70 research outputs found

    Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores.

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    Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

    Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

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    Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk

    Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells

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    Background and Aims: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV‐specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity‐enhanced T Cell receptor with an anti‐CD3 T Cell‐activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus‐derived peptides presented by human leukocyte antigen (HLA). Approach and Results: ImmTAV molecules specific for HLA‐A*02:01‐restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV‐Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging‐based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV‐Env can redirect T cells from healthy and HBV‐infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV‐Env redirection of T cells induced cytolysis of antigen‐positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. Conclusions: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non‐HBV‐specific T cells, bypassing exhausted HBV‐specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials

    THE COMMUNITY LEVERAGED UNIFIED ENSEMBLE (CLUE) IN THE 2016 NOAA/HAZARDOUS WEATHER TESTBED SPRING FORECASTING EXPERIMENT

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    One primary goal of annual Spring Forecasting Experiments (SFEs), which are coorganized by NOAA’s National Severe Storms Laboratory and Storm Prediction Center and conducted in the National Oceanic and Atmospheric Administration’s (NOAA) Hazardous Weather Testbed, is documenting performance characteristics of experimental, convection-allowing modeling systems (CAMs). Since 2007, the number of CAMs (including CAM ensembles) examined in the SFEs has increased dramatically, peaking at six different CAM ensembles in 2015. Meanwhile, major advances have been made in creating, importing, processing, verifying, and developing tools for analyzing and visualizing these large and complex datasets. However, progress toward identifying optimal CAM ensemble configurations has been inhibited because the different CAM systems have been independently designed, making it difficult to attribute differences in performance characteristics. Thus, for the 2016 SFE, a much more coordinated effort among many collaborators was made by agreeing on a set of model specifications (e.g., model version, grid spacing, domain size, and physics) so that the simulations contributed by each collaborator could be combined to form one large, carefully designed ensemble known as the Community Leveraged Unified Ensemble (CLUE). The 2016 CLUE was composed of 65 members contributed by five research institutions and represents an unprecedented effort to enable an evidence-driven decision process to help guide NOAA’s operational modeling efforts. Eight unique experiments were designed within the CLUE framework to examine issues directly relevant to the design of NOAA’s future operational CAM-based ensembles. This article will highlight the CLUE design and present results from one of the experiments examining the impact of single versus multicore CAM ensemble configurations

    THE COMMUNITY LEVERAGED UNIFIED ENSEMBLE (CLUE) IN THE 2016 NOAA/HAZARDOUS WEATHER TESTBED SPRING FORECASTING EXPERIMENT

    Get PDF
    One primary goal of annual Spring Forecasting Experiments (SFEs), which are coorganized by NOAA’s National Severe Storms Laboratory and Storm Prediction Center and conducted in the National Oceanic and Atmospheric Administration’s (NOAA) Hazardous Weather Testbed, is documenting performance characteristics of experimental, convection-allowing modeling systems (CAMs). Since 2007, the number of CAMs (including CAM ensembles) examined in the SFEs has increased dramatically, peaking at six different CAM ensembles in 2015. Meanwhile, major advances have been made in creating, importing, processing, verifying, and developing tools for analyzing and visualizing these large and complex datasets. However, progress toward identifying optimal CAM ensemble configurations has been inhibited because the different CAM systems have been independently designed, making it difficult to attribute differences in performance characteristics. Thus, for the 2016 SFE, a much more coordinated effort among many collaborators was made by agreeing on a set of model specifications (e.g., model version, grid spacing, domain size, and physics) so that the simulations contributed by each collaborator could be combined to form one large, carefully designed ensemble known as the Community Leveraged Unified Ensemble (CLUE). The 2016 CLUE was composed of 65 members contributed by five research institutions and represents an unprecedented effort to enable an evidence-driven decision process to help guide NOAA’s operational modeling efforts. Eight unique experiments were designed within the CLUE framework to examine issues directly relevant to the design of NOAA’s future operational CAM-based ensembles. This article will highlight the CLUE design and present results from one of the experiments examining the impact of single versus multicore CAM ensemble configurations

