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Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules.

In this study, we examined the potential use of N-acetyl-L-cysteine (NAC) in association with a polyvalent antivenom and as stand-alone therapy to reduce the acute local and systemic effects induced by Lachesis muta muta venom in rats. Male Wistar rats (300–350 g) were exposed to L. m. muta venom (1.5 mg/kg – i.m.) and subsequently treated with anti-Bothrops/Lachesis serum (antivenom:venom ratio 1:3 ‘v/w’ – i.p.) and NAC (150 mg/kg – i.p.) separately or in association; the animals were monitored for 120 min to assess changes in temperature, locomotor activity, local oedema formation and the prevalence of haemorrhaging. After this time, animals were anesthetized in order to collect blood samples through intracardiac puncture and then euthanized for collecting tissue samples; the hematological-biochemical and histopathological analyses were performed through conventional methods. L. m. muta venom produced pronounced local oedema, subcutaneous haemorrhage and myonecrosis, with both antivenom and NAC successfully reducing the extent of the myonecrotic lesion when individually administered; their association also prevented the occurrence of subcutaneous haemorrhage. Venom-induced creatine kinase (CK) release was significantly prevented by NAC alone or in combination with antivenom; NAC alone failed to reduce the release of hepatotoxic (alanine aminotransferase) and nephrotoxic (creatinine) serum biomarkers induced by L. m. muta venom. Venom induced significant increase of leucocytes which was also associated with an increase of neutrophils, eosinophils and monocytes; antivenom and NAC partially reduced these alterations, with NAC alone significantly preventing the increase of eosinophils whereas neither NAC or antivenom prevented the increase in monocytes. Venom did not induce changes in the erythrogram parameters. In the absence of a suitable antivenom, NAC has the potential to reduce a number of local and systemic effects caused by L. m. muta venom.

The modern food system is characterised by 1) unsustainable agricultural practices, heavily dependent on agrochemical inputs and leaking large amounts of reactive nitrogen (N) whilst degrading soils, and 2) the consumption of energy-rich but nutrient-poor foods, contributing to non-communicable diseases related to malnutrition. Substituting cereals with low-input, protein- and fibre-rich legumes in the production of mainstream foods offers a promising solution to both issues. Chickpea ( Cicer arietinum ) is a legumi- nous crop that can be grown with little or no synthetic N fertiliser. We performed life cycle assessment (LCA) to compare the environmental footprint of pasta made from chickpeas with conventional pasta made from durum wheat ( Triticum durum ) from cradle to fork. Two functional units were used, an 80g serving of pasta, and a Nutrient Density Unit (NDU). Environmental burdens per serving were smaller for chickpea pasta across at least 10 of the 16 impact categories evaluated. Global warming, resource use minerals and metals, freshwater eutrophication, marine eutrophication, and terrestrial eutrophication burdens were smaller than those of durum wheat pasta by up to 45%, 55%, 50%, 86%, and 76%, respec- tively. Cooked chickpea pasta contains 1.5 more protein, 3.2 times more fibre and 8 times more essential fatty acids than cooked durum wheat pasta per kcal energy content. Thus, the environmental advantage of chickpea pasta extended to 15 of the 16 impact categories when footprints were compared per unit of nutrition. Global warming, resource use and eutrophication burdens per NDU were 79–95% smaller for chickpea pasta than for durum wheat pasta. The one major trade-off was land use, where chickpea pasta had a burden 200% higher per serving, or 17% higher per NDU, than wheat pasta. We conclude that there is high potential to simultaneously improve the environmental sustainability and nutritional quality of food chains through simple substitution of cereals with legumes in staple foods such as pasta. Breeding and agronomic management improvements for legumes could reduce the yield gap with cereals, miti- gating the land use penalty. Meanwhile, the higher protein content of chickpea pasta could contribute towards wider environmental benefits via animal protein substitution in diets, and merits further investi- gation. Consumers who look for the traditional taste and texture of wheat pasta can achieve these aspects by cooking the chickpea pasta al dente and combining it with a typical pasta sauce, which will hide its subtle nutty taste.

