients determining the total speed of several important reactions has been widely adopted when modeling the D-region of the ionosphere. Experimental opportunities for obtaining effective recombination coefficients are rather limited. One of the methods to estimate effective recombination coefficients uses the phenomenon of a solar eclipse. The basis of this method is the idea of Appleton about similarity of the behavior of the linear inductive circuit and variations of the electron concentration in the ionosphere on a fixed height in the absence of the transport processes, the change in the rate of formation of electrons in time and the disappearance of free electrons due to recombination. By analogy with the time constant of the electric circuit Appleton called the reaction of the ionosphere on the process of ionization in the ionosphere as "sluggishness" with a characteristic time constant τ, which is also called the "relaxation time" or "time constant of the ionosphere". During 11 August 1999, 1 August 2008, 11 June 2011, 20 March 2015 solar eclipses at the partial reflection facility of the observatory "Tumanny" (69.0N, 35.7E observations of the amplitudes of reflections of ordinary and extraordinary waves have been carried out. Using the obtained data the two-dimensional (time, height distribution of the electron density ne at altitudes of the D-region ionosphere has been calculated. This has made it possible to obtain the behavior of the electron concentration in time at selected altitudes (temporal profiles of electron density at selected altitudes. Using the obtained experimental profiles, the effective recombination coefficients on the heights of the D-region ionosphere have been evaluated. Transport processes of plasma (for example, propagation of acoustic

  8. Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

    Directory of Open Access Journals (Sweden)

    Nathalie Kin

    2015-05-01

    Full Text Available Human coronavirus OC43 (HCoV-OC43 is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses.

  9. Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

    NARCIS (Netherlands)

    Houwelingen, van A.M.M.L.; Saeger, de T.; Rusanova, T.; Waalwijk, C.; Beekwilder, M.J.

    2008-01-01

    To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin

  10. Comparison of therapeutic efficacy and clinical parameters between recombinant human thyroid stimulating hormone and thyroid hormone withdrawal in high-dose radioiodine treatment with differentiated thyroid cancer

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Se Hun; Na, Chang Ju; Kim, Jeong Hun; Han, Yeon Hee; KIm, Hee Kwon; Jeong, Hwan Jeong; Sohn, Myung Hee; Lim, Seok Tae [Dept. of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju (Korea, Republic of)

    2015-06-15

    High-dose radioiodine treatment (HD-RIT) after injection of recombinant human thyroid stimulating hormone (rh-TSH) has become widely used. This study compared the therapeutic efficacy of HD-RIT and clinical parameters between rh-TSH supplement and thyroid hormone withdrawal (THW) after total thyroidectomy in patients with differentiated thyroid cancer. We retrospectively reviewed 266 patients (47 male and 219 female; age, 49.0 ± 10.9 years) with differentiated thyroid cancer detected from September 2011 to September 2012. Patients comprised THW (217, 81.6 %) and rh-TSH (49, 18.4 %). Inclusion criteria were: first HD-RIT; any TN stage; absence of distant metastasis. To evaluate the complete ablation of the remnant thyroid tissue or metastasis, we reviewed stimulated serum thyroglobulin (sTg), I-123 whole-body scan (RxWBS) on T4 off-state, and thyroid ultrasonography (US) or [F-18]-fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT) 6–8 months after HD-RIT. We defined a complete ablation state when all three of the follow-up conditions were satisfied; <2.0 ng/ml of the sTg, I-123 RxWBS (−), and thyroid US or F-18 FDG PET/CT (−). If one of the three was positive, ablation was considered incomplete. We also compared various clinical biomarkers (body weight, body mass index, liver and kidney function) between THW and rh-TSH groups. The rates of complete ablation were 73.7 % (160/217) for the THW group and 73.5 % (36/49) for the rh-TSH group. There was no significant difference between the two groups (p = 0.970). The follow-up aspartate transaminase (p = 0.001) and alanine transaminase (p = 0.001) were significantly higher in the THW group. The renal function parameters of blood urea nitrogen (p = 0.001) and creatinine (p = 0.005) tended to increase in the THW group. The change of body weight was + Δ0.96 (±1.9) kg for the THW group and was decreased by -Δ1.39 (±1.5) kg for the rh-TSH group. The change

  11. Interaction of Reactive Gas Flows and Ceramics at High Temperature - Experimental Methods for the Measurement of Species Recombination during Planetary Entry

    Science.gov (United States)

    2006-02-01

    Species Recombination during Planetary Entry Marianne BALAT-PICHELIN Laboratoire Procédés, Matériaux et Energie Solaire , PROMES-CNRS, UPR 8521 rue du...PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Laboratoire Procédés, Matériaux et Energie Solaire , PROMES-CNRS, UPR 8521 rue du four solaire 66120...four solaire 66120 Font-Romeu Odeillo France Tél : +33 468 307 768 Fax : +33 468 302 940 balat@promes.cnrs.fr 1. INTRODUCTION During the

  12. CRMAGE: CRISPR Optimized MAGE Recombineering

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

  13. East and central farming and forest region and Atlantic basin diversified farming region: LRRs N and S

    Science.gov (United States)

    Brad D. Lee; John M. Kabrick

    2017-01-01

    The central, unglaciated US east of the Great Plains to the Atlantic coast corresponds to the area covered by LRR N (East and Central Farming and Forest Region) and S (Atlantic Basin Diversified Farming Region). These regions roughly correspond to the Interior Highlands, Interior Plains, Appalachian Highlands, and the Northern Coastal Plains.

  14. High resolution spectral signatures of X-ray emission following charge exchange recombination between highly charged iron and neutral helium, molecular hydrogen and molecular nitrogen: A comparison between theory and experiment

    Science.gov (United States)

    Brown, Gregory V.; Cumbee, Renata; Gu, Liyi; Kelley, Richard L.; Kilbourne, Caroline; Leutenegger, Maurice A.; Porter, Frederick S.; Beiersdorfer, Peter

    2017-08-01

    We have used the LLNL electron beam ion trap EBIT-I and a NASA/GSFC quantum microcalorimeter to measure the X-ray emission following charge exchange recombination between highly charged Fe25+ and Fe 26+ and neutral helium, molecular hydrogen, and molecular nitrogen. The ~ 5 eV energy resolution of the microcalorimeter has made it possible to measure and resolve n to 1 K-shell transitions from up to n = 14. We compare the measurements to a model based on the Landau-Zener theory and also the models found in SPEX and APEC. Our results include relative intensities of the 1P1 resonance line to the 3S1 forbidden line, commonly referred to as lines w and z. These results are especially useful for interpreting spectra from celestial sources measured with XARM's Resolve and ATHENA's X-IFU. These data have also proved useful in the interpretation of Hitomi's SXS spectrum of the Perseus cluster.Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

  15. Greenhouse gas emissions and global warming potential of traditional and diversified tropical rice rotation systems.

    Science.gov (United States)

    Weller, Sebastian; Janz, Baldur; Jörg, Lena; Kraus, David; Racela, Heathcliff S U; Wassmann, Reiner; Butterbach-Bahl, Klaus; Kiese, Ralf

    2016-01-01

    Global rice agriculture will be increasingly challenged by water scarcity, while at the same time changes in demand (e.g. changes in diets or increasing demand for biofuels) will feed back on agricultural practices. These factors are changing traditional cropping patterns from double-rice cropping to the introduction of upland crops in the dry season. For a comprehensive assessment of greenhouse gas (GHG) balances, we measured methane (CH4 )/nitrous oxide (N2 O) emissions and agronomic parameters over 2.5 years in double-rice cropping (R-R) and paddy rice rotations diversified with either maize (R-M) or aerobic rice (R-A) in upland cultivation. Introduction of upland crops in the dry season reduced irrigation water use and CH4 emissions by 66-81% and 95-99%, respectively. Moreover, for practices including upland crops, CH4 emissions in the subsequent wet season with paddy rice were reduced by 54-60%. Although annual N2 O emissions increased two- to threefold in the diversified systems, the strong reduction in CH4 led to a significantly lower (P < 0.05) annual GWP (CH4  + N2 O) as compared to the traditional double-rice cropping system. Measurements of soil organic carbon (SOC) contents before and 3 years after the introduction of upland crop rotations indicated a SOC loss for the R-M system, while for the other systems SOC stocks were unaffected. This trend for R-M systems needs to be followed as it has significant consequences not only for the GWP balance but also with regard to soil fertility. Economic assessment showed a similar gross profit span for R-M and R-R, while gross profits for R-A were reduced as a consequence of lower productivity. Nevertheless, regarding a future increase in water scarcity, it can be expected that mixed lowland-upland systems will expand in SE Asia as water requirements were cut by more than half in both rotation systems with upland crops. © 2015 John Wiley & Sons Ltd.

  16. Energy auditing of diversified rice-wheat cropping systems in Indo-gangetic plains

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhary, V.P.; Gangwar, B.; Pandey, D.K.; Gangwar, K.S. [Project Directorate for Cropping Systems Research, Modipuram, Meerut 250110 (U.P.) (India)

    2009-09-15

    The field investigations were carried out for energy use analysis in terms of different input requirements and outputs harvested under the diversified rice-wheat cropping systems at the research farm of Project Directorate for Cropping Systems Research, Modipuram, Meerut, India during the year 2000-2004. The experiments were conducted on rice (Oryza sativa L.)-wheat (Triticum aestivum L. emend. Fiori and Paol) system involving 8 sequences using diversification, furrow irrigated raised bed system (FIRB) of sowing wheat, use of summer period for deep ploughing or raising legume crops for seed or green manure to study the energy dynamics of different diversified cropping systems. Results revealed that total energy use was highest in rice-potato-wheat (i.e. 77,601 MJ/ha in flat bed and 75,697 MJ/ha in raised bed) followed by rice-wheat-sesbania (i.e. 48,770 MJ/ha in flat and 47,830 MJ/ha in raised bed) and rice-wheat-greengram (i.e. 48,414 MJ/ha in flat and 47,482 MJ/ha in raised bed). In overall, the raised bed sowing of wheat in the cropping system consumed 6-11% less fertilizer energy than flat bed while saved up to 4.2% energy through irrigation. The total output energy of the system was recorded significantly higher in rice-potato-wheat system (i.e. 222,836 MJ/ha in flat bed and 218,065 MJ/ha in raised bed) in comparison to rice-wheat-greengram (i.e. 177,477 MJ/ha in flat bed and 175,125 MJ/ha in raised bed), rice-wheat-sesbania (i.e. 172,000 MJ/ha in flat bed and 168,919 MJ/ha in raised bed) and rice-wheat system (i.e. 156,085 MJ/ha in flat bed and 151,862 MJ/ha in raised bed). The significantly higher net return of energy was obtained in rice-potato-wheat system as compared to other systems. This system required about 75% more input energy but provided about 42% more output energy compared to conventional rice-wheat system. About 10% higher output energy was obtained through growing greengram in summer for grain and foliage incorporation while 14% gain obtained

  17. Surface recombination velocity of silicon wafers by photoluminescence

    Science.gov (United States)

    Baek, D.; Rouvimov, S.; Kim, B.; Jo, T.-C.; Schroder, D. K.

    2005-03-01

    Photoluminescence (PL) and optical reflection measurements, obtained in the two-wavelength SiPHER PL instrument, are used to determine the surface recombination velocity of silicon wafers. Local measurements and contour maps are possible allowing surface recombination maps to be displayed. This instrument also allows doping and trap density measurements. Surface recombination velocities from 10 to 106cm/s can be measured on low or high resistivity polished and epitaxial wafers.

  18. Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

    Science.gov (United States)

    Nakamura, Miki; Suetsugu, Atsushi; Hasegawa, Kousuke; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Hoffman, Robert M

    2017-12-01

    The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Production of human recombinant bone morphogenetic protein-2A by high density culture of Escherichia coli with stationary dissolved oxygen fed-batch condition.

    Science.gov (United States)

    Li, M; Chen, C; Pu, Q; Chen, S

    1998-01-01

    The optimization of cultivation condition in 500 ml shake flasks was carried out to produce recombinant human bone morphogenetic protein-2A (BMP-2A) in recombinant Escherichia coli YK537/pDH-B2m, followed by a 5L fermenter batch and condition-controlled fed-batch culture to obtain BMP-2A. The comparison of these two methods indicated that cultivation by keeping dissolved oxygen at 30%-40% and limiting glucose concentration could obtain BMP-2A 2.78 g/L broth, the final cell density was OD600 53 (dry cell weight 21.2 g/L), and expressed BMP-2A was 25% of the total amount of protein in E.coli. The critical fermentation conditions included: (1) keeping appropriate dissolved oxygen concentration in the process; (2) limiting glucose concentration; (3) taking heat induction at the middle-log phase and maintaining 42 degrees C for 4 hours; (4) controlling specific growth rate around 0.3 h-1 in the duration of growth.