    The peatland map of Europe

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    Based on the ‘European Mires Book’ of the International Mire Conservation Group (IMCG), this article provides a composite map of national datasets as the first comprehensive peatland map for the whole of Europe. We also present estimates of the extent of peatlands and mires in each European country individually and for the entire continent. A minimum peat thickness criterion has not been strictly applied, to allow for (often historically determined) country-specific definitions. Our ‘peatland’ concept includes all ‘mires’, which are peatlands where peat is being formed. The map was constructed by merging national datasets in GIS while maintaining the mapping scales of the original input data. This ‘bottom-up’ approach indicates that the overall area of peatland in Europe is 593,727 km². Mires were found to cover more than 320,000 km² (around 54 % of the total peatland area). If shallow-peat lands (< 30 cm peat) in European Russia are also taken into account, the total peatland area in Europe is more than 1,000,000 km2, which is almost 10 % of the total surface area. Composite inventories of national peatland information, as presented here for Europe, may serve to identify gaps and priority areas for field survey, and help to cross-check and calibrate remote sensing based mapping approaches

    Problems of nature protectionin Poland in the context of common agricultural policy

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    Celem artykułu jest syntetyczna analiza potrzeb polskiej przyrody w kontekście instrumentów proponowanych przez Komisję Europejską w ramach wspólnej polityki rolnej na lata 2014-2020. W pracy przedstawiono atuty przyrodnicze obszarów wiejskich w Polsce, zhierarchizowano główne zagrożenia przyrody na nich występujące oraz przedstawiono nowe spojrzenie na rolę rolnika jako dostarczyciela dóbr publicznych. Omówiono główne potrzeby w zakresie ochrony przyrody obszarów wiejskich w Polsce w kontekście oddziaływania wspólnej polityki rolnej. Przedstawiono potencjalne możliwości poprawy sytuacji z wykorzystaniem instrumentów tejże polityki.The aim of this paper is to synthetically analyse the needs of nature protection in Poland in the context of instruments proposed by the European Commission within the common agricultural Policy for the years 2014-2020. Chief natural assets of rural areas in Poland are shown, main threats to nature in these areas are hierarchized and a new look at the farmer as a provider of public goods is presented. Main needs of nature protection in rural areas in Poland are discussed in the context of the impact of common agricultural policy. Potential possibilities of improving the situation with the use of instruments of this policy are presented

    History and development of studies on wetland identification and protection at the institute of technology and life sciences

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    Celem pracy jest ukazanie ewolucji podejścia do ekosystemów mokradłowych w działalności badawczej Instytutu Melioracji i Użytków Zielonych, przekształconego w 2009 r. w Instytut Technologiczno-Przyrodniczy. Ewolucję tę kształtowała sytuacja polityczna i gospodarcza Polski. Co interesujące, największy postęp w badaniach podstawowych, dotyczących siedlisk i gleb hydrogenicznych, nastąpił w okresie realizacji badawczych programów rządowych, zorientowanych na melioracje wielkich kompleksów bagiennych Polski w dolinach Biebrzy, Narwi, Noteci i na Polesiu. Zmniejszenie presji produkcyjnej spowodowało ukształtowanie się podejścia ekologicznego do obszarów mokradłowych. Jednocześnie zaowocowało wieloma pracami o charakterze inwentaryzacyjnym i porządkującym istniejące dane. Praca ma charakter przeglądowy. Ukazano w niej poszczególne fazy orientacji badawczej Instytutu i zilustrowano je odpowiednimi pozycjami literatury. Zakresem pracy nie objęto badań nad właściwościami i przemianami chemicznymi gleb hydrogenicznych i semihydrogenicznych.The aim of this study was to show the evolving approach to wetland ecosystems in the research activity of the Institute for Land Reclamation and Grassland Farming transformed into the Institute of Technology and Life Sciences in 2009. The evolution was shaped by political and economic situation in Poland. Interestingly, the greatest progress in basic research of hydrogenic habitats and soils took place in the period of realization of government projects focussed on the reclamation of large bog complexes in the Biebrza, Narew and Noteć River valleys and in Polesie. Weakening of productive pressure facilitated more ecological approach to wetlands. This also resulted in many studies of inventory character which ordered the existing data. This paper is a review that shows particular phases of research interests at the Institute illustrated with appropriate references. The paper does not involve studies on chemical properties and transformations in hydrogenic and semi-hydrogenic soils
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