Antimicrobial resistance (AMR) in bacteria is a frequent and widespread phenomenon. The European Food Safety Authority (EFSA) reports that multidrug resistant (MDR) Escherichia coli is considered an important hazard to public health. The lack of data on the correlation between the administration of antibiotics to pigs and the diffusion of MDR E. coli necessitates an in-depth study. The aims of our study were first of all to determine the presence of MDR and/or extended spectrum β-lactamase (ESβL) E. coli isolated from feces and carcasses of pigs; and second, to evaluate the correlation between antibiotic resistance and the antibiotic treatment administrated to the animals considered. The examined E. coli was isolated from 100 fecal swabs and 100 carcass sponges taken from farms and slaughterhouses located in Reggio Emilia province in Italy. The MDR isolates were tested following the protocol defined by EUCAST (2015). Subsequently, a real-time PCR and an endpoint-PCR were used for the genomic analysis. Data highlighted 76.5% of MDR E. coli with a marked presence of the ampicillin (AMP)-streptomycin (STRE)-tetracycline (TETRA) pattern. Moreover, 13 isolates were ESβL producers, and the blaCTXM gene was the most frequently observed in genomic analysis. Results confirm the complexity of the AMR phenomenon showing a partial correlation between the administration of antibiotics and the resistance observed. Pigs destined to the production of Protected Designation of Origin items are colonized by bacteria resistant to a wide range of antibiotic classes even if data are encouraging for colistin and third generation cephalosporin. Furthermore, in-depth study focused on food production could be useful in a view of high safety standards for consumers.

Escherichia coli is a ubiquitous organism capable of forming a biofilm. This is an important virulence factor and is critical in certain diseases and in the development of antibiotic resistance, which is increased by biofilm synthesis. In the present study, the potential health risk associated with handling and consumption of foods of animal origin contaminated with E. coli-producing biofilm was evaluated. We analyzed the ability of 182 E. coli strains isolated from pork, poultry, and beef, purchased in three different supermarkets in the area of the "Italian Food Valley" (Parma, northern Italy), to form biofilms. Positive strains were also tested for the presence of 12 biofilm-associated genes. Moreover, the 182 E. coli were characterized for antibiotic resistance, presence of multidrug resistance, extended-spectrum β-lactamase strains, and phylogenetic diversity through PCR. Twenty-five percent of the isolates produced biofilm. The majority showed weak adherence, five were moderate, and three were strong producers. E. coli with a strong adherence capability (three of three) harbored eight biofilm-associated genes, while weak and moderate producers harbored only five (frequencies ranging from 80 to 100%). Multidrug resistance was observed in 20 biofilm-producing E. coli, and 15 of these belonged to phylogenetic group D. Among nonbiofilm producers, the percentage of strains belonging to phylogenetic groups B2 and D was approximately 40%, highlighting a potential health risk for consumers and people handling contaminated products. The present study underlines the importance of monitoring the prevalence and characteristics of E. coli contaminating retail meat in relation to the potential virulence highlighted here.

Made available in DSpace on 2019-10-04T12:14:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Bemisia tuberculata Bondar, 1923 (Hemiptera: Aleyrodidae) is one of the most important pest of cassava Manihot esculenta Crantz in Brazil. However, there is a lack of information about the bioecology and control to this species. An alternative for managing whitefly populations is to develop genetically resistant varieties. To improve the understanding about the bioecology and to find new sources of resistance in cassava were evaluated biological and demographics parameters of B. tuberculata on six genotypes (MEcu 72, Santa Helena, IAC-Caapora, IPR-UniAo, Caiua, and Baianinha) under controlled conditions (25 +/- 2 degrees C, 12L/D). The average of survival rate, longevity and oviposition of B. tuberculata fed on MEcu 72 were the lowest. The nymphs of B. tuberculata were difficulty in development and feed on this genotype. The fecundity life table showed a significant impact of the MEcu 72 on the potential growth of B. tuberculata. When fed on Santa Helena, the females were a similar size to those fed on genotypes that are more susceptible, however, they were the lowest oviposition rates. Considering the parameters evaluated, the results from this study revealed important levels of resistance to the B. tuberculata on the genotypes MEcu 72 and Santa Helena. Univ Estadual Paulista, Access Via Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Oeste Parana, Rua Pernambuco 1777, BR-85960000 Marechal Candido Rodondo, PR, Brazil Univ Estadual Oeste Parana, Rua Univ 2069, BR-85819110 Cascavel, PR, Brazil Embrapa Mandioca & Furticultura, Rua Embrapa S-N, BR-44380000 Cruz Das Almas, BA, Brazil Univ Fed Pelotas, Campus Univ S-N, BR-96160000 Capao Do Leao, RS, Brazil Univ Estadual Paulista, Access Via Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil CAPES: 001