  20. Analysis of Adaptive Evolution in Lyssavirus Genomes Reveals Pervasive Diversifying Selection during Species Diversification

    Directory of Open Access Journals (Sweden)

    Carolina M. Voloch

    2014-11-01

    Full Text Available Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G, RNA-dependent RNA polymerase (L and polymerase (P genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.

  1. A survey of Populus PIN-FORMED family genes reveals their diversified expression patterns.

    Science.gov (United States)

    Liu, Bobin; Zhang, Jin; Wang, Lin; Li, Jianbo; Zheng, Huanquan; Chen, Jun; Lu, Mengzhu

    2014-06-01

    The plant hormone auxin is a key regulator of plant development, and its uneven distribution maintained by polar intercellular auxin transport in plant tissues can trigger a wide range of developmental processes. Although the roles of PIN-FORMED (PIN) proteins in intercellular auxin flow have been extensively characterized in Arabidopsis, their roles in woody plants remain unclear. Here, a comprehensive analysis of PIN proteins in Populus is presented. Fifteen PINs are encoded in the genome of Populus, including four PIN1s, one PIN2, two PIN3s, three PIN5s, three PIN6s, and two PIN8s. Similar to Arabidopsis AtPIN proteins, PtPINs share conserved topology and transmembrane domains, and are either plasma membrane- or endoplasmic reticulum-localized. The more diversified expansion of the PIN family in Populus, comparing to that in Arabidopsis, indicates that some auxin-regulated developmental processes, such as secondary growth, may exhibit unique features in trees. More importantly, different sets of PtoPINs have been found to be strongly expressed in the roots, leaves, and cambium in Populus; the dynamic expression patterns of selected PtoPINs were further examined during the regeneration of shoots and roots. This genome-wide analysis of the Populus PIN family provides important cues for their potential roles in tree growth and development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  2. Whole-farm mating disruption to manage Grapholita molesta (Lepidoptera: Tortricidae) in diversified New Jersey orchards.

    Science.gov (United States)

    Tollerup, Kris E; Rucker, Ann; Shearer, Peter W

    2012-10-01

    Fruit orchards in New Jersey are usually isolated from neighboring farms and diversified, often containing separate plantings of peach (Prunus spp.) and apple (Malus spp.). These crops can suffer significant damage from oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae). This study evaluated the effect of managing G. molesta by using sex pheromone-based mating disruption applied to both peaches and apples (whole-farm mating disruption) rather than treating either crop alone. In year 1 of the experiment, G. molesta mating disruption applied to the adjacent peach and apple blocks provided better control than treating peaches or apples alone. During year 2, treating these adjacent blocks or only treating apples controlled G. molesta equally well. G. molesta populations were so low at the end of year 2 that mating disruption was not applied against this pest during year 3. This allowed us to determine whether applying mating disruption for two consecutive years controlled G. molesta well enough that it eliminated the need mating disruption for three consecutive years. The mean cumulative number of G. molesta captured in plots where both peaches and apples had been treated did not exceed two moths per trap in the third year of this experiment. In contrast, G. molesta capture rebounded during August in peaches and apples that had not been treated with mating disruption the previous 2 yr. Implications for managing G. molesta by using mating disruption as a "whole-farm" tactic as well applying it for two consecutive years and not a third year are discussed.

  3. Perspective on the energy future of the Northeast United States. [Reduce demand and develop diversified supplies

    Energy Technology Data Exchange (ETDEWEB)

    Brainard, J.; Davitian, H.; Goettle, R. IV; Palmedo, P.F.

    1976-06-01

    This study was undertaken to examine the implications of alternative energy futures for the northeastern U.S. First, the past and present energy supply and demand patterns for the U.S. and the northeast region are reviewed. Then, on the basis of detailed analyses of present and possible future supply and demand activities, scenarios for the years 1985 and 2000 are constructed and compared to examine the implications of various policies that will affect future supply and demand activities. Economic and environmental consequences are also discussed. The principal findings of the study are these: (1) conservation measures can reduce fuel and resource requirements in the northeast by over 30 percent; (2) oil imports are likely to continue to be a major energy resource for the northeast since only if strong conservation measures are combined with large increases in U.S. energy supplies is there apt to be a substantial decline in oil imports to the region; (3) a shift to coal and other alternate energy supplies, coupled with increased conservation, could compensate for a curtailment in the use of nuclear power in the region; (4) new resource technologies are capable of supplying up to 20 percent of the region's energy requirements in 2000; (5) no single supply technology or single conservation strategy taken alone can reduce the region's increasing dependence on foreign oil. Rather, the creation of an acceptable energy system for the region will require efforts in many directions in terms both of reducing demand and developing reliable, diversified supplies.

  4. Financing the Commerce – Creating Capacities and Diversifying the Offer within the National Financial Field

    Directory of Open Access Journals (Sweden)

    Irina-Stefana CIBOTARIU

    2010-08-01

    Full Text Available Romania has always aimed for creating a friendly financial sector for the exporters,which has to be able to contribute to an increased national competitiveness. In order to have a relevant strategic answer for this sector, the main objectives, such as increasingthe budget resources to finance the exports and activities that have been carried out for these, the easier access to credits for the export, structured dialogue and a better communication and cooperation between banks, insurance companies and exporters orincreased management ability of the risk have to be carried out within strategic sectors. The Romanian vision on financing the Romanian exports is represented by the contribution for an increased national competitiveness. The globalization process of theworld economy has strengthened the competition of the international trade, especially on diversifying the financial facilities and payments. Within this context, successful promotion of the Romanian exports has depended not only on quality, price, delivery conditions and warranties, but also on a competitive financial offer, in order to credit the importers.

  5. A diversified approach to evaluate biostimulation and bioaugmentation strategies for heavy-oil-contaminated soil.

    Science.gov (United States)

    Lladó, S; Solanas, A M; de Lapuente, J; Borràs, M; Viñas, M

    2012-10-01

    A diversified approach involving chemical, microbiological and ecotoxicity assessment of soil polluted by heavy mineral oil was adopted, in order to improve our understanding of the biodegradability of pollutants, microbial community dynamics and ecotoxicological effects of various bioremediation strategies. With the aim of improving hydrocarbon degradation, the following bioremediation treatments were assayed: i) addition of inorganic nutrients; ii) addition of the rhamnolipid-based biosurfactant M(AT10); iii) inoculation of an aliphatic hydrocarbon-degrading microbial consortium (TD); and iv) inoculation of a known hydrocarbon-degrading white-rot fungus strain of Trametes versicolor. After 200 days, all the bioremediation assays achieved between 30% and 50% total petroleum hydrocarbon (TPH) biodegradation, with the T. versicolor inoculation degrading it the most. Biostimulation and T. versicolor inoculation promoted the Brevundimonas genus concurrently with other α-proteobacteria, β-proteobacteria and Cytophaga-Flexibacter-Bacteroides (CFB) as well as Actinobacteria groups. However, T. versicolor inoculation, which produced the highest hydrocarbon degradation in soil, also promoted autochthonous Gram-positive bacterial groups, such as Firmicutes and Actinobacteria. An acute toxicity test using Eisenia fetida confirmed the improvement in the quality of the soil after all biostimulation and bioaugmentation strategies. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Patterns and role of diversifying selection in the evolution of Toxoplasma gondii SAG5 locus.

    Science.gov (United States)

    Elsheikha, Hany M; Zhao, Xiangrong

    2008-06-01

    The higher intergenotypic polymorphism of the surface antigen genes 5 (SAG5)A, SAG5C, and SAG5E in Toxoplasma gondii was proposed to be the outcome of positive selection pressure favoring variation within these loci. However, the exact nature and magnitude of this selection is not completely known. To address this issue, the amino acids on which natural selection may operate were identified by comparing the ratios of nonsynonymous and synonymous substitutions (pN/pS) of homologous DNA sequences in strains belonging to the three major genotypes of T. gondii. Both positive and negative selections were detected and are likely to have contributed to shaping the patterns of nucleotide substitution and polymorphism in SAG5 genes. Several sites identified in SAG5 loci as likely to be under positive selection suggesting that diversifying selection may have promoted divergence in these genes. Also, it was noted that the SAG5 genetic loci contain many areas that exhibit signs of purifying selection; some of these areas might be the attractive candidates for drug targets. Phylogenetic analysis using the neighbor-joining and maximum parsimony methods grouped the SAG5 sequences of T. gondii strains into three distinct statistically well-supported evolutionary lineages. These findings carry important implications for human and veterinary toxoplasmosis epidemiology and may provide important insights into the pathways through which virulence has evolved in T. gondii.

  7. Diversifying Employment Opportunities of Urban Planning Graduates in the Period of Uncertainty

    Directory of Open Access Journals (Sweden)

    Abdul Razak Jaffar

    2014-11-01

    Full Text Available Several countries, including Malaysia, have set a long term target of establishing a more educated workforce. This thrust on the massification of higher education has resulted in a new problem-graduate unemployment. The growing problem of graduate unemployment in Malaysia is widely debated in the media and blogs. The Higher Education Ministry, in its Graduate Tracer Study Report 2011, reported that 24 per cent of them have not found a job after six months of graduating. The employment scenario of the urban planning graduates has changed over the years from catering the needs of the public sector, to catering the needs of the private sector and to fulfilling the need of the One Stop Centre or the OSC. The advent of the liberalization of urban planning services challenges urban planning schools in Malaysia to produce planners not only to cater for local needs but also with the capability of exporting their skills and services internationally. Given the diverse scope of urban planning it is a paradox that graduates of urban planning should converge on the conventional urban planning organizations for employment. The paper will highlight some preliminary findings on the employment prospect of urban planning graduates in the immediate future and the possibility of diversifying employment opportunities of urban planning graduates.

  8. Graded Recombination Layers for Multijunction Photovoltaics

    KAUST Repository

    Koleilat, Ghada I.

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. © 2012 American Chemical Society.

  9. High plasma tumor necrosis factor (TNF)-alpha concentrations and a sepsis-like syndrome in patients undergoing hyperthermic isolated limb perfusion with recombinant TNF-alpha, interferon-gamma, and melphalan

    NARCIS (Netherlands)

    Zwaveling, JH; Maring, JK; Clarke, FL; vanGinkel, RJ; Limburg, PC; Hoekstra, HJ; Girbes, ARJ; Schraffordt Koops, H.

    Objectives: To describe the postoperative course of patients who underwent hyperthermic isolated limb perfusion with recombinant tumor necrosis factor (TNF)-alpha and melphalan after pretreat ment with recombinant interferon-gamma as treatment for recurrent melanoma, primary nonresectable

  10. High efficiency ZnO-based dye-sensitized solar cells with a 1H,1H,2H,2H-perfluorodecyltriethoxysilane chain barrier for cutting on interfacial recombination

    Science.gov (United States)

    Xie, Yahong; Zhou, Xiaofeng; Mi, Hongyu; Ma, Junhong; Yang, Jianya; Cheng, Jian

    2018-03-01

    Charge recombination at the ZnO photoanode/electrolyte interface is one of the major limitations for high performance dye-sensitized solar cells (DSSCs) toward their theoretical power conversion efficiency (PCE). Here, we proposed an efficient approach for reducing this interfacial losses and consequently facilitating charge transfer by decorating a hydrophobic thin-film on the surface of the dye-coated zinc oxide photoanode via 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES) hexane solution immersing. As a result, a high PCE of 8.22% was obtained, which far exceeded the efficiency of 5.40% in a conventional DSSC without PFDTES treatment. Furthermore, PFDTES treatment also largely elongated the lifetime of photogenerated electrons, and maintained a good photo-response at the photoelectrode. This work provides a comprehensive explanation of electron injection, transfer and recombination at the ZnO photoanode/electrolyte interface, and a promising strategy to explore high efficiency ZnO-based DSSCs.

