Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling.

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg(677)Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-alpha production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-kappaB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-kappaB by human TLR2Arg(677)Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.

Transgenic plant factories for the production of biopolymers and plateform chemicals

In the mid-to long-term, resource constraints will force society to cover a significant share of the future demand for fuels, materials and chemicals by renewable resources. This trend is already visible in the increasing conversion of carbohydrates and plant oils to fuels, chemicals, and polymers. In this perspective, we discuss current efforts and ideas to produce platform chemicals and polymers directly in transgenic plants.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

Human Capital, the Structure of Production, and Growth

Recent decisions by the Spanish national competition authority (TDC) mandate payment systems to include only two costs when setting their domestic multilateral interchange fees (MIF): a fixed processing cost and a variable cost for the risk of fraud. This artificial lowering of MIFs will not lower consumer prices, because of uncompetitive retailing; but it will however lead to higher cardholders fees and, likely, new prices for point of sale terminals, delaying the development of the immature Spanish card market. Also, to the extent that increased cardholders fees do not offset the fall in MIFs revenue, the task of issuing new cards will be underpaid relatively to the task of acquiring new merchants, causing an imbalance between the two sides of the networks. Moreover, the pricing scheme arising from the decisions will cause unbundling and underprovision of those services whose costs are excluded. Indeed, the payment guarantee and the free funding period will tend to be removed from the package of services currently provided, to be either provided by third parties, by issuers for a separate fee, or not provided at all, especially to smaller and medium-sized merchants. Transaction services will also suffer the consequences that the TDC precludes pricing them in variable terms.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

Scientific production on workplace bullying/harassment in dissertations and theses in the Brazilian scenario

OBJECTIVE To analyze scientific production about workplace bullying and harassment in dissertations and theses in Brazil, with emphasis on the year of publication; educational institution; area of knowledge; professional and academic background of the authors; keywords used; and concept map organization. METHOD Bibliometric study with a quantitative approach with a sample consisting of 57 papers, 5 theses and 52 dissertations, published between 2002 and 2012. RESULTS It was found that 2012 was the year with the highest number of publications in this topic area. The region that stood out was the Southeast. The institution with the highest number of publications was the Federal University of Santa Catarina. There was a predominance of dissertations and most publications were produced by researchers focused on a multidisciplinary perspective. CONCLUSION Expanding the views regarding bullying in order to disseminate scientific production was proposed, promoting further advancement of debates and raising pertinent questions.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Growing at the production frontier. European aggregate growth, 1870-1914

The view of a 1870-1913 expanding European economy providing increasing welfare to everybody has been challenged by many, then and now. We focus on the amazing growth that was experienced, its diffusion and its sources, in the context of the permanent competition among European nation states. During 1870-193 the globalized European economy reached a silver age . GDP growth was quite rapid (2.15% per annum) and diffused all over Europe. Even discounting the high rates of population growth (1.06%), per capita growth was left at a respectable 1.08%. Income per capita was rising in every country, and the rates of improvement were quite similar. This was a major achievement after two generations of highly localized growth, both geographically and socially. Growth was based on the increased use of labour and capital, but a good part of growth (73 per cent for the weighted average of the best documented European countries) came out of total factor productivity efficiency gains resulting from not well specified ultimate sources of growth. This proportion suggests that the European economy was growing at full capacity at its production frontier. It would have been very difficult to improve its performance. Within Europe, convergence was limited, and it only was in motion after 1900. What happened was more the end of the era of big divergence rather than an era of convergence.

Spinning welfare: The gains from process innovation in cotton and car production

Economists and economic historians want to know how much better life is today than in the past.Fifty years ago economic historians found surprisingly small gains from 19th century US railroads,while more recently economists have found relatively large gains from electricity, computers and cellphones. In each case the implicit or explicit assumption is that researchers were measuring the valueof a new good to society. In this paper we use the same techniques to find the value to society ofmaking existing goods cheaper. Henry Ford did not invent the car, and the inventors of mechanisedcotton spinning in the industrial revolution invented no new product. But both made existing productsdramatically cheaper, bringing them into the reach of many more consumers. That in turn haspotentially large welfare effects. We find that the consumer surplus of Henry Ford s production linewas around 2% by 1923, 15 years after Ford began to implement the moving assembly line, while themechanisation of cotton spinning was worth around 6% by 1820, 34 years after its initial invention.Both are large: of the same order of magnitude as consumer expenditure on these items, and as largeor larger than the value of the internet to consumers. On the social savings measure traditionally usedby economic historians, these process innovations were worth 15% and 18% respectively, makingthem more important than railroads. Our results remind us that process innovations can be at least asimportant for welfare and productivity as the invention of new products.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice.

A Escrita em Língua Francesa no 2º Ciclo do Ensino Secundário em Cabo Verde: Análise do Manual Escolar e de Concepções e Práticas dos Supervisores

A relevância do ensino da escrita na educação em línguas estrangeiras decorre do papel que ela assume no desenvolvimento expressivo e cognitivo do aluno, sendo fundamental para a sua integração em diversos contextos sociais, incluindo o contexto escolar. No entanto, o processo de aprendizagem da escrita não ocorre espontaneamente e requer um ensino explícito e sistematizado, o que nem sempre acontece. O estudo efectuado centra-se em concepções e práticas de escrita em língua estrangeira – Francês – no contexto do ensino secundário em Cabo Verde. O programa de Língua Francesa para este nível de ensino reconhece a importância da escrita, mas não a perspectiva como processo nas propostas de actividades de produção textual que apresenta. O estudo incide na análise do manual adoptado e nas concepções de supervisores de estágio, no sentido de compreender em que medida num e noutro caso se atende a uma concepção actualizada da escrita. Teve como objectivos: 1. Caracterizar as propostas de aprendizagem da escrita num manual escolar de Francês do 2º ciclo do ensino secundário; 2. Caracterizar as concepções de escrita de um grupo de supervisores de estágio de Francês, da universidade e da escola; 3. Comparar as concepções de escrita patentes no manual analisado com as concepções de escrita dos supervisores; 4. Caracterizar práticas supervisivas de promoção do ensino da escrita. Os resultados obtidos na análise do manual evidenciam o predomínio de uma concepção redutora das práticas de escrita, essencialmente concebida como produto, em detrimento da escrita como processo, com provável reflexo nas práticas pedagógicas dos professores. As concepções dos supervisores aproximam-se de uma visão mais actual da escrita, mas verifica-se algum desfasamento entre essas concepções e as suas percepções das práticas supervisivas, nomeadamente no que diz respeito a alguma falta de apoio aos estagiários em termos de informação sobre a escrita e análise de práticas à luz de referenciais teóricos. Concluindo, verifica-se a necessidade de maior formação neste domínio, que possibilite um olhar crítico sobre os manuais e as práticas escolares, por referência a uma concepção actualizada do que significa ensinar e aprender a escrever.

Organic Newsletter, January 2, 2008

A newsletter produced by Iowa Department of Agriculture and Land Stewardship about the changes of organic farming and raising livestock in Iowa.

A note on the stading stock and production of microplankton off the coast of Peru as determined by particle counting

Analiza el método de conteo de particulas usado para determinar las distribución de particulas suspendidas en un rango de 2 -102 um