Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species

During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.     

New approaches for education and training in veterinary public health: the SAPUVET projects

Continued contact between humans and animals, in combination with the ever-increasing movement of human and animal populations that is one effect of globalization, contributes to the spread of diseases, often with detrimental effects on public health. This has led professionals involved in both animal health and public health to recognize veterinary public health (VPH) as a key area for their activities to address the human-animal interface. Veterinarians, a profession with major involvement in this field, are in need of specific knowledge and skills to prevent and control public-health problems. As a result, VPH must be directly integrated into veterinary educational programs. At present, only few veterinary schools have specific VPH programs; in most institutions, VPH does not feature as a specific subject in either undergraduate or post-graduate curricula. SAPUVET and SAPUVETNET II are network projects supported by the ALFA program of the European Union (EU). Their main objectives are to reach a common understanding between European and Latin American universities in the definition of the areas in which VPH is important in their respective countries, and to design a harmonized training program for veterinarians in VPH, by making use of new technological applications and innovative teaching methodologies. The elaboration of educational material in combination with case studies presenting real-life problems provides a basis to apply the knowledge acquired on VPH. It is envisaged that the material and modules developed during the two projects will be integrated into the veterinary curricula of the participating universities, as well as in other partner organizations.     

The influences of forest stand management on biotic and abiotic risks of damage

? This article synthesizes and reviews the available information on the effects of forestry practices on the occurrence of biotic and abiotic hazards, as well as on stand susceptibility to these damaging agents, concentrating on mammal herbivores, pest insects, pathogenic fungi, wind and fire.

? The management operations examined are site selection, site preparation, stand composition, regeneration method, cleaning and weed control, thinning and pruning, and harvesting. For each of these operations we have examined how they influence the occurrence of biotic and abiotic damaging agents, the susceptibility of European forests, and describe the ecological processes that may explain these influences.

? Overall, we find that the silvicultural operations that have the largest influence on both biotic and abiotic risks to European forest stands are closely related to species composition and the structure of the overstorey. Four main processes that drive the causal relationships between stand management and susceptibility have been identified: effect on local microclimate, provision of fuel and resources to biotic and abiotic hazards, enhancement of biological control by natural enemies and changes in individual tree physiology and development.

? The review demonstrates an opportunity to develop silvicultural methods that achieve forest management objectives at the same time as minimising biotic and abiotic risks.

     

Modeling spatial variability and pattern of rust and brown eye spot in coffee agroecosystem

The advent of Precision Agriculture has made possible the analysis of complex spatial patterns of plant disease epidemiology considering statements of integrated disease management. The objective of this work was to use geostatistics, statistics and geographical information systems to characterize the structure and magnitude of spatial dependency of rust (Hemileia vastatrix) and brown eye spot (Cercospora coffeicola) incidence and severity in coffee agroecosystem cultivated with Catuai Vermelho IAC-99 (Coffea arabica L.). Evaluations of incidence and severity of rust and brown eye spot were accomplished at 67 georeferenced points arranged in 6.6202 ha of coffee crop, in the years of 2005, 2006 and 2007. Exponential models of covariance enabled the characterization of the magnitude and structure of rust and brown eye spot spatial variability in the evaluated dates. Ordinary block kriging presented satisfactory performance to map rust and brown eye spot outbreaks based on kriging error coefficients. Kriged maps enabled the visualization of intensity of rust and brown eye spot in each evaluation date. Assessments of incidence and severity presented highly statistical correlation based on linear regression models, also confirmed by the spatial variability of kriging maps. Kriging maps of rust and brown eye spot enabled to observe that intensity of disease was dispersed in foci patterns along the coffee plantation, indicating that the current strategy of disease control based in total area may be replaced by site specific disease management, with less environmental impact and sustainability of coffee crop, according to statements of integrated disease management and precision agriculture.     

Microbial induced nitrous oxide emissions from an arable soil during winter

Nitrous oxide (N₂O) release rates were measured from an fertilized and unfertilized plot on silty loam (Gleyic Luvisol) cropped with winter wheat. Rates were estimated using a closed soil cover box technique throughout a continuous investigation period of 12 months. The 12 months of investigation were separated into the cropping period (March to November) and the winter period (December to February). Soil management and all N-applications were made during the cropping period. The application of 220 kg N to the soil induced significantly higher N₂O losses throughout the cropping season compared to the unfertilized soil. No significant differences were found during winter, where 70% of the annual N₂O emissions were found. The temporal changes of the N₂O emission rates on both soils were highly correlated (r=0.96; P≤0.001), and could be attributed to temporal changes in soil temperature (r=0.65; P≤0.01) resulting from freezing and thawing cycles. In order to decide whether the N₂O production can be attributed to microbial or non-microbial processes in soil, the time courses of the N₂O emissions from a γ-ray sterilized and a non-sterilized soil were compared in a laboratory experiment, where the freezing and thawing cycles were simulated according to field conditions. The results indicated, that microbial processes were responsible for N₂O production in thawing and even frozen soils.     