  11. Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

    Directory of Open Access Journals (Sweden)

    Joseph A Ross

    2011-07-01

    Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

  12. Bias-Variance Tradeoffs in Recombination Rate Estimation.

    Science.gov (United States)

    Stone, Eric A; Singh, Nadia D

    2016-02-01

    In 2013, we and coauthors published a paper characterizing rates of recombination within the 2.1-megabase garnet-scalloped (g-sd) region of the Drosophila melanogaster X chromosome. To extract the signal of recombination in our high-throughput sequence data, we adopted a nonparametric smoothing procedure, reducing variance at the cost of biasing individual recombination rates. In doing so, we sacrificed accuracy to gain precision-precision that allowed us to detect recombination rate heterogeneity. Negotiating the bias-variance tradeoff enabled us to resolve significant variation in the frequency of crossing over across the garnet-scalloped region. Copyright © 2016 by the Genetics Society of America.

  13. Heterogeneous recombination among Hepatitis B virus genotypes.

    Science.gov (United States)

    Castelhano, Nadine; Araujo, Natalia M; Arenas, Miguel

    2017-10-01

    The rapid evolution of Hepatitis B virus (HBV) through both evolutionary forces, mutation and recombination, allows this virus to generate a large variety of adapted variants at both intra and inter-host levels. It can, for instance, generate drug resistance or the diverse viral genotypes that currently exist in the HBV epidemics. Concerning the latter, it is known that recombination played a major role in the emergence and genetic diversification of novel genotypes. In this regard, the quantification of viral recombination in each genotype can provide relevant information to devise expectations about the evolutionary trends of the epidemic. Here we measured the amount of this evolutionary force by estimating global and local recombination rates in >4700 HBV complete genome sequences corresponding to nine (A to I) HBV genotypes. Counterintuitively, we found that genotype E presents extremely high levels of recombination, followed by genotypes B and C. On the other hand, genotype G presents the lowest level, where recombination is almost negligible. We discuss these findings in the light of known characteristics of these genotypes. Additionally, we present a phylogenetic network to depict the evolutionary history of the studied HBV genotypes. This network clearly classified all genotypes into specific groups and indicated that diverse pairs of genotypes are derived from a common ancestor (i.e., C-I, D-E and, F-H) although still the origin of this virus presented large uncertainty. Altogether we conclude that the amount of observed recombination is heterogeneous among HBV genotypes and that this heterogeneity can influence on the future expansion of the epidemic. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started......-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site....

  15. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

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

    Directory of Open Access Journals (Sweden)

    You Bang-Jau

    2011-07-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    Lee, Meng-Shiou; Hseu, You-Cheng; Lai, Guan-Hua; Chang, Wen-Te; Chen, Hsi-Jien; Huang, Chi-Hung; Lee, Meng-Shiunn; Wang, Min-Ying; Kao, Jung-Yie; You, Bang-Jau; Lin, Wen- Hsin; Lien, Yi-Yang; Lin, Ming-Kuem

    2011-07-23

    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. 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. 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.

  18. Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

    DEFF Research Database (Denmark)

    Næsted, Henrik; Kramhøft, Birte; Lok, F.

    2006-01-01

    of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams a-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 k...... nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants. (c) 2005 Elsevier Inc. All rights reserved....

  19. Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant

    DEFF Research Database (Denmark)

    Mathiesen, Christian K.; Prentoe, Jannick; Meredith, Luke W.

    2015-01-01

    requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE: Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing......UNLABELLED: Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells......, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B...

  20. Recombination in Eukaryotic Single Stranded DNA Viruses

    Directory of Open Access Journals (Sweden)

    Philippe Roumagnac

    2011-09-01

    Full Text Available Although single stranded (ss DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  1. Polyploidization increases meiotic recombination frequency in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Rehmsmeier Marc

    2011-04-01

    Full Text Available Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

  2. Biological Activities of Recombinant Liver X Receptor β- Ligand ...

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, high- quality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  3. Pollen Ingestion by Chrysoperla externa (Hagen) Adults in a Diversified Organic Agroecosystem.

    Science.gov (United States)

    Andrade, K A; Aguiar-Menezes, E L; Gonçalves-Esteves, V; Mendonça, C B F; Vieira, G R M; Melo, S J; Magalhães, J L A; Melo, G J B

    2018-02-01

    Chrysoperla externa (Hagen) larvae prey on pest insects and mites in agroecosystems, and adults mainly feed on pollen, nectar, and honeydew. Therefore, preserving this lacewing in crop systems depends on having plants that provide these resources. The objectives of this research were to identify pollen grains ingested by Ch. externa adults collected in a diversified organic agroecosystem and to evaluate whether there is a difference in the amount of ingested pollen grains between males and females. The adults of Ch. externa were collected in four different crops during 13 months in Seropédica, state of Rio de Janeiro, Brazil, using a collecting net. The adults were killed and underwent acetolysis, in order to recover the pollen in the gut. A total of 37,441 pollen grains from 19 Angiospermae families were found, besides 16 Pteridophyte spores. Among the recognized pollen grains, those of Poaceae were the majority, both in frequency of occurrence (87.5%) and in quantity (33496), and were found and recovered in every month of collection. Females and males ingested, respectively, 71.9 and 28.1% of the total number of Angiospermae pollen grains consumed by both sexes. The highest number of Poaceae pollens was obtained from the females (72.1% of the total number of Poaceae pollen, recovered from females + males). Taken as a whole, this study showed that adults of Ch. externa find possibilities to maintain throughout the year, in different crops, but the main source of pollen to males and females was Poaceae plants.

  4. SUSTAINABLE DIVERSIFIED AGRICULTURE AND LAND MANAGEMENT IN THE HIMALAYA: IMPLICATIONS FOR CLIMATE CHANGE ADAPTATION AND MITIGATION

    Directory of Open Access Journals (Sweden)

    R. M. Bajracharya

    2016-08-01

    Full Text Available The soil and land resources play a vital role in sustaining the local livelihoods of rural communities in the Himalaya. Most of the arable land has already been brought under cultivation, hence the ever-increasing demand for food and fiber has left farmers with no choice but to intensify agriculture. However, producing more crops and greater quantities of food, fiber and other materials on the same parcel of land can to soil fertility and productivity decline with overall degradation of land quality. Therefore, ways and means to intensify agriculture to enhance productivity without degrading the soil and land resource base have become imperative. Agro-forestry, agro-slivi-pastoral systems, and the adoption of a variety of crop, soil and water management and conservation practices offer potential to deliver multiple benefits without sacrificing the very resource upon which the human population depends. Presented herein are findings on approaches to sustainable intensification of agriculture and land management related to soil OM management and C sequestration for multiple benefits, and, agro-forestry as a crop diversification strategy with both livelihood, and climate change adaptation/mitigation benefits. The results indicate that sustainable soil management practices could lead to significant SOC accumulations (4-8 t/ha over 6 yrs. SOC and soil C stocks tend to increase with elevation due to cooler climate and slow decomposition rates. Carbon stocks for the 3 LU types was in the order CF>AF/LH>AG, suggesting that diversified cropping practices including agro-forestry have good potential sequester C while providing livelihood opportunities and climate adaptive capacity for local farming communities. Biochar amendment increased growth of both coffee plants and radish with mixed grass/weed biochar being most effective. Biochar application also significantly decreased emission of GHGs, especially N2O.

  5. The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

    Science.gov (United States)

    Hobbhahn, Nina; Johnson, Steven D; Bytebier, Benny; Yeung, Edward C; Harder, Lawrence D

    2013-11-01

    The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production. The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species. Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification. Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or

  6. Expression of recombinant Streptokinase from local Egyptian ...

    African Journals Online (AJOL)

    We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian ...

  7. Recombination mechanisms and doping density in silicon

    Science.gov (United States)

    Passari, Luigi; Susi, Enrichetta

    1983-07-01

    Carrier recombination in silicon is analyzed as a function of doping density. Two mechanisms are identified: one for the low concentration range and one, of the Auger type, for the high concentration range. Disagreements with the theoretical predictions for the Auger process are discussed and empirical laws connecting lifetime to dopant concentration are determined.

  8. Purification of human recombinant granulocyte colony stimulating ...

    African Journals Online (AJOL)

    In Escherichia coli, recombinant proteins were produced either as three dimensionally folded forms or as unfolded forms, inclusion body (IB). The formation of IB was a frequent consequence of high-level protein production and inadequacy of folding agents namely chaperones in the cytoplasm. The structure of the protein in ...

  9. Conservation of monasteries by adaptive reuse: diversified program as a source of inspiration in past and future?

    OpenAIRE

    Lens, Karen

    2014-01-01

    Adaptive reuse has proven to be an important strategy in conserving historical buildings. Nevertheless, reprogramming underused religious heritage sites in Western Europe is a sensitive subject because of its sacred aspects, history and connection to the surroundings. They represent strong intangible qualities. What can we learn from this original hybrid structures for adaptive reuse focusing on four monasteries and their diversified programs that made them selfsufficient? White Sisters an...

  10. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  11. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  12. Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

    2017-01-15

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific {sup 13}C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

  13. Pneumococcal Capsule Synthesis Locus cps as Evolutionary Hotspot with Potential to Generate Novel Serotypes by Recombination.

    Science.gov (United States)

    Mostowy, Rafal J; Croucher, Nicholas J; De Maio, Nicola; Chewapreecha, Claire; Salter, Susannah J; Turner, Paul; Aanensen, David M; Bentley, Stephen D; Didelot, Xavier; Fraser, Christophe

    2017-10-01

    Diversity of the polysaccharide capsule in Streptococcus pneumoniae-main surface antigen and the target of the currently used pneumococcal vaccines-constitutes a major obstacle in eliminating pneumococcal disease. Such diversity is genetically encoded by almost 100 variants of the capsule biosynthesis locus, cps. However, the evolutionary dynamics of the capsule remains not fully understood. Here, using genetic data from 4,519 bacterial isolates, we found cps to be an evolutionary hotspot with elevated substitution and recombination rates. These rates were a consequence of relaxed purifying selection and positive, diversifying selection acting at this locus, supporting the hypothesis that the capsule has an increased potential to generate novel diversity compared with the rest of the genome. Diversifying selection was particularly evident in the region of wzd/wze genes, which are known to regulate capsule expression and hence the bacterium's ability to cause disease. Using a novel, capsule-centered approach, we analyzed the evolutionary history of 12 major serogroups. Such analysis revealed their complex diversification scenarios, which were principally driven by recombination with other serogroups and other streptococci. Patterns of recombinational exchanges between serogroups could not be explained by serotype frequency alone, thus pointing to nonrandom associations between co-colonizing serotypes. Finally, we discovered a previously unobserved mosaic serotype 39X, which was confirmed to carry a viable and structurally novel capsule. Adding to previous discoveries of other mosaic capsules in densely sampled collections, these results emphasize the strong adaptive potential of the bacterium by its ability to generate novel antigenic diversity by recombination. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. Resequencing at ≥40-Fold Depth of the Parental Genomes of a Solanum lycopersicum × S. pimpinellifolium Recombinant Inbred Line Population and Characterization of Frame-Shift InDels That Are Highly Likely to Perturb Protein Function.

    Science.gov (United States)

    Kevei, Zoltan; King, Robert C; Mohareb, Fady; Sergeant, Martin J; Awan, Sajjad Z; Thompson, Andrew J

    2015-03-24

    A recombinant in-bred line population derived from a cross between Solanum lycopersicum var. cerasiforme (E9) and S. pimpinellifolium (L5) has been used extensively to discover quantitative trait loci (QTL), including those that act via rootstock genotype, however, high-resolution single-nucleotide polymorphism genotyping data for this population are not yet publically available. Next-generation resequencing of parental lines allows the vast majority of polymorphisms to be characterized and used to progress from QTL to causative gene. We sequenced E9 and L5 genomes to 40- and 44-fold depth, respectively, and reads were mapped to the reference Heinz 1706 genome. In L5 there were three clear regions on chromosome 1, chromosome 4, and chromosome 8 with increased rates of polymorphism. Two other regions were highly polymorphic when we compared Heinz 1706 with both E9 and L5 on chromosome 1 and chromosome 10, suggesting that the reference sequence contains a divergent introgression in these locations. We also identified a region on chromosome 4 consistent with an introgression from S. pimpinellifolium into Heinz 1706. A large dataset of polymorphisms for the use in fine-mapping QTL in a specific tomato recombinant in-bred line population was created, including a high density of InDels validated as simple size-based polymerase chain reaction markers. By careful filtering and interpreting the SnpEff prediction tool, we have created a list of genes that are predicted to have highly perturbed protein functions in the E9 and L5 parental lines. Copyright © 2015 Kevei et al.

  15. Dissociative recombination of HCl+

    Science.gov (United States)

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

    2017-08-01

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

  16. Microbial factories for recombinant pharmaceuticals

    National Research Council Canada - National Science Library

    Ferrer-Miralles, Neus; Domingo-Espín, Joan; Corchero, José Luis; Vázquez, Esther; Villaverde, Antonio

    2009-01-01

    ...-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production...