Made available in DSpace on 2021-06-25T10:56:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Several secondary metabolites are associated with plant resistance against herbivores. Cassava genotypes present a wide variety of metabolites with insecticidal potential, but little is known about the identity of these compounds. The present work was conducted for the identification of insecticidal molecules present in cassava genotypes. To this purpose, we firstly evaluated the development of the generalist herbivore Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) fed with leaves from six cassava genotypes (IAC-14, IAC-90, IAC-12, IAC-Caapora, Baianinha and MEcu 72). Following bioassays, we measured the levels of two anti-herbivory related groups of compounds (phenolics and steroidal saponins) in all genotypes using colorimetric methods. The metabolic fingerprinting (GC–MS) of three contrasting cassava genotypes selected by their biological interferences over S. frugiperda was performed. The most unsuitable nutritional indices were observed for larvae fed with MEcu 72. Lower reproductive indices were observed for adults in which larval stages were fed with Baianinha or MEcu 72. Steroidal saponin content was similar in all genotypes, but phenolic content was 25% higher in MEcu 72. GC–MS metabolite fingerprinting of MEcu 72, Baianinha and IAC-Caapora resulted in the annotation of 53 metabolites in which 20 presented different relative abundance among the evaluated genotypes. Molecules such as myo-inositol-2-monophosphate, p-coumaric acid, chlorogenic acid, p-hydroxybenzoic acid, octadecadienoic acid and n-hexacosane were more abundant in MEcu 72 or Baianinha than in IAC-Caapora. The possible roles of these molecules in the development of S. frugiperda are discussed. School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Departamento de Ciências da Produção Agrícola Setor de Fitossanidade Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Via de Acesso Prof. Paulo Donato Castellane s/n School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Departamento de Ciências da Produção Agrícola Setor de Fitossanidade Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Via de Acesso Prof. Paulo Donato Castellane s/n CAPES: Finance Code 001

At present, the central role played by arginine in the modulation of the inflammatory cellular responses is well-recognized, and many pro-inflammatory stimuli are known to modulate the expression and activity of its transmembrane transporters. In this regard, we have addressed the effects of bacterial flagellin from Pseudomonas aeruginosa (FLA-PA) on the uptake of the amino acid in human epithelial respiratory cells. Among the arginine transporters, only ATB0,+, y+L, and y+ were operative in bronchial epithelial Calu-3 cells under control conditions; however, only the expression and activity of ATB0,+ were stimulated upon incubation with flagellin, whereas those of systems y+L and y+ were not stimulated. As a result, this induction, in turn, led to an increase in the intracellular content of arginine without making any change to its metabolic pathway. In addition, flagellin upregulated the amount of other amino acids substrates of ATB0,+, in particular, all the essential amino acids, such as valine, isoleucine, and leucine, along with the non-essential glutamine. At the molecular level, these effects were directly referable to the stimulation of a toll-like receptor-5 (TLR5) signaling pathway and to the induction of nuclear factor-κB (NF-κB) transcription factor. An induction of ATB0,+ expression has been observed also in EpiAirway™, a model of primary human normal tracheal-bronchial epithelial cells that mimics the in vitro pseudostratified columnar epithelium of the airways. In this tissue model, the incubation with flagellin is associated with the upregulation of messenger RNAs (mRNAs) for the chemokine IL-8 and for the cytokines IL-6 and interleukin-1β (IL-1β); as for the latter, a marked secretion in the extracellular medium was also observed due to the concomitant activation of caspase-1. The overall findings indicate that, in human respiratory epithelium, flagellin promotes cellular responses associating the increase of intracellular amino acids through ATB0,+ with the activation of the inflammasome. Given the role of the ATB0,+ transporter as a delivery system for bronchodilators in human airway epithelial cells, its induction under inflammatory conditions gains particular relevance in the field of respiratory pharmacology.

Lentil is a staple in many diets around the world and growing in popularity as a quick-cooking, nutritious, plant-based source of protein in the human diet. Lentil varieties are usually grown close to where they were bred. Future climate change scenarios will result in increased temperatures and shifts in lentil crop production areas, necessitating expanded breeding efforts. We show how we can use a daylength and temperature model to identify varieties most likely to succeed in these new environments, expand genetic diversity, and give plant breeders additional knowledge and tools to help mitigate these changes for lentil producers., This research was conducted as part of the ‘Application of Genomics to Innovation in the Lentil Economy (AGILE)' project funded by Genome Canada and managed by Genome Prairie. We are grateful for the matching financial support from the Saskatchewan Pulse Growers, Western Grains Research Foundation, the Government of Saskatchewan, and the University of Saskatchewan. We acknowledge the support from our international partners: University of Basilicata (UNIBAS) in Italy; Institute for Sustainable Agriculture (IAS) in Spain; Center for Agriculture Research in the Dry Areas (ICARDA) in Morocco, India and Bangladesh; Local Initiatives for Biodiversity, Research and Development (LI-BIRD) in Nepal; and United States Department of Agriculture (USDA CRIS Project 5348-21000-017-00D) in the USA, for conducting field experiments in their respective countries.