Posidonia oceanica (L.) Delile Dampens Cell Migration of Human Neuroblastoma Cells

Neuroblastoma (NB) is a common cancer in childhood, and lethal in its high-risk form, primarily because of its high metastatic potential. Targeting cancer cell migration, and thus preventing metastasis formation, is the rationale for more effective cancer therapy against NB. Previous studies have described the leaf extract from Posidonia oceanica marine plant (POE) as an antioxidant, anti-inflammatory agent and inhibitor of cancer cell migration. This study aims to examine the POE anti-migratory role in human SH-SY5Y neuroblastoma cells and the underlying mechanisms of action. Wound healing and gelatin zymography assays showed that POE at early times inhibits cell migration and reduces pro-MMP-2 release into culture medium. By monitoring expression level of key autophagy markers by Western blot assay, a correlation between POE-induced cell migration inhibition and autophagy activation was demonstrated. Cell morphology and immunofluorescence analyses showed that POE induces neurite formation and neuronal differentiation at later times. These results suggest POE might act against cell migration by triggering early nontoxic autophagy. The POE-induced cellular morphological change toward cell differentiation might contribute to prolonging the phytocomplex anti-migratory effect to later times. Overall, these results encourage future in vivo studies to test POE applicability in neuroblastoma treatment.     

Rapid Solid-Liquid Dynamic Extraction (RSLDE): a New Rapid and Greener Method for Extracting Two Steviol Glycosides (Stevioside and Rebaudioside A) from Stevia Leaves

Stevioside and rebaudioside A are the main diterpene glycosides present in the leaves of the Stevia rebaudiana plant, which is used in the production of foods and low-calorie beverages. The difficulties associated with their extraction and purification are currently a problem for the food processing industries. The objective of this study was to develop an effective and economically viable method to obtain a high-quality product while trying to overcome the disadvantages derived from the conventional transformation processes. For this reason, extractions were carried out using a conventional maceration (CM) and a cyclically pressurized extraction known as rapid solid-liquid dynamic extraction (RSLDE) by the Naviglio extractor (NE). After only 20 min of extraction using the NE, a quantity of rebaudioside A and stevioside equal to 1197.8 and 413.6 mg/L was obtained, respectively, while for the CM, the optimum time was 90 min. From the results, it can be stated that the extraction process by NE and its subsequent purification developed in this study is a simple, economical, environmentally friendly method for producing steviol glycosides. Therefore, this method constitutes a valid alternative to conventional extraction by reducing the extraction time and the consumption of toxic solvents and favouring the use of the extracted metabolites as food additives and/or nutraceuticals. As an added value and of local interest, the experiment was carried out on stevia leaves from the Benevento area (Italy), where a high content of rebaudioside A was observed, which exhibits a sweet taste compared to stevioside, which has a significant bitter aftertaste.     

The peach (Prunus persica) defensin PpDFN1 displays antifungal activity through specific interactions with the membrane lipids

Ppdfn1 is a defensin gene previously identified in peach (Prunus persica). The biological role of Ppdfn1 was investigated by analysing its expression profile in leaves, flowers and fruits, either inoculated with the Monilinia laxa fungal pathogen or mock‐inoculated. Ppdfn1 expression was highest in flowers and, in fruits, did not vary upon M. laxa inoculation. To characterize the PpDFN1 antifungal activity, the recombinant mature peptide was expressed in Escherichia coli and purified; recombinant PpDFN1 displays antifungal activity against Botrytis cinerea, M. laxa and Penicillium expansum, with IC₅₀ values of 15·1, 9·9 and 1·1 μg mL^?1, respectively. Treatment of fungal hyphae with FITC‐labelled PpDFN1 indicated that the peptide is not internalized by fungal hyphae, but localizes on their external cell surface. At this site, PpDFN1 is capable of membrane destabilization and permeabilization, as demonstrated by SYTOX Green fluorescence uptake by the treated mycelia. Using artificial lipid monolayers, it was shown that PpDFN1 interacts with sphingolipid‐containing membranes; however the strongest interaction occurs with monolayers composed of lipids extracted from sensitive fungi, such as P. expansum. These data suggest that the lipid composition of fungal membranes is of key relevance for defensin specificity.