  17. Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures.

    Science.gov (United States)

    Cecchini, Sylvain; Virag, Tamas; Kotin, Robert M

    2011-08-01

    The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 10(6) cells/ml, ≥ 10(16) purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.

  18. Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to Cisplatin and poly (adp-ribose polymerase inhibition.

    Directory of Open Access Journals (Sweden)

    Francesca Cavallo

    Full Text Available Testicular Germ Cell Tumors (TGCT and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose polymerase (PARP inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.

  19. Economics, energy, and environmental assessment of diversified crop rotations in sub-Himalayas of India.

    Science.gov (United States)

    Singh, Raman Jeet; Meena, Roshan Lal; Sharma, N K; Kumar, Suresh; Kumar, Kuldeep; Kumar, Dileep

    2016-02-01

    Reducing the carbon footprint and increasing energy use efficiency of crop rotations are the two most important sustainability issues of the modern agriculture. Present study was undertaken to assess economics, energy, and environmental parameters of common diversified crop rotations (maize-tomato, and maize-toria-wheat) vis-a-vis traditional crop rotations like maize-wheat, maize + ginger and rice-wheat of the north-western Himalayan region of India. Results revealed that maize-tomato and maize + ginger crop rotations being on par with each other produced significantly higher system productivity in terms of maize equivalent yield (30.2-36.2 t/ha) than other crop rotations (5.04-7.68 t/ha). But interestingly in terms of energy efficiencies, traditional maize-wheat system (energy efficiency 7.9, human energy profitability of 177.8 and energy profitability of 6.9 MJ/ha) was significantly superior over other systems. Maize + ginger rotation showed greater competitive advantage over other rotations because of less consumption of non-renewable energy resources. Similarly, maize-tomato rotation had ability of the production process to exploit natural resources due to 14-38% less use of commercial or purchased energy sources over other crop rotations. Vegetable-based crop rotations (maize + ginger and maize-tomato) maintained significantly the least carbon footprint (0.008 and 0.019 kg CO2 eq./kg grain, respectively) and the highest profitability (154,322 and 274,161 Rs./ha net return, respectively) over other crop rotations. As the greatest inputs of energy and carbon across the five crop rotations were nitrogen fertilizer (15-29% and 17-28%, respectively), diesel (14-24% and 8-19%, respectively) and irrigation (10-27% and 11-44%, respectively), therefore, alternative sources like organic farming, conservation agriculture practices, soil and water conservation measures, rain water harvesting etc. should be encouraged to reduce dependency of direct energy and external

  20. Comparison of the Genetic Recombination Rates of Human Immunodeficiency Virus Type 1 in Macrophages and T Cells†

    OpenAIRE

    Chen, Jianbo; Rhodes, Terence D.; Hu, Wei-Shau

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) exhibits a high level of genetic variation generated by frequent mutation and genetic recombination during reverse transcription. We have measured HIV-1 recombination rates in T cells in one round of virus replication. It was recently proposed that HIV-1 recombines far more frequently in macrophages than in T cells. In an attempt to delineate the mechanisms that elevate recombination, we measured HIV-1 recombination rates in macrophages at three dif...

  1. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    Energy Technology Data Exchange (ETDEWEB)

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  2. Recombinant antithrombin: production and role in cardiovascular disorder.

    Science.gov (United States)

    Levy, J H; Weisinger, A; Ziomek, C A; Echelard, Y

    2001-08-01

    Plasma-derived antithrombin (AT) concentrates have been used for the management of hereditary and acquired deficiencies since the early 1980s. Recombinant versions of other blood factors and their derivatives are increasingly becoming available, providing a safe and abundant supply of these important therapeutics. However, the complexity of the AT molecule and the large doses often required for supplementation treatments preclude the use of traditional cell culture bioreactors for recombinant production. The development of a very efficient expression system has been necessary for the cost-efficient recombinant production of AT. Transgenic production, with its ability to yield high levels of heterologous protein and its scale-up flexibility, is an attractive alternative to plasma fractionation. Purification of recombinant AT from the milk of transgenic dairy goats has been developed to provide a homogeneous, well-defined, and abundant supply of this factor. This article describes the production of recombinant AT and aspects of clinical applications of this molecule to cardiovascular disorders.

  3. Justified chauvinism: advances in defining meiotic recombination through sperm typing.

    Science.gov (United States)

    Carrington, Mary; Cullen, Michael

    2004-04-01

    Sperm typing offers an efficient means of studying the quantitative and qualitative aspects of meiotic recombination that are virtually unapproachable by pedigree analysis. Since the initial development of the technique >10 years ago, several salient findings based on empirically derived recombination data have been described. The precise rates and distributions of recombination have been reported for specific regions of the genome, serving as the prototype for high-resolution genome-wide recombination patterns. Identification and characterization of molecular genetic events, such as unequal crossing over, gene conversion and crossover asymmetry, are under close inspection for the first time as a result of this technology. The influence of these phenomena on the evolution of the genome is of primary interest from a scientific and medical perspective. In this article, we review the novel discoveries in mammalian meiotic recombination that have been revealed through sperm typing.

  4. Recombination-Driven Genome Evolution and Stability of Bacterial Species.

    Science.gov (United States)

    Dixit, Purushottam D; Pang, Tin Yau; Maslov, Sergei

    2017-09-01

    While bacteria divide clonally, horizontal gene transfer followed by homologous recombination is now recognized as an important contributor to their evolution. However, the details of how the competition between clonality and recombination shapes genome diversity remains poorly understood. Using a computational model, we find two principal regimes in bacterial evolution and identify two composite parameters that dictate the evolutionary fate of bacterial species. In the divergent regime, characterized by either a low recombination frequency or strict barriers to recombination, cohesion due to recombination is not sufficient to overcome the mutational drift. As a consequence, the divergence between pairs of genomes in the population steadily increases in the course of their evolution. The species lacks genetic coherence with sexually isolated clonal subpopulations continuously formed and dissolved. In contrast, in the metastable regime, characterized by a high recombination frequency combined with low barriers to recombination, genomes continuously recombine with the rest of the population. The population remains genetically cohesive and temporally stable. Notably, the transition between these two regimes can be affected by relatively small changes in evolutionary parameters. Using the Multi Locus Sequence Typing (MLST) data, we classify a number of bacterial species to be either the divergent or the metastable type. Generalizations of our framework to include selection, ecologically structured populations, and horizontal gene transfer of nonhomologous regions are discussed as well. Copyright © 2017 by the Genetics Society of America.

  5. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [Univ. of Georgia, Athens, GA (United States)

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  6. CATALYTIC RECOMBINER FOR A NUCLEAR REACTOR

    Science.gov (United States)

    King, L.D.P.

    1960-07-01

    A hydrogen-oxygen recombiner is described for use with water-boiler type reactors. The catalyst used is the wellknown platinized alumina, and the novelty lies in the structural arrangement used to prevent flashback through the gas input system. The recombiner is cylindrical, the gases at the input end being deflected by a baffle plate through a first flashback shield of steel shot into an annular passage adjacent to and extending the full length of the housing. Below the baffle plate the gases flow first through an outer annular array of alumina pellets which serve as a second flashback shield, a means of distributing the flowing gases evenly and as a means of reducing radiation losses to the walls. Thereafter the gases flow inio the centrally disposed catalyst bed where recombination is effected. The steam and uncombined gases flow into a centrally disposed cylindrical passage inside the catalyst bod and thereafter out through the exit port. A high rate of recombination is effected.

  7. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    Elizabeth A Specht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for molecular pharming in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  8. Algae-based oral recombinant vaccines

    Science.gov (United States)

    Specht, Elizabeth A.; Mayfield, Stephen P.

    2014-01-01

    Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity. PMID:24596570

  9. Recombination and chiasmata: few but intriguing discrepancies.

    Science.gov (United States)

    Sybenga, J

    1996-06-01

    The paradigm that meiotic recombination and chiasmata have the same basis has been challenged, primarily for plants. High resolution genetic mapping frequently results in maps with lengths far exceeding those based on chiasma counts. In addition, recombination between specific homoeologous chromosomes derived from interspecific hybrids is sometimes much higher than can be explained by meiotic chiasma frequencies. However, almost the entire discrepancy disappears when proper care is taken of map inflation resulting from the shortcomings of the mapping algorithm and classification errors, the use of dissimilar material, and the difficulty of accurately counting chiasmata. Still, some exchanges, especially of short interstitial segments, cannot readily be explained by normal meiotic behaviour. Aberrant meiotic processes involving segment replacement or insertion can probably be excluded. Some cases of unusual recombination are somatic, possibly premeiotic exchange. For other cases, local relaxation of chiasma interference caused by small interruptions of homology disturbing synaptonemal complex formation is proposed as the cause. It would be accompanied by a preference for compensating exchanges (negative chromatid interference) resulting from asymmetry of the pairing chromatid pairs, so that one side of each pair preferentially participates in pairing. Over longer distances, the pairing face may switch, causing the normal random chromatid participation in double exchanges and the relatively low frequency of short interstitial exchanges. Key words : recombination frequency, map length, chiasmata, discrepancy, chromatid interference.

  10. Cloning, recombinant expression and characterization of a new ...

    African Journals Online (AJOL)

    Jane

    2011-06-13

    Jun 13, 2011 ... time-of-flight/time-of-flight; HPLC, high-performance liquid chromatography. ... amylase in engineering microorganism may be a better production .... concentration as test substrate for the recombinant APGA1. Hydrolysis test ...

  11. Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

    Science.gov (United States)

    Weller, Sandra K.; Sawitzke, James A.

    2015-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

  12. Recombination does not hinder formation or detection of ecological species of Synechococcus inhabiting a hot spring cyanobacterial mat

    Directory of Open Access Journals (Sweden)

    Melanie Crystal Melendrez

    2016-01-01

    Full Text Available Recent studies of bacterial speciation have claimed to support the biological species concept—that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B´ of Synechococcus in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B´ lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B´ lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes, and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal

  13. High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs.

    Science.gov (United States)

    Lu, Dan; Li, Qiuyan; Wu, Zhibin; Shang, Shengzhe; Liu, Shen; Wen, Xiao; Li, Zhiyuan; Wu, Fangfang; Li, Ning

    2014-01-01

    Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ) to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and morphology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1) produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34 ± 24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272 ± 26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1), with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (ppigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.

  14. Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior

    OpenAIRE

    Ryan, Tomás J; Kopanitsa, Maksym V.; Indersmitten, Tim; Nithianantharajah, Jess; Afinowi, Nurudeen O; Pettit, Charles; Stanford, Lianne E.; Sprengel, Rolf; Saksida, Lisa M.; Bussey, Timothy J.; O'Dell, Thomas J.; Grant, Seth G.N.; Komiyama, Noboru H.

    2012-01-01

    Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C-terminal domains (CTDs). To identify shared ancestral functions and diversified subunit-specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock-in mice and analyzed the mice's biochemist...

  15. Efficient Inference of Recent and Ancestral Recombination within Bacterial Populations.

    Science.gov (United States)

    Mostowy, Rafal; Croucher, Nicholas J; Andam, Cheryl P; Corander, Jukka; Hanage, William P; Marttinen, Pekka

    2017-05-01

    Prokaryotic evolution is affected by horizontal transfer of genetic material through recombination. Inference of an evolutionary tree of bacteria thus relies on accurate identification of the population genetic structure and recombination-derived mosaicism. Rapidly growing databases represent a challenge for computational methods to detect recombinations in bacterial genomes. We introduce a novel algorithm called fastGEAR which identifies lineages in diverse microbial alignments, and recombinations between them and from external origins. The algorithm detects both recent recombinations (affecting a few isolates) and ancestral recombinations between detected lineages (affecting entire lineages), thus providing insight into recombinations affecting deep branches of the phylogenetic tree. In simulations, fastGEAR had comparable power to detect recent recombinations and outstanding power to detect the ancestral ones, compared with state-of-the-art methods, often with a fraction of computational cost. We demonstrate the utility of the method by analyzing a collection of 616 whole-genomes of a recombinogenic pathogen Streptococcus pneumoniae, for which the method provided a high-resolution view of recombination across the genome. We examined in detail the penicillin-binding genes across the Streptococcus genus, demonstrating previously undetected genetic exchanges between different species at these three loci. Hence, fastGEAR can be readily applied to investigate mosaicism in bacterial genes across multiple species. Finally, fastGEAR correctly identified many known recombination hotspots and pointed to potential new ones. Matlab code and Linux/Windows executables are available at https://users.ics.aalto.fi/~pemartti/fastGEAR/ (last accessed February 6, 2017). © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. Photoionization and electron-ion recombination of P II

    Science.gov (United States)

    Nahar, Sultana N.