Anthracnose, caused by the fungal pathogen Colletotrichum lentis, is a severe disease of lentil (Lens culinaris) causing premature defoliation, necrotic stem lesions that lead to plant wilting and death in susceptible varieties. Two races of C. lentis (0 and 1) have been described so far. Race 0 is the most virulent one and limited genetic resistance is available to date. To address this scarcity of resistance, we screened a germplasm collection covering different Lens spp. originating from different countries for C. lentis race 0 resistance. Leaf and stem damage and plant mortality were assessed on seedlings inoculated under controlled conditions. A significant variability was observed among accession. Most of the collection studied was highly susceptible, but some levels of resistance were identified in about 15% of the accessions. The highest levels of resistance were identified in L. ervoides accessions PI572330, PI572334 and PI572338. Moderate resistance was also identified in 10 L. culinaris ssp. culinaris accessions but not in the remaining species studied. Selected accessions showed potential to integrate several breeding programs.

The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties (n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects (n = 46) were: 19 healthy subjects included in the control group, 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response.

SummaryLentil (Lens culinarisMedik.) is cultivated under a wide range of environmental conditions, which led to diverse phenological adaptations and resulted in a decrease in genetic variability within breeding programs due to reluctance in using genotypes from other environments.We phenotyped 324 genotypes across nine locations over three years to assess their phenological response to the environment of major lentil production regions and to predict days from sowing to flowering (DTF) using a photothermal model.DTF was highly influenced by the environment and is sufficient to explain adaptation. We were able to predict DTF reliably in most environments using a simple photothermal model, however, in certain site-years, results suggest there may be additional environmental factors at play. Hierarchical clustering of principal components revealed the presence of eight groups based on the responses of DTF to contrasting environments. These groups are associated with the coefficients of the photothermal model and revealed differences in temperature and photoperiod sensitivity.Expanding genetic diversity is critical to the success of a breeding program; understanding adaptation will facilitate the use of exotic germplasm. Future climate change scenarios will result in increase temperature and/or shifts in production areas, we can use the photothermal model to identify genotypes most likely to succeed in these new environments.

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney, in the lung, OCTs mediate inhaled drugs&rsquo, transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines, here, we studied OCTs in EpiAirway&trade, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP+) at the apical and basolateral side of monolayers and protein expression through Western Blot analysis. OCTs and OCTNs expression, along with that of Amino acid Transporter B0,+ (ATB0,+)transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway&trade, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side, there, the most abundant carrier is, instead, SLC6A14/ATB0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

[Background] Pea powdery mildew incited by Erysiphe pisi represents a major constraint for pea crops worldwide. Crop protection is largely based on chemical control, although recently a renewed interest in the discovery of natural products as alternatives to synthetic fungicides application has emerged. Thus, 12 bioactive plant and fungal metabolites belonging to different class of natural compounds were evaluated, together with a commercial fungicide, at different concentrations on detached pea leaves for their potential to inhibit spore germination and subsequent stages of fungal growth. The most effective metabolites were tested at different concentrations in planta under controlled conditions to evaluate the level of control achieved by treatments before, concurrently and after pathogen inoculation. Pathogen development was macroscopically scored on whole plants as percentage of disease severity and area under the disease progress curve., [Results] Cavoxin, inuloxin C and sphaeropsidin A strongly inhibited E. pisi germination and haustoria formation and reduced colony size. This effect was dose dependent. These results were further confirmed in whole plants by spraying the metabolites on plant leaves for preventive or curative control, which reduced fungal developmental of E. pisi at levels comparable with those obtained by application of the fungicide., [Conclusions] Cavoxin, inuloxin C and sphaeropsidin A have potential as alternatives to synthetic fungicides for the control of crop pathogens of economic importance such as powdery mildew. © 2019 Society of Chemical Industry, Research was supported by Programme STAR 2017 (M.M.) (UniNa and Compagnia di San Paolo, Italy). Dr. E. Barilli and M.J. González Bernal are also greatly indebted to the AGL2017‐82907‐R project co‐financed by FEDER for financial support.

Toxoplasma gondii is considered one of the most important food-borne parasitic zoonoses globally and sheep are important intermediate hosts of the parasite. Meat and milk from infected sheep are considered an important source of infection for humans. Here, the authors evaluated T. gondii infection in the Italian Cornigliese sheep breed using meat juice ELISA, and in vitro assay for followed by Real Time-PCR and PCR-RFLP. Twenty-one hearts were collected at slaughter. Meat juice serology was carried out on all samples, while eleven hearts with the highest antibody titres were subjected to acid-peptic digestion and seeding onto Vero cells. DNA was extracted at three different time points following seeding. PCR-positive samples were then genotyped by PCR-RFLP. All the meat juice samples were positive for IgG antibodies against p30 protein of T. gondii. Five of the 11 samples, seeded onto Vero cells, were positive in PCR made on DNA extracted after 21days of culture and the PCR-RFLP revealed a Type-II or Type II variant profile at 9/10 loci. Two out of five samples showed an increase in terms of parasite growth by comparing the Cq values at three different time points. To the authors' knowledge, this is the first report of in vitro cultivation of T. gondii from muscle tissue of naturally-infected sheep. In vitro assays may be a promising alternative to bioassays and further studies are necessary in order to improve assay performance and to identify possible early markers of parasite proliferation.

[Background] Pea (Pisum sativum) is one of the most important temperate grain legumes in the world, and its production is severely constrained by the pea aphid (Acyrthosiphon pisum). Wild relatives, such as P. fulvum, are valuable sources of allelic diversity to improve the genetic resistance of cultivated pea species against A. pisum attack. To unravel the genetic control underlying resistance to the pea aphid attack, a quantitative trait loci (QTL) analysis was performed using the previously developed high density integrated genetic linkage map originated from an intraspecific recombinant inbred line (RIL) population (P. fulvum: IFPI3260 × IFPI3251)., [Results] We accurately evaluated specific resistance responses to pea aphid that allowed the identification, for the first time, of genomic regions that control plant damage and aphid reproduction. Eight QTLs associated with tolerance to pea aphid were identified in LGs I, II, III, IV and V, which individually explained from 17.0% to 51.2% of the phenotypic variation depending on the trait scored, and as a whole from 17.0% to 88.6%. The high density integrated genetic linkage map also allowed the identification of potential candidate genes co‐located with the QTLs identified., [Conclusions] Our work shows how the survival of P. fulvum after the pea aphid attack depends on the triggering of a multi‐component protection strategy that implies a quantitative tolerance. The genomic regions associated with the tolerance responses of P. fulvum during A. pisum infestation have provided six potential candidate genes that could be useful in marker‐assisted selection (MAS) and genomic assisted breeding (GAB) after functional validation in the future. © 2019 Society of Chemical Industry, Authors are greatly indebted to AGL2017‐82907‐R project for financial support.

Made available in DSpace on 2021-06-25T11:22:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T14:36:13Z : No. of bitstreams: 1 S0085-56262020000300208.pdf: 1243473 bytes, checksum: 41ce7786684918750dbc33289d42c204 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Vatiga manihotae (Drake 1922) (Hemiptera: Tingidae) is an important cassava pest due to the great potential damage and the increasing population in recent years. However, few studies about bioecology and control of the cassava lace bug have been conducted and their results don't provide adequate control strategies. An alternative to maintain the population below economic injury levels is through the adoption of host plant resistance. To improve the understanding about the bioecology and find new sources of resistance in cassava, the biological parameters and demographics of V. manihotae on five cassava genotypes (Santa Helena, MEcu 72, Col 22, Clone 02 and Clone 03) under controlled conditions (25 +/- 2 degrees C, 14L/10D) were evaluated. Duration and viability of the stages, the period egg-adult, sex ratio, the pre-oviposition period, fecundity and longevity of male and female was determined, and a fertility life table was prepared. Insects fed on MEcu 72 showed increased nymphal, egg-adult, and pre-oviposition periods and reduced female fecundity and longevity in comparison to the other genotypes. Demographic parameters (Ro, r(m), T, DT) showed a significant impact on the growth potential of V. manihotae fed on MEcu 72 indicating that the populations would diminish over time. The combined effect of these parameters indicated that MEcu 72 has resistance on V. manihotae specimens hindering their development. Univ Sao Paulo, Escola Super Agr Luiz de Queiroz ESALQ, Programa Posgrad Entomol & Acarol, Piracicaba, SP, Brazil Univ Estadual Paulista, Jaboticabal, SP, Brazil Univ Estadual Oeste Parana, Marechal Candid Rondon, PR, Brazil Univ Estadual Londrina, Londrina, Parana, Brazil Univ Estadual Oeste Parana, Cascavel, PR, Brazil Univ Estadual Paulista, Jaboticabal, SP, Brazil CAPES: 001

El objetivo de este estudio fue desarrollar un método de cría de Bemisia tuberculata en plantas de yuca para la producción y el mantenimiento continuo de una colonia. La población inicial de B. tuberculata se obtuvo a partir de hojas recogidas de la planta con alta infestación de ninfas. Los adultos obtenidos fueron transferidos de la taza de plástico para jaulas de tul, en diferentes densidades. La densidad de 125 adultos de B. tuberculata por hojas fue el más adecuado para la cría. El método que se propone en este estudio ha demostrado ser eficaz y se recomienda, ya que el nivel de la población se mantuvo estable durante los experimentos.

The development of microbial antibiotic resistance has become one of the biggest threats to global health and the search for new molecules active against resistant pathogenic strains is a challenge that must be tackled. In many cases nosocomial infections are caused by bacteria characterized by multi-drug resistance patterns and by their ability to produce biofilms. These properties lead to the persistence of pathogens in the hospital environment. This paper reports the synthesis and characterization of three thiosemicarbazone derivatives based on a compound containing the cinnamaldehyde natural scaffold but possessing different logPow values. These molecules are then used as ligands to prepare complexes of the Cu(II) and Zn(II) ions. All these compounds, ligands and complexes, were screened in vitro on stains of Escherichia coli and Klebsiella pneumoniae for their antibacterial activity. Despite their molecular similarity they revealed variegated behaviors. Only two of them present interesting antimicrobial properties and have also been studied to verify their stability in solution. The compound with the lowest partition coefficient is the most promising. The minimal bactericidal concentration on K. pneumoniae and E. coli of these substances are very interesting and demonstrate that the use of metalloantibiotics is a promising device to fight antibiotic resistance.

Background : Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, are second in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic, myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action of the main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine, gyroxine and conxulxin. The present study had the objective of analyzing by histology and fractal dimension liver samples of Wistar rats experimentally poisoned with venom of the snake Crotalus durissus terrificus . The hypothesis is that the venom of Crotalus durissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantified by the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions induced by the venom. Materials, Methods & Results : Ninety rats were distributed into different groups and treated with: control group (GC, n = 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30) crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommended by the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration and submitted to histological analysis and fractal dimension (DF) using the ImageJ ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normality was verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance (ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, however necrosis, cytoplasmic and nuclear vacuolization and absence of inflammatory infiltrate were observed in 8 h (M2) and 24 h (M3) in GV. In addition to the lesions found in the GV, mononuclear inflammatory infiltrate was evident always (2 h, 8 h and 24 h) in the GVS. The lesions of necrosis, cytoplasmic and nuclear vacuolization, considered of greater severity were visualized in M3 (24 h) in both GV and GVS. There was an increase in DF for the same changes in GV and GVS over time, but with no difference between them, but with a significant difference compared to CG. In this study, lesions evidenced in the liver were not minimized by the application of antiofidic serum at the recommended dose and during the time of observation of the animals. Discussion : This study agrees with other authors about the hepatotoxicity of crotalic venom in relation to histological findings and the results indicate an increase in DF for the findings of vacuolization and necrosis, proving to be an efficient method for the quantitative evaluation of morphological changes induced by venom without observer interference. In addition, non-protection of the liver by antiofidic serum was evident. It is concluded that Crotalus durissus terrificus venom causes important hepatotoxic effects in the first 24 h after experimental intoxication in rats; DF is effective in the quantitative morphological evaluation of liver changes and was characterized mainly by vacuolization and necrosis; the antiofidic serum did not protect the liver from lesions induced by crotalic venom according to the dose of venom and antiofidic serum and time of observation recommended in this study.

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F8:9) population was developed. The RIL was genotyped through Diversity Arrays Technology PL’s DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM−1. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control., Thais Aznar-Fernandez was supported by a FPI fellowship from the Spanish Ministry of Economy and Competitiveness co-financed by FEDER. Financial support by Spanish projects AGL2014-52871-R and AGL2017-82907-R is acknowledged.

BACKGROUND Rusts are a noxious group of plant diseases affecting major economically important crops. Crop protection is largely based on chemical control. There is a renewed interest in the discovery of natural products as alternatives to synthetic fungicides for control. In this study we tested two fungal metabolites, namely cyclopaldic acid and epi-epoformin, for their effectiveness in reducing early stages of development of two major rust fungi from the genera Puccinia and Uromyces, P. triticina and U. pisi. Spore germination and appressorium formation were assessed on pretreated detached leaves under controlled conditions. Cyclopaldic acid and epi-epoformin were also tested in infected plants in order to evaluate the level of control achieved by treatments both before and after inoculation. RESULTS Cyclopaldic acid and epi-epoformin were strongly effective in inhibiting fungal germination and penetration of both rust species studied. This effect was not dose dependent. These results were further confirmed in planta by spraying the metabolites on plant leaves, which reduced fungal developmental of U. pisi and P. triticina at values comparable with those obtained by application of the fungicide. CONCLUSION Our results further demonstrate the potential of fungal metabolites as natural alternatives to synthetic fungicides for the control of crop pathogens of economic importance as rusts. © 2016 Society of Chemical Industry

© 2020 by the authors., Grass pea (Lathyrus sativus) is an annual legume crop that is currently underutilized but has the potential for reintroduction into Mediterranean rain-fed farming systems. In this study, we compared the adaptation of breeding lines in multi-environment field testing, which had wide variation for precocity, grain yield and broomrape infection. Heritability-adjusted genotype plus genotype-by-environment interaction (HA-GGE) biplot and non-metric multidimensional scaling (NMDS) were performed to determine the effect on genotype (G), environment (E) and G × E interaction on grain yield, precocity and broomrape infection. Precocity was associated with reduced broomrape infection, and this with increased grain yield. Step-wise regression analysis revealed that the broomrape infection had the highest influence on grain yield, whereas precocity had a lower effect. Rain and humidity and mild temperatures before and during flowering were the climatic factors most influential on broomrape. Accessions with a shorter growth cycle suffered lower broomrape infection and were more productive in the environments with a high broomrape incidence. Accessions with longer growth cycle suffered overall higher broomrape infection and were therefore more productive in the environments with low or moderate broomrape incidence., This research was funded by the Spanish Agencia Estatal Investigación (AEI) grant AGL2017-82019., Publisher’s version

A plant can combine physical and chemical tools to interact with other organisms. Some are designed for pollinator attraction (i.e., colors and volatile organic compounds-VOCs), others can act to discourage herbivores (i.e., non-glandular trichomes). Few studies fully address available tools in a single species, notwithstanding, this information can be pivotal in understanding new interactions out of the home range. We characterized flower traits, emission profiles of constitutive compounds from flowers and leaves, micro-morphology of the glandular trichomes, and listed flower visitors of two Mexican bird-pollinated Salvia species (S. blepharophylla and S. greggii), growing in an Italian botanical garden. Flowers were highly variable in their morphometric characteristics. In both species, four trichome morphotypes with similar histochemistry and distribution were documented for leaves and flowers except the calyx abaxial side. The vegetative emission profiles were qualitatively more complex than the floral ones, however, common compounds occurring in high relative percentages were &beta, caryophyllene and germacrene D. Floral bouquets were dominated by limonene and &beta, pinene in S. greggii and by 1,8-cineole in S. blepharophylla. Two potential (non-bird) pollinators were especially abundant: small bees belonging to the genus Lasioglossum and large bees belonging to the species Xylocopa violacea. Our study highlights the plasticity of these plants, as well as tools that can be conveniently used to establish novel interactions.

European legislation stipulates that food no longer intended for human consumption, due to commercial reasons, manufacturing problems, or some defect, can be used in pet feed. However, the presence of former foodstuffs in pet diets could constitute a public health issue because pets can act as reservoirs of antimicrobial resistance genes. In this study, for the first time, biological hazards due to the presence of Escherichia coli and Salmonella in former foodstuff meat products were evaluated. Among the 112 samples of packaged fresh meat (poultry, pork, and beef) collected from cold storage warehouses of a mass market retailer, the overall prevalence of E. coli and Salmonella, the prevalence of strains with multidrug resistance, the phenotypic and genotypic characteristics of strains that produce extended-spectrum β-lactamase, and the presence of biofilm producers were assessed. A high prevalence of E. coli was observed in former foodstuffs of poultry (100%), pork (100%), and beef (93.3%). Salmonella Derby and Salmonella Typhimurium were found in 11.5% of the poultry samples, and Salmonella Typhimurium was found in 13.3% of the pork samples. Salmonella was not recovered from the beef samples. Multidrug resistance was found in E. coli and Salmonella isolates from poultry, pork, and beef. Overall, 18.2% of E. coli isolates and 20% of Salmonella isolates were resistant to various types of antibiotics with various mechanisms of action. Salmonella isolates from pork are an important source of extended-spectrum β-lactamase production. Both E. coli and Salmonella were carriers of antibiotic resistance marker genes ( blaTEM, blaSHV, and blaCTX-M) associated with β-lactamase production in poultry and pork. Approximately 30% of the E. coli isolates from the various types of meat were phenotypically biofilm producers, and all carried the fimH gene. These findings indicate that the use of former foodstuff meat products in pet diets can represent a risk for public health.

Salmonella is the second cause of foodborne infection in humans in the USA and Europe. Pigs represent the second most important reservoir for the pathogen and the consumption of pork meat is a major risk factor for human salmonellosis. Here, we evaluated the virulence patterns of eleven Salmonella isolated from pigs (carcasses and faces) bred in intensive farms in the north of Italy. The two serotypes identified were S. Typhimurium and its monophasic variant 1,4,5,12:i:-. None of the isolates was an ESBL producer, as confirmed also by PCR. However, the presence of a multidrug resistant pattern was evident, with all the isolates being resistant to at least to five antimicrobial agents belonging to various classes. Moreover, six out of eleven isolates showed important resistance profiles, such as resistance against colistin and ciprofloxacin, with nine to twelve recorded resistances. The isolates were negative for the biofilm synthesis test, while four different virulotypes were characterized. All the isolates showed the presence of invA, hilA, stn, ssrA, sipC. One sample also harbored ssaR and spvC genes. One strain was positive for all the virulence genes tested and was resistant to 12 antimicrobial agents. The present study contributes new data to the surveillance program for antibiotic resistance. Furthermore, the presence of eleven highly virulent isolates poses concern for human health in relation to their diffusion in the environment.

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone cause a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increases the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and massive endoplasmic reticulum vacuolization that precede a nonapoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) is reproduced in normal human endothelial cells (HUVEC) at concentrations double those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent. © 2012 American Chemical Society.

Pisum fulvum, a wild relative of pea is an important source of allelic diversity to improve the genetic resistance of cultivated species against fungal diseases of economic importance like the pea rust caused by Uromyces pisi. To unravel the genetic control underlying resistance to this fungal disease, a recombinant inbred line (RIL) population was generated from a cross between two P. fulvum accessions, IFPI3260 and IFPI3251, and genotyped using Diversity Arrays Technology. A total of 9,569 high-quality DArT-Seq and 8,514 SNPs markers were generated. Finally, a total of 12,058 markers were assembled into seven linkage groups, equivalent to the number of haploid chromosomes of P. fulvum and P. sativum. The newly constructed integrated genetic linkage map of P. fulvum covered an accumulated distance of 1,877.45 cM, an average density of 1.19 markers cM−1 and an average distance between adjacent markers of 1.85 cM. The composite interval mapping revealed three QTLs distributed over two linkage groups that were associated with the percentage of rust disease severity (DS%). QTLs UpDSII and UpDSIV were located in the LGs II and IV respectively and were consistently identified both in adult plants over 3 years at the field (Córdoba, Spain) and in seedling plants under controlled conditions. Whenever they were detected, their contribution to the total phenotypic variance varied between 19.8 and 29.2. A third QTL (UpDSIV.2) was also located in the LGIVand was environmentally specific as was only detected for DS % in seedlings under controlled conditions. It accounted more than 14% of the phenotypic variation studied. Taking together the data obtained in the study, it could be concluded that the expression of resistance to fungal diseases in P. fulvum originates from the resistant parent IFPI3260., Authors are greatly indebted to AGL2017-82907-R and AGL2014-52871-R projects co-financed by FEDER for financial support.

Resistance to new generation cephalosporins is an important public health problem globally, in terms of economic and social costs, morbidity and mortality. Βeta-lactamase enzymes are mainly responsible for the antibiotic resistance of Gram negative bacteria and extended-spectrum-β-lactamases (ESβLs) are one of the major determinants of resistance against oxymino-cephalosporins in Enterobacteriaceae. Food-producing animals represent one of the sources of antibiotic resistant bacteria, including pigs. Here we analysed the presence of E. coli resistant to III generation cephalosporins isolated from different matrices collected from intensively bred pigs. A total of 498 E. coli were isolated from faeces and carcasses of pigs at slaughterhouse as well as from pork meat and sausages. Among these, 73 were phenotypically confirmed to be ESβL producers. Genetic analysis revealed that all except two harboured at least one of the three selected genes: blaCTX-M, blaTEM, and blaSHV. Furthermore, six of the E. coli ESβL isolated from faeces and carcasses swabs, were also able to produce biofilm, highlighting the virulence potential of these strains. The presence of Multi-Drug-Resistance patterns (MDR) recorded by the 73 ESβL E. coli was significant (60% of the strains were resistant to more than six antibiotics in MIC test). Results from the present study show that the transmission of resistant bacteria is possible along the food chain, including production of pork, one the most highly consumed meats around the world. Transmission is possible through the ingestion of raw meat products, and following cross-contamination between raw and cooked foods during preparation. The potential risk for human health demonstrated here, associated with the consumption of pork contaminated with bacterial strains characterized by multidrug resistance patterns, and the ability to produce ESβL and biofilm, is cause for concern. It is imperative to study future control strategies to avoid or limit as much as possible the transmission of these highly pathogenic strains through food consumption and/or contact with the environment.

Made available in DSpace on 2015-02-02T12:39:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-10-01Bitstream added on 2015-02-02T13:08:09Z : No. of bitstreams: 1 S0102-09352014000501413.pdf: 188986 bytes, checksum: dfc0ce913d38f7036ffd5b1a5340415d (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O presente estudo teve como objetivo quantificar os níveis de citocinas pró-inflamatórias, entre as quais TNF-α, interleucina-1β (IL-1β), IL-6, e anti-inflamatórias, como IL-10, interferon-γ (INF-γ), bem como comparar o efeito do tratamento convencional com o