    2017-08-01

    A study of the inverse processes of photoionization and electron-ion recombination of P II is reported. Phosphorus, a little studied cosmic element, requires atomic parameters such as those presented here for spectral analysis. The unified method of Nahar and Pradhan, which incorporates two methods of recombination - radiative recombination (RR) and dielectronic recombination (DR) - and the interference between them, is used to obtain the total electron-ion recombination. This method implements the framework of the {R}-matrix close-coupling approximation. The present results include the partial photoionization cross-sections σPI(Jπ) leaving the residual ion in the ground level and level-specific recombination rate coefficients, αRC(Jπ), of 475 fine-structure levels of P II with n ≤10. In photoionization of the ground and many excited levels, a sharp resonance is found to form at the ionization threshold from couplings of relativistic fine-structure channels. These, with other resonances in the near-threshold energy region, yield a slight curvature, in contrast to typical smooth decay, at a very low temperature of about 330 K in the total recombination rate coefficient αRC. The presence of other Rydberg and Seaton resonances in the photoionization cross-section introduces features in the level-specific recombination rate coefficients and a DR bump at high temperature at 105 K for the total recombination rate coefficient. Considerable interference between RR and DR is noted around 6700 K. The recombination spectrum with respect to photoelectron energy αRC(E) is also presented. The results are expected to provide accurate models for astrophysical plasmas up to ˜1 MK.

  17. Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

    Directory of Open Access Journals (Sweden)

    Marie Touchon

    2009-01-01

    Full Text Available The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re- annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species, including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an

  18. Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

    Science.gov (United States)

    Touchon, Marie; Hoede, Claire; Tenaillon, Olivier; Barbe, Valérie; Baeriswyl, Simon; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane; Bouchier, Christiane; Bouvet, Odile; Calteau, Alexandra; Chiapello, Hélène; Clermont, Olivier; Cruveiller, Stéphane; Danchin, Antoine; Diard, Médéric; Dossat, Carole; Karoui, Meriem El; Frapy, Eric; Garry, Louis; Ghigo, Jean Marc; Gilles, Anne Marie; Johnson, James; Le Bouguénec, Chantal; Lescat, Mathilde; Mangenot, Sophie; Martinez-Jéhanne, Vanessa; Matic, Ivan; Nassif, Xavier; Oztas, Sophie; Petit, Marie Agnès; Pichon, Christophe; Rouy, Zoé; Ruf, Claude Saint; Schneider, Dominique; Tourret, Jérôme; Vacherie, Benoit; Vallenet, David; Médigue, Claudine; Rocha, Eduardo P C; Denamur, Erick

    2009-01-01

    The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

  19. Evidence of recombination within human alpha-papillomavirus

    Directory of Open Access Journals (Sweden)

    Carvajal-Rodríguez Antonio

    2007-03-01

    Full Text Available Abstract Background Human papillomavirus (HPV has a causal role in cervical cancer with almost half a million new cases occurring each year. Presence of the carcinogenic HPV is necessary for the development of the invasive carcinoma of the genital tract. Therefore, persistent infection with carcinogenic HPV causes virtually all cervical cancers. Some aspects of the molecular evolution of this virus, as the putative importance of recombination in its evolutionary history, are an opened current question. In addition, recombination could also be a significant issue nowadays since the frequency of co-infection with more than one HPV type is not a rare event and, thus, new recombinant types could be currently being generated. Results We have used human alpha-PV sequences from the public database at Los Alamos National Laboratory to report evidence that recombination may exist in this virus. A model-based population genetic approach was used to infer the recombination signal from the HPV DNA sequences grouped attending to phylogenetic and epidemiological information, as well as to clinical manifestations. Our results agree with recently published ones that use a different methodology to detect recombination associated to the gene L2. In addition, we have detected significant recombination signal in the genes E6, E7, L2 and L1 at different groups, and importantly within the high-risk type HPV16. The method used has recently been shown to be one of the most powerful and reliable procedures to detect the recombination signal. Conclusion We provide new support to the recent evidence of recombination in HPV. Additionally, we performed the recombination estimation assuming the best-fit model of nucleotide substitution and rate variation among sites, of the HPV DNA sequence sets. We found that the gene with recombination in most of the groups is L2 but the highest values were detected in L1 and E6. Gene E7 was recombinant only within the HPV16 type. The

  20. Dissociation of recombinant prion autocatalysis from infectivity

    OpenAIRE

    Noble, Geoffrey P; Supattapone, Surachai

    2015-01-01

    Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication – that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and ...

  1. Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives

    Directory of Open Access Journals (Sweden)

    Anett Stephan

    2017-12-01

    Full Text Available Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA and U. S. Department of Agriculture (USDA as food-processing antibacterials through the GRAS (generally recognized as safe regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins—including biopharmaceuticals, vaccines, and biomaterials—but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.

  2. Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli; Renaturacao em altas pressoes hidrostaticas de proteinas recombinantes agregadas em corpos de inclusao produzidos em Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Keli Nunes

    2009-07-01

    The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

  3. Protection against H7N3 high pathogenicity avian influenza in chickens immunized with a recombinant fowlpox and an inactivated avian influenza vaccines

    Science.gov (United States)

    Beginning on June 2012, an H7N3 highly pathogenic avian influenza (HPAI) epizootic was reported in the State of Jalisco (Mexico), with some 22.4 million chickens that died, were slaughtered on affected farms or were preemptively culled on neighboring farms. In the current study, layer chickens were ...

  4. Rectification of artificial molecular recombination with the use of high fidelity enzyme in the amplification of 16S rDNA sequences from Stool sample

    Directory of Open Access Journals (Sweden)

    Vijay Nema

    2012-06-01

    Full Text Available Reliance on routinely used taq polymerases for amplification may generate spurious sequences, especially in metagenomic studies utilizing complex mixtures of various DNA templates. Use of high fidelity enzymes and verification of the sequences using various software tools before submission to the databases ensures better quality and confidence.

  5. The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA' suppresses growth of a highly metastatic pancreatic carcinoma cell line

    NARCIS (Netherlands)

    Bruell, D; Stocker, M; Huhn, M; Redding, N; Kupper, M; Schumacher, P; Paetz, A; Bruns, CJ; Haisma, HJ; Fischer, R; Finnern, R; Barth, S

    2003-01-01

    Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the

  6. Three Decades of Recombinant DNA.

    Science.gov (United States)

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  7. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  8. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted; Lundin, Luisa

    2012-01-01

    contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in bioreactors...... are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale....

  9. Use of a highly sensitive recombinant hepatoma cell method to determine dioxin concentrations in samples of fish and crab from a hotspot area.

    Science.gov (United States)

    Lin, Ding-Yan; Shy, Cherng-Gueih; Chen, Fu-An; Wang, Ya-Fan; Chen, Kuan-Chung; Hsieh, Lien-Te; Tsai, Feng-Yuan; Tsou, Tsui-Chun; Chao, How-Ran

    2013-06-01

    A new and easy fast-screening test (the Ad-DR (adenoviral vector-dioxin response) bioassay) for dioxins in biological samples from highly dioxin-contaminated areas was developed. The aryl-hydrocarbon-receptor (AhR) reporter system was utilized to transport a dioxin-responsive-element (DRE) via an adenovirus vector into rat hepatoma (H4IIE) cells before each experiment; these DRE-H4IIE cells were utilized in the Ad-DR bioassay. Biological extracts were simultaneously analyzed by the Ad-DR bioassay and high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). A good correlation was found between the results of the HRGC/HRMS assay and those of the Ad-DR bioassay (R(2) = 0.920, p fish or crab caught in the abandoned pentachlorophenol plant (AP) was extremely high compared with the BEQ in fish or crab caught in two rivers nearby this abandoned plant. Dioxins were more heavily bioaccumulated in fish viscera than in fish muscles or in the whole fish. Two-way analysis of variance tests identified the significant effects of fish collection site, fish or crab tissue sample and the interaction between them on dioxin levels in the tissues of these aquatic animals. In conclusion, the Ad-DR bioassay is a useful tool to determine dioxin levels in samples of fish and crab. Compared with fish tissues, where a sample is taken (in the PCP plant or nearby rivers) is the most important factor to determine bioaccumulation of dioxins in fish.

  10. High Frequency of Mosaicism among Patients with Neurofibromatosis Type 1 (NF1) with Microdeletions Caused by Somatic Recombination of the JJAZ1 Gene

    OpenAIRE

    Kehrer-Sawatzki, H; Kluwe, L; Sandig, C.; Kohn, M.; Wimmer, K.; Krammer, U.; Peyrl, A.; Jenne, D E; Hansmann, I; Mautner, V.-F.

    2004-01-01

    Detailed analyses of 20 patients with sporadic neurofibromatosis type 1 (NF1) microdeletions revealed an unexpected high frequency of somatic mosaicism (8/20 [40%]). This proportion of mosaic deletions is much higher than previously anticipated. Of these deletions, 16 were identified by a screen of unselected patients with NF1. None of the eight patients with mosaic deletions exhibited the mental retardation and facial dysmorphism usually associated with NF1 microdeletions. Our study demonstr...

  11. Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation.

    Science.gov (United States)

    Chida, Yasuhito; Takagi, Keiichi; Terada, Satoshi

    2013-12-01

    Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5 Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.

  12. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  13. Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice

    Directory of Open Access Journals (Sweden)

    Fatih M. Uckun

    2015-04-01

    Full Text Available In high-risk remission B-precursor acute lymphoblastic leukemia (BPL patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT even after the use of very intensive total body irradiation (TBI-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand × soluble TRAIL (“CD19L–sTRAIL” fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI combined with CD19L–sTRAIL was highly effective against (1 xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2 radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT.

  14. Crude glycerol from biodiesel as a carbon source for production of a recombinant highly thermostable β-mannanase by Pichia pastoris.

    Science.gov (United States)

    Luo, Zhangcai; Miao, Jing; Luo, Wei; Li, Guoying; Du, Yao; Yu, Xiaobin

    2017-10-12

    To explore an efficient use of crude glycerol for the production of a highly thermostable β-mannanase (ReTMan26) by Pichia pastoris X33. Cell growth was significantly inhibited by 4 and 6% (w/v) crude glycerol in 250 ml shake-flasks and in 5 l bioreactor batch cultures, respectively, but not affected by pure glycerol at the same concentrations. For further study, the impact of various impurities in crude glycerol on the cell growth of, and ReTMan26 production by, Pichia pastoris was investigated. Salts and methanol did not exert an inhibitory effect, but ≥ 0.2% and 0.3% (w/v) soap in shake-flask and bioreactor cultures, respectively, inhibited fermentation. Under identical conditions, the biomass and ReTMan26 activity produced by high-cell-density fermentation using 5% crude glycerol (glycerol at 80%, w/w) were slightly higher than those using 4% (w/v) pure glycerol. Non-pretreated ≤ 5% (w/v) crude glycerol could be effectively utilized for industrial production of ReTMan26, and the total production costs using crude glycerol were ~ 4.2% lower than those using pure glycerol.

  15. The development of a highly specific radiochemical compound based on labeled 99mtc recombinant molecules for targeted imaging of cells with the overexpression of Her-2 / neu

    Directory of Open Access Journals (Sweden)

    Olga D. Bragina

    2017-01-01

    Full Text Available Currently, there is a urgent need to search for new diagnostic methods that allow us to reveal malignant tumors with the overexpression of Her-2/neu with high accuracy. In recent years radioisotope methods have been actively developing to identify specific tumor targets, with antibodies being the “targeting” module.The purpose of the study. Creation of a chemically stable radiochemical compound for the imaging of cells with the overexpression of Her-2/neu.Materials and methods. The study was conducted using two human adenocarcinoma cell lines with expression (BT-474 and without expression (MCF-7 Her-2/neu. The specificity of the binding of the test complex with Her-2/neu receptor was determined by direct radiometric and planar scintigraphy. To evaluate the differences in quantitative characteristics between the groups a non-parametric Mann – Whitney test was used.Results. The yield of the labeled complex was more than 91% and the radiochemical frequency was more than 94%. When performing a visual scintigraphic evaluation, a much higher accumulation rate of the studied radiopharmaceutical preparation (RFP was observed in the culture of cells with overexpression of the surface Her-2/neu receptor. Direct radiometric results also demonstrated a higher accumulation of RFPs in the human BT-474 mammary adenocarcinoma cell line with Her-2/neu overexpression in comparison with the control group.Conclusion. Preclinical studies demonstrated high stability of the test compound, as well as its accumulation in the group of cells with Her-2/neu overexpression

  16. RECOMBINANT HORSERADISH PEROXIDASE FOR ANALYTICAL APPLICATIONS

    Directory of Open Access Journals (Sweden)

    А.M. Egorov

    2012-08-01

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

  17. High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica Using Table Sugar as Carbon Source.

    Science.gov (United States)

    Charoenrat, Theppanya; Antimanon, Sompot; Kocharin, Kanokarn; Tanapongpipat, Sutipa; Roongsawang, Niran

    2016-12-01

    The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.

  18. Recombination dynamics in coalesced a-plane GaN ELO structures investigated by high spatially and ps-time-resolved cathodoluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bastek, B.; Bertram, F.; Christen, J. [Institute of Experimental Physics, Otto-von-Guericke-University Magdeburg (Germany); Wernicke, T.; Weyers, M. [Ferdinand-Braun-Institut fuer Hoechstfrequenztechnik, Berlin (Germany); Kneissl, M. [Institute of Solid State Physics, Technical University, Berlin (Germany)

    2008-07-01

    The characteristic epitaxial lateral overgrowth (ELO) domains of fully coalesced a-plane GaN layers were directly imaged by highly spatially and spectrally resolved cathodoluminescence microscopy (CL) at 5 K. The patterned layers were grown by MOVPE on r-plane sapphire substrate and stripe masks oriented in the [01 anti 10] direction. In the area of coherent growth (I) the broad basal plane stacking fault (BSF) emission centered at 3.41 eV dominates the spectra. Also in the region (II) of coalescence the BSF luminescence dominates, however, the intensity increases by one order of magnitude compared to area (I). In complete contrast, in the stripes associated with the laterally grown domains (III) in [0001] direction, exclusively an intense and sharp (D{sup 0},X) emission at 3.475 eV is observed. ps-time-resolved CL of the free excitons (FX) recorded from this domains (III) decays bi-exponentially. The initial lifetime of 180 ps is primarily given by the capture of FX by impurities to form bound excitons (BE). With rising temperature this capture time constant decreases as T{sup -1/4} and reaches a minimum of 104 ps at T=60 K. Above 60 K, i.e. when FX starts to dominate the BEs, the lifetime increases rapidly to a value of 240 ps for 300 K.

  19. High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs.

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    Dan Lu

    Full Text Available Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and morphology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1 produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34 ± 24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272 ± 26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1, with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (p<0.001 and their villus height to crypt depth ratio in the intestine were increased due to the significant decrease of crypt depth in the duodenum, jejunum, and ileum (p<0.001. Together, we successfully generated rhLZ transgenic cloned pigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.

  20. Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs

    Science.gov (United States)

    Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

    2015-01-01

    ABSTRACT Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against

  1. The role of recombination in the emergence of a complex and dynamic HIV epidemic

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    Morgenstern Burkhard

    2010-03-01

    Full Text Available Abstract Background Inter-subtype recombinants dominate the HIV epidemics in three geographical regions. To better understand the role of HIV recombinants in shaping the current HIV epidemic, we here present the results of a large-scale subtyping analysis of 9435 HIV-1 sequences that involve subtypes A, B, C, G, F and the epidemiologically important recombinants derived from three continents. Results The circulating recombinant form CRF02_AG, common in West Central Africa, appears to result from recombination events that occurred early in the divergence between subtypes A and G, followed by additional recent recombination events that contribute to the breakpoint pattern defining the current recombinant lineage. This finding also corrects a recent claim that G is a recombinant and a descendant of CRF02, which was suggested to be a pure subtype. The BC and BF recombinants in China and South America, respectively, are derived from recent recombination between contemporary parental lineages. Shared breakpoints in South America BF recombinants indicate that the HIV-1 epidemics in Argentina and Brazil are not independent. Therefore, the contemporary HIV-1 epidemic has recombinant lineages of both ancient and more recent origins. Conclusions Taken together, we show that these recombinant lineages, which are highly prevalent in the current HIV epidemic, are a mixture of ancient and recent recombination. The HIV pandemic is moving towards having increasing complexity and higher prevalence of recombinant forms, sometimes existing as "families" of related forms. We find that the classification of some CRF designations need to be revised as a consequence of (1 an estimated > 5% error in the original subtype assignments deposited in the Los Alamos sequence database; (2 an increasing number of CRFs are defined while they do not readily fit into groupings for molecular epidemiology and vaccine design; and (3 a dynamic HIV epidemic context.

  2. The role of recombination in the emergence of a complex and dynamic HIV epidemic.

    Science.gov (United States)

    Zhang, Ming; Foley, Brian; Schultz, Anne-Kathrin; Macke, Jennifer P; Bulla, Ingo; Stanke, Mario; Morgenstern, Burkhard; Korber, Bette; Leitner, Thomas

    2010-03-23

    Inter-subtype recombinants dominate the HIV epidemics in three geographical regions. To better understand the role of HIV recombinants in shaping the current HIV epidemic, we here present the results of a large-scale subtyping analysis of 9435 HIV-1 sequences that involve subtypes A, B, C, G, F and the epidemiologically important recombinants derived from three continents. The circulating recombinant form CRF02_AG, common in West Central Africa, appears to result from recombination events that occurred early in the divergence between subtypes A and G, followed by additional recent recombination events that contribute to the breakpoint pattern defining the current recombinant lineage. This finding also corrects a recent claim that G is a recombinant and a descendant of CRF02, which was suggested to be a pure subtype. The BC and BF recombinants in China and South America, respectively, are derived from recent recombination between contemporary parental lineages. Shared breakpoints in South America BF recombinants indicate that the HIV-1 epidemics in Argentina and Brazil are not independent. Therefore, the contemporary HIV-1 epidemic has recombinant lineages of both ancient and more recent origins. Taken together, we show that these recombinant lineages, which are highly prevalent in the current HIV epidemic, are a mixture of ancient and recent recombination. The HIV pandemic is moving towards having increasing complexity and higher prevalence of recombinant forms, sometimes existing as "families" of related forms. We find that the classification of some CRF designations need to be revised as a consequence of (1) an estimated > 5% error in the original subtype assignments deposited in the Los Alamos sequence database; (2) an increasing number of CRFs are defined while they do not readily fit into groupings for molecular epidemiology and vaccine design; and (3) a dynamic HIV epidemic context.

  3. A cheap, simple high throughput method for screening native Helicobacter pylori urease inhibitors using a recombinant Escherichia coli, its validation and demonstration of Pistacia atlantica methanolic extract effectivity and specificity.

    Science.gov (United States)

    Amar, Natalie; Peretz, Avi; Gerchman, Yoram

    2017-02-01

    Helicobacter pylori is the most frequent and persistent bacterial infection worldwide, and a risk factor for active gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Although combined antibiotics treatment is effective cases of antibiotic resistance are reported at an alarming rate. The H. pylori urease enzyme is essential for the bacteria establishment in the gastric mucosa, resulting urease inhibitors being sought after as effective and specific anti- H. pylori treatment. To-date, screening assays are based mostly on the analog plant urease enzyme but difference in properties of the plant and bacterial enzymes hamper these efforts. We have developed a screening assay based on recombinant Escherichia coli expressing native H. pylori urease, and validated this assay using thiourea and a methanolic extract of Pistacia atlantica. The assay demonstrated the thiourea and the extract to be potent urease inhibitors, with the extract having strong bacteriostatic activity against clinical isolates of H. pylori, including such with antibiotic resistance. The extract was also found to be neutral toward common probiotic bacteria, supporting its specificity and compatibility with digestive system desired microflora and suggesting it could be a good source for anti-H. pylori compounds. The assay has proven to be cheap, simple and native alternative to the plant enzyme based assay and could allow for high throughput screening for new urease inhibitors and could expedite screening and development of novel, better H. pylori remedies helping us to combat this infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors

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    Huixuan Liang

    2012-09-01

    Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs and intermediate neural progenitor cells (NPCs. Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1cre line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs.

  5. Development of Diversified Tourism Destination Products – A Case Study of Tourism Destination, Municipality of Sofia, Bulgaria

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    Elena PETKOVA

    2017-03-01

    Full Text Available In this paper, it is argued that there is a variety of products and accordingly a diversity of types of tourism in the municipality of Sofia, Bulgaria: urban and "non-urban", mass and specialized, tourism based on natural and anthropogenic, on tangible and intangible resources. In this regard, diverse tourism products of the destination may be offered to its visitors, which to a greater extent meets their various needs and contributes to the sustainable tourism development. Thus, the aim of the paper is to reveal whether tourism professionals in Sofia are aware of the possibilities for combining various types of tourism and promoting the diversified destination tourism product among local and foreign visitors.

  6. Diversifying Selection Between Pure-Breed and Free-Breeding Dogs Inferred from Genome-Wide SNP Analysis.

    Science.gov (United States)

    Pilot, Małgorzata; Malewski, Tadeusz; Moura, Andre E; Grzybowski, Tomasz; Oleński, Kamil; Kamiński, Stanisław; Fadel, Fernanda Ruiz; Alagaili, Abdulaziz N; Mohammed, Osama B; Bogdanowicz, Wiesław

    2016-08-09

    Domesticated species are often composed of distinct populations differing in the character and strength of artificial and natural selection pressures, providing a valuable model to study adaptation. In contrast to pure-breed dogs that constitute artificially maintained inbred lines, free-ranging dogs are typically free-breeding, i.e., unrestrained in mate choice. Many traits in free-breeding dogs (FBDs) may be under similar natural and sexual selection conditions to wild canids, while relaxation of sexual selection is expected in pure-breed dogs. We used a Bayesian approach with strict false-positive control criteria to identify FST-outlier SNPs between FBDs and either European or East Asian breeds, based on 167,989 autosomal SNPs. By identifying outlier SNPs located within coding genes, we found four candidate genes under diversifying selection shared by these two comparisons. Three of them are associated with the Hedgehog (HH) signaling pathway regulating vertebrate morphogenesis. A comparison between FBDs and East Asian breeds also revealed diversifying selection on the BBS6 gene, which was earlier shown to cause snout shortening and dental crowding via disrupted HH signaling. Our results suggest that relaxation of natural and sexual selection in pure-breed dogs as opposed to FBDs could have led to mild changes in regulation of the HH signaling pathway. HH inhibits adhesion and the migration of neural crest cells from the neural tube, and minor deficits of these cells during embryonic development have been proposed as the underlying cause of "domestication syndrome." This suggests that the process of breed formation involved the same genetic and developmental pathways as the process of domestication. Copyright © 2016 Pilot et al.

  7. Diversifying Selection Between Pure-Breed and Free-Breeding Dogs Inferred from Genome-Wide SNP Analysis

    Directory of Open Access Journals (Sweden)

    Małgorzata Pilot

    2016-08-01

    Full Text Available Domesticated species are often composed of distinct populations differing in the character and strength of artificial and natural selection pressures, providing a valuable model to study adaptation. In contrast to pure-breed dogs that constitute artificially maintained inbred lines, free-ranging dogs are typically free-breeding, i.e., unrestrained in mate choice. Many traits in free-breeding dogs (FBDs may be under similar natural and sexual selection conditions to wild canids, while relaxation of sexual selection is expected in pure-breed dogs. We used a Bayesian approach with strict false-positive control criteria to identify FST-outlier SNPs between FBDs and either European or East Asian breeds, based on 167,989 autosomal SNPs. By identifying outlier SNPs located within coding genes, we found four candidate genes under diversifying selection shared by these two comparisons. Three of them are associated with the Hedgehog (HH signaling pathway regulating vertebrate morphogenesis. A comparison between FBDs and East Asian breeds also revealed diversifying selection on the BBS6 gene, which was earlier shown to cause snout shortening and dental crowding via disrupted HH signaling. Our results suggest that relaxation of natural and sexual selection in pure-breed dogs as opposed to FBDs could have led to mild changes in regulation of the HH signaling pathway. HH inhibits adhesion and the migration of neural crest cells from the neural tube, and minor deficits of these cells during embryonic development have been proposed as the underlying cause of “domestication syndrome.” This suggests that the process of breed formation involved the same genetic and developmental pathways as the process of domestication.

  8. Treatment with high-dose recombinant human hyaluronidase-facilitated subcutaneous immune globulins in patients with juvenile dermatomyositis who are intolerant to intravenous immune globulins: a report of 5 cases.

    Science.gov (United States)

    Speth, Fabian; Haas, Johannes-Peter; Hinze, Claas H

    2016-09-13

    High-dose intravenous immune globulins (IVIg) are frequently used in refractory juvenile dermatomyositis (JDM) but are often poorly tolerated. High-dose recombinant human hyaluronidase-facilitated subcutaneous immune globulins (fSCIg) allow the administration of much higher doses of immune globulins than conventional subcutaneous immune globulin therapy and may be an alternative to IVIg. The safety and efficacy of fSCIg therapy in JDM is unknown. In this retrospective case series, five patients with steroid-refractory severe JDM were treated with high-dose fSCIg due to IVIg adverse effects (severe headaches, nausea, vomiting, difficult venous access). Peak serum IgG levels, muscle enzymes, the childhood myositis assessment scale and adverse effects were retrieved for at least 6 months following intiation of fSCIg. Data were analyzed by descriptive statistics. Patients initially received fSCIg 1 g/kg every 14 days, resulting in median IgG peak levels of 1901 mg/dl (1606-2719 mg/dl), compared to median IgG peak and trough levels while previously receiving IVIg of 2741 mg/dl (2429-2849 mg/dl) and 1351 mg/dl (1156-1710 mg/dl). Additional antirheumatic therapies consisted of low-dose glucocorticoid therapy, methotrexate, mycophenolate mofetil and/or rituximab. Two patients maintained clinically inactive disease and three patients had only a partial treatment response. In the three patients with partial treatment response, fSCIg 1 g/kg was then given on days 1 and 6 of every 28-day cycle resulting in IgG peak levels of between 2300-2846 mg/dl (previously 1606-1901 mg/dl on the biweekly regimen), resulting in clinically inactive disease in two of the three patients. There were no relevant adverse effects that limited continuation of fSCIg treatment. High-dose fSCIg is well-tolerated in patients with JDM and high peak serum IgG levels can be achieved which may be important for treatment success. High-dose fSCIg may therefore be an alternative to high-dose IVIg

  9. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems.

    Science.gov (United States)

    Rozwadowski, Kevin; Yang, Wen; Kagale, Sateesh

    2008-11-17

    profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression. We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

  10. Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections.

    Science.gov (United States)

    Berhe, G; Minet, C; Le Goff, C; Barrett, T; Ngangnou, A; Grillet, C; Libeau, G; Fleming, M; Black, D N; Diallo, A

    2003-01-01

    A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

  11. Development of a Dual Recombinant Vaccine To Protect Small Ruminants against Peste-des-Petits-Ruminants Virus and Capripoxvirus Infections

    OpenAIRE

    Berhe, G.; Minet, C.; Le Goff, C.; Barrett, T; Ngangnou, A.; Grillet, C.; Libeau, G.; Fleming, M; Black, D. N.; Diallo, A.

    2003-01-01

    A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

  12. Interplay of recombination and selection in the genomes of Chlamydia trachomatis

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    Dean Deborah

    2011-05-01

    Full Text Available Abstract Background Chlamydia trachomatis is an obligate intracellular bacterial parasite, which causes several severe and debilitating diseases in humans. This study uses comparative genomic analyses of 12 complete published C. trachomatis genomes to assess the contribution of recombination and selection in this pathogen and to understand the major evolutionary forces acting on the genome of this bacterium. Results The conserved core genes of C. trachomatis are a large proportion of the pan-genome: we identified 836 core genes in C. trachomatis out of a range of 874-927 total genes in each genome. The ratio of recombination events compared to mutation (ρ/θ was 0.07 based on ancestral reconstructions using the ClonalFrame tool, but recombination had a significant effect on genetic diversification (r/m = 0.71. The distance-dependent decay of linkage disequilibrium also indicated that C. trachomatis populations behaved intermediately between sexual and clonal extremes. Fifty-five genes were identified as having a history of recombination and 92 were under positive selection based on statistical tests. Twenty-three genes showed evidence of being under both positive selection and recombination, which included genes with a known role in virulence and pathogencity (e.g., ompA, pmps, tarp. Analysis of inter-clade recombination flux indicated non-uniform currents of recombination between clades, which suggests the possibility of spatial population structure in C. trachomatis infections. Conclusions C. trachomatis is the archetype of a bacterial species where recombination is relatively frequent yet gene gains by horizontal gene transfer (HGT and losses (by deletion are rare. Gene conversion occurs at sites across the whole C. trachomatis genome but may be more often fixed in genes that are under diversifying selection. Furthermore, genome sequencing will reveal patterns of serotype specific gene exchange and selection that will generate important

  13. Frequent intra-subtype recombination among HIV-1 circulating in Tanzania.

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    Ireen E Kiwelu

    Full Text Available The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR of 38 (28-50 sequences per subject. Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84% subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60% subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69% to 36 (82% over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design.

  14. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  15. Lineage specific recombination rates and microevolution in Listeria monocytogenes

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    Nightingale Kendra K

    2008-10-01

    Full Text Available Abstract Background The bacterium Listeria monocytogenes is a saprotroph as well as an opportunistic human foodborne pathogen, which has previously been shown to consist of at least two widespread lineages (termed lineages I and II and an uncommon lineage (lineage III. While some L. monocytogenes strains show evidence for considerable diversification by homologous recombination, our understanding of the contribution of recombination to L. monocytogenes evolution is still limited. We therefore used STRUCTURE and ClonalFrame, two programs that model the effect of recombination, to make inferences about the population structure and different aspects of the recombination process in L. monocytogenes. Analyses were performed using sequences for seven loci (including the house-keeping genes gap, prs, purM and ribC, the stress response gene sigB, and the virulence genes actA and inlA for 195 L. monocytogenes isolates. Results Sequence analyses with ClonalFrame and the Sawyer's test showed that recombination is more prevalent in lineage II than lineage I and is most frequent in two house-keeping genes (ribC and purM and the two virulence genes (actA and inlA. The relative occurrence of recombination versus point mutation is about six times higher in lineage II than in lineage I, which causes a higher genetic variability in lineage II. Unlike lineage I, lineage II represents a genetically heterogeneous population with a relatively high proportion (30% average of genetic material imported from external sources. Phylograms, constructed with correcting for recombination, as well as Tajima's D data suggest that both lineages I and II have suffered a population bottleneck. Conclusion Our study shows that evolutionary lineages within a single bacterial species can differ considerably in the relative contributions of recombination to genetic diversification. Accounting for recombination in phylogenetic studies is critical, and new evolutionary models that

  16. Influenza Vaccine, Inactivated or Recombinant

    Science.gov (United States)

    ... die from flu, and many more are hospitalized.Flu vaccine can:keep you from getting flu, make flu ... What is inactivated or recombinant influenza vaccine?A dose of flu vaccine is recommended every flu season. Children 6 months through 8 years of age may need two ...

  17. Molecular Mechanism for Genetic Recombination

    Science.gov (United States)

    Sobell, Henry M.

    1972-01-01

    Symmetry considerations of proteinnucleic acid interaction suggest the existence of an alternate branched configuration for DNA induced by binding specific structural proteins to symmetrically arranged polynucleotide base sequences. The concept that such sequences exist at the ends of genes or operons leads to a molecular model for genetic recombination in eukaryotic cells. PMID:4115953

  18. Genetic recombination and molecular evolution.

    Science.gov (United States)

    Charlesworth, B; Betancourt, A J; Kaiser, V B; Gordo, I

    2009-01-01

    Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill-Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequence polymorphism and divergence data from the noncrossing over dot chromosome of Drosophila to discriminate between two of the major forms of HR effects: selective sweeps and background selection. This chromosome shows reduced levels of silent variability and reduced effectiveness of selection. We show that neither model fits the data on variability. We propose that, in large genomic regions with restricted recombination, HR effects among nonsynonymous mutations undermine the effective strength of selection, so that their background selection effects are weakened. This modified model fits the data on variability and also explains why variability in very large nonrecombining genomes is not completely wiped out. We also show that HR effects of this type can produce an individual selection advantage to recombination, as well as greatly reduce the mean fitness of nonrecombining genomes and genomic regions.

  19. Recombination in immunoglobulin gene loci

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    Komisarenko S. V.

    2009-02-01

    Full Text Available Gene network of the lymphoid cell differentiation coordinates precisely the recombination process in immunoglobulin gene loci. In our opinion, cellular microRNAs can contribute to the allelic exclusion through microRNA-directed DNA methylation and participate in retargeting recombinases activity from the gene loci of heavy immunoglobulin chains to the gene loci of light chains

  20. Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling.

    Directory of Open Access Journals (Sweden)

    Dawson Fogen

    Full Text Available To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C, respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M that are similar to SAVSBPM18. Although SBP(A18C binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation-a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C tags in excess, two SBP(A18C tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

  1. The Contribution of the Self-Efficacy of Curriculum Development Team and Curriculum Document Quality to the Implementation of Diversified Curriculum in Indonesia

    Science.gov (United States)

    Susilana, Rudi; Asra; Herlina

    2014-01-01

    The aim of this study is to describe how the self-efficacy of curriculum development team (CDT) and curriculum document quality contributed to the implementation of diversified curriculum in elementary schools. This research is a survey study using descriptive method. Schools were the unit of analysis while respondents selected from the schools…

  2. Recombination hotspots and population structure in Plasmodium falciparum.

    Science.gov (United States)

    Mu, Jianbing; Awadalla, Philip; Duan, Junhui; McGee, Kate M; Joy, Deirdre A; McVean, Gilean A T; Su, Xin-zhuan

    2005-10-01

    Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD) is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge) chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations.

  3. Recombination hotspots and population structure in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Jianbing Mu

    2005-10-01

    Full Text Available Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations.

  4. Ground beetle (Coleoptera: Carabidae) diversity, activity density, and community structure in a diversified agroecosystem.

    Science.gov (United States)

    Hummel, J D; Dosdall, L M; Clayton, G W; Harker, K N; O'Donovan, J T

    2012-02-01

    Diversity and abundance of ground beetles (Coleoptera: Carabidae) can be enhanced in vegetable and field intercropping systems, but the complexity of polycultures precludes the application of generalized assumptions of effects for novel intercropping combinations. In a field experiment conducted at Lacombe and Ellerslie, Alberta, Canada, in 2005 and 2006, we investigated the effects of intercropping canola (Brassica napus L.) with wheat (Triticum aestivum L.) on the diversity and community structure of carabid beetles, and on the activity density responses of individual carabid species. Shannon-Wiener diversity index scores and species evenness increased significantly as the proportion of wheat comprising total crop plant populations increased in one site-year of the study, indicating a positive response to enhanced crop plant species evenness in the intercrops, and in that same site-year, ground beetle communities in intercrops shifted to more closely approximate those in wheat monocultures as the percentage of wheat in the intercrops increased. Individual carabid species activity densities showed differing responses to intercropping, although activity densities of some potential root maggot (Delia spp.) (Diptera: Anthomyiidae) predators were greater in intercrops with high proportions of wheat than in canola monocultures. The activity density of Pterostichus melanarius (Illiger), the most abundant species collected, tended to be greater in canola monocultures than high-wheat intercrops or wheat monocultures. We conclude that intercrops of canola and wheat have the potential to enhance populations of some carabid species, therefore possibly exerting increased pressure on some canola insect pests.

  5. Initiation of meiotic recombination in Ustilago maydis

    National Research Council Canada - National Science Library

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

    2013-01-01

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

  6. GARD: a genetic algorithm for recombination detection

    National Research Council Canada - National Science Library

    Kosakovsky Pond, Sergei L; Posada, David; Gravenor, Michael B; Woelk, Christopher H; Frost, Simon D W

    2006-01-01

    .... We developed a likelihood-based model selection procedure that uses a genetic algorithm to search multiple sequence alignments for evidence of recombination breakpoints and identify putative recombinant sequences...

  7. Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

    Science.gov (United States)

    2010-01-01

    Background Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale. Results Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor. Conclusions Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first

  8. Creating Diversified Response Profiles from a Single Quenchometric Sensor Element by Using Phase-Resolved Luminescence

    Directory of Open Access Journals (Sweden)

    Elizabeth C. Tehan

    2015-01-01

    Full Text Available We report a new strategy for generating a continuum of response profiles from a single luminescence-based sensor element by using phase-resolved detection. This strategy yields reliable responses that depend in a predictable manner on changes in the luminescent reporter lifetime in the presence of the target analyte, the excitation modulation frequency, and the detector (lock-in amplifier phase angle. In the traditional steady-state mode, the sensor that we evaluate exhibits a linear, positive going response to changes in the target analyte concentration. Under phase-resolved conditions the analyte-dependent response profiles: (i can become highly non-linear; (ii yield negative going responses; (iii can be biphasic; and (iv can exhibit super sensitivity (e.g., sensitivities up to 300 fold greater in comparison to steady-state conditions.

  9. Is the segmented plasma excitation recombination laser a recombination laser

    Energy Technology Data Exchange (ETDEWEB)

    Apollonov, V.V.; Sirotkin, A.A. (Institut Obshchei Fiziki, Moscow (USSR))

    1989-10-01

    The role of plasmachemical reactions in the formation of active media in lasers with a sectional plasma source for metal vapor is investigated. It is shown that the population of ionic levels in Cd II and Zn II occurs under recharging with He(+) and in the process of Penning ionization. It is found that these processes are more efficient than recombination and electron impact. 13 refs.

  10. Bacterial tower of Babel --How cheating and lying diversify bacterial communication

    Science.gov (United States)

    Eldar, Avigdor

    2012-02-01

    In microbial ``quorum sensing'' (QS) communication systems, microbes produce and respond to a signaling molecule, enabling a cooperative response at high cell densities. Many species of bacteria show fast, intraspecific, evolutionary divergence of their QS pathway specificity---signaling molecules activate cognate receptors in the same strain but fail to activate, and sometimes inhibit, those of other strains. Despite many molecular studies, it has remained unclear how a signaling molecule and receptor can coevolve, what maintains diversity, and what drives the evolution of cross-inhibition. Here I use mathematical analysis to show that when QS controls the production of extracellular enzymes ---``public goods''---diversification can readily evolve. Coevolution is positively selected by cycles of alternating ``cheating'' receptor mutations and ``cheating immunity'' signaling mutations. The maintenance of diversity and the evolution of cross-inhibition between strains are facilitated by facultative cheating between the competing strains. My results suggest a role for complex social strategies in the long-term evolution of QS systems. More generally, my model of QS divergence suggests a form of kin recognition where different kin types coexist in unstructured populations.

  11. Virus encoded MHC-like decoys diversify the inhibitory KIR repertoire.

    Directory of Open Access Journals (Sweden)

    Paola Carrillo-Bustamante

    Full Text Available Natural killer (NK cells are circulating lymphocytes that play an important role in the control of viral infections and tumors. Their functions are regulated by several activating and inhibitory receptors. A subset of these receptors in human NK cells are the killer immunoglobulin-like receptors (KIRs, which interact with the highly polymorphic MHC class I molecules. One important function of NK cells is to detect cells that have down-regulated MHC expression (missing-self. Because MHC molecules have non polymorphic regions, their expression could have been monitored with a limited set of monomorphic receptors. Surprisingly, the KIR family has a remarkable genetic diversity, the function of which remains poorly understood. The mouse cytomegalovirus (MCMV is able to evade NK cell responses by coding "decoy" molecules that mimic MHC class I. This interaction was suggested to have driven the evolution of novel NK cell receptors. Inspired by the MCMV system, we develop an agent-based model of a host population infected with viruses that are able to evolve MHC down-regulation and decoy molecules. Our simulations show that specific recognition of MHC class I molecules by inhibitory KIRs provides excellent protection against viruses evolving decoys, and that the diversity of inhibitory KIRs will subsequently evolve as a result of the required discrimination between host MHC molecules and decoy molecules.

  12. Virus Encoded MHC-Like Decoys Diversify the Inhibitory KIR Repertoire

    Science.gov (United States)

    Carrillo-Bustamante, Paola; Keşmir, Can; de Boer, Rob J.

    2013-01-01

    Natural killer (NK) cells are circulating lymphocytes that play an important role in the control of viral infections and tumors. Their functions are regulated by several activating and inhibitory receptors. A subset of these receptors in human NK cells are the killer immunoglobulin-like receptors (KIRs), which interact with the highly polymorphic MHC class I molecules. One important function of NK cells is to detect cells that have down-regulated MHC expression (missing-self). Because MHC molecules have non polymorphic regions, their expression could have been monitored with a limited set of monomorphic receptors. Surprisingly, the KIR family has a remarkable genetic diversity, the function of which remains poorly understood. The mouse cytomegalovirus (MCMV) is able to evade NK cell responses by coding “decoy” molecules that mimic MHC class I. This interaction was suggested to have driven the evolution of novel NK cell receptors. Inspired by the MCMV system, we develop an agent-based model of a host population infected with viruses that are able to evolve MHC down-regulation and decoy molecules. Our simulations show that specific recognition of MHC class I molecules by inhibitory KIRs provides excellent protection against viruses evolving decoys, and that the diversity of inhibitory KIRs will subsequently evolve as a result of the required discrimination between host MHC molecules and decoy molecules. PMID:24130473

  13. Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

    Science.gov (United States)

    Siddique, Bhuiyan Sharmin; Kinoshita, Shigeharu; Wongkarangkana, Chaninya; Asakawa, Shuichi; Watabe, Shugo

    2016-06-01

    Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

  14. Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot.

    Directory of Open Access Journals (Sweden)

    Frédéric Baudat

    2007-06-01

    Full Text Available In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.

  15. Suppression of auger recombination in ""giant"" core/shell nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Garcia Santamaria, Florencio [Los Alamos National Laboratory; Vela, Javier [Los Alamos National Laboratory; Schaller, Richard D [Los Alamos National Laboratory; Hollingsworth, Jennifer A [Los Alamos National Laboratory; Klimov, Victor I [Los Alamos National Laboratory; Chen, Yongfen [NON LANL

    2009-01-01

    Many potential applications of semiconductor nanocrystals are hindered by nonradiative Auger recombination wherein the electron-hole (exciton) recombination energy is transferred to a third charge carrier. This process severely limits the lifetime and bandwidth of optical gain, leads to large nonradiative losses in light emitting diodes and photovoltaic cells, and is believed to be responsible for intermittency ('blinking') of emission from single nanocrystals. The development of nanostructures in which Auger recombination is suppressed has been a longstanding goal in colloidal nanocrystal research. Here, we demonstrate that such suppression is possible using so-called 'giant' nanocrystals that consist of a small CdSe core and a thick CdS shell. These nanostructures exhibit a very long biexciton lifetime ({approx}10 ns) that is likely dominated by radiative decay instead of non-radiative Auger recombination. As a result of suppressed Auger recombination, even high-order multiexcitons exhibit high emission efficiencies, which allows us to demonstrate optical amplification with an extraordinarily large bandwidth (>500 me V) and record low excitation thresholds.

  16. Diversifying selection on flavanone 3-hydroxylase and isoflavone synthase genes in cultivated soybean and its wild progenitors.

    Directory of Open Access Journals (Sweden)

    Hao Cheng

    Full Text Available Soybean isoflavone synthase (IFS and flavanone 3-hydroxylase (F3H are two key enzymes catalyzing the biosynthesis of isoflavonoids and flavonoids, both of which play diverse roles in stress responses. However, little is known about the evolutionary pattern of these genes in cultivated soybean and its wild progenitors. Herein, we investigated the nucleotide polymorphisms in Isoflavone synthase (IFS1, IFS2 and Flavanone 3-hydroxylase (F3H2 genes from 33 soybean accessions, including 17 cultivars (Glycine max and 16 their wild progenitors (Glycine soja. Our data showed that the target genes shared the levels of nucleotide polymorphism with three reference genes involved in plant-microbe interactions, but possessed a much higher nucleotide polymorphism than other reference genes. Moreover, no significant genetic differentiation was found between cultivated soybean and its wild relatives in three target genes, despite of considering bottleneck and founder effect during domestication. These results indicate that IFS and F3H genes could have experienced gene introgressions or diversifying selection events during domestication process. Especially, F3H2 gene appears to evolve under positive selection and enjoy a faster evolutionary rate than IFS1 and IFS2 genes.

  17. Conserved and Diversified Gene Families of Monovalent Cation/H+ Antiporters from Algae to Flowering Plants

    Directory of Open Access Journals (Sweden)

    Salil eChanroj

    2012-02-01

    Full Text Available All organisms have evolved strategies to regulate ion and pH homeostasis in response to developmental and environmental cues. One strategy is mediated by cation-proton antiporters (CPA. CPA1 genes found in bacteria, fungi, metazoa and plants have been functionally-characterized; though roles of plant CPA2 genes in KEA (K+-efflux antiporter and CHX (cation/H+ exchanger families are largely unknown. Phylogenetic analysis showed that three clades of the Na+-H+ exchanger (NHX family have been conserved from single-celled alga to Arabidopsis. These are i plasma membrane-bound SOS1/AtNHX7 that share ancestry with prokaryote NhaP, ii endosomal AtNHX5/6 that is part of the eukaryote Intracellular-NHE clade, and iii a vacuolar NHX clade (AtNHX1-4 specific to plants. Early diversification of KEA genes possibly from ancestral genes of a cyanobacterium is suggested for three K+-efflux antiporter clades (KEA/Kef seen in all plants. Intriguingly, the CHX gene family blossomed from a few members in early land plants to >40 genes in legumes. Homologs from spirogyra or moss share high similarity with guard cell-specific AtCHX20, suggesting that AtCHX20 and its relatives (AtCHX16-19 are founders of the family. Evolutionary analysis suggests pollen-expressed CHX genes appeared later in monocots and early eudicots. AtCHX proteins have been localized to intracellular and plasma membrane of plants, and shown to mediate K+ transport and pH homeostasis. Thus KEA genes are conserved from green algae to angiosperms, and their presence in red algae and secondary endosymbionts suggest a role in plastids. In contrast, AtNHX1-4 subtype evolved in ancestral plants to handle ion homeostasis of vacuoles in all cell types. The strong presence of CHX genes in land plants, but not in metazoa or fungi, would infer a role of ion and pH homeostasis at dynamic endomembranes to support vegetative and reproductive success of flowering plants.

  18. Recombinant viral vaccines for enzootic bovine leucosis.

    Science.gov (United States)

    Daniel, R C; Gatei, M H; Good, M F; Boyle, D B; Lavin, M F

    1993-10-01

    Recently published studies on the development and use of recombinant vaccinia virus (VV) vaccines incorporating either the complete envelope (env) gene or only a fragment of the env gene consisting of the coding sequence for the env glycoprotein 51 (gp51) and part of gp30 of the bovine leukaemia virus (BLV) are described. It has been reported that vaccination of sheep with recombinant VV vaccines containing the complete env gene appears to protect sheep against challenge infection with BLV. The evidence for this protection is based on the lack of persistence of high titres of anti-gp51 antibodies compared with unvaccinated BLV infected controls, on the enhanced CD4 proliferative responses to specific BLV gp51 synthetic peptides in the vaccinated sheep, and on the inability to detect BLV pro-virus by polymerase chain reaction in the vaccinated sheep after 4 months following challenge infection compared with continual detection in unvaccinated sheep over a 16 month trial period. It has been suggested that cell-mediated immune responses may be an important aspect of protective immunity against BLV infection and it has been reported that large tracts of amino acid sequences within the env and pol genes are highly conserved in different isolates from different countries which is of importance in designing peptide derived vaccines.

  19. Microbial factories for recombinant pharmaceuticals

    OpenAIRE

    Domingo-Espín Joan; Ferrer-Miralles Neus; Corchero José; Vázquez Esther; Villaverde Antonio

    2009-01-01

    Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, po...

  20. Production systems for recombinant antibodies.

    Science.gov (United States)

    Schirrmann, Thomas; Al-Halabi, Laila; Dübel, Stefan; Hust, Michael

    2008-05-01

    Recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. The increasing demand for antibodies with regards to amount and quality resulted in the development of a variety of recombinant production systems employing gram-negative and gram-positive bacteria, yeast and filamentous fungi, insect cell lines as well as mammalian cell lines. More recently, antibodies were also successfully produced in transgenic plants and animals. Currently, the production of recombinant antibodies for therapy is performed in mammalian cell lines to reduce the risk of immunogenicity caused by non-human post-translational modifications, in particular glycosylation. However, novel strategies already allow human-like glycosylation patterns in yeast, insect cell lines and transgenic plants. Furthermore, therapeutic strategies not requiring glycosylation of the Fc portion have been conceived, most prominently using bispecific antibodies or scFv fusion proteins, which can be produced in bacteria. Here, we review all current antibody production systems considering their advantages and limitations with respect to intended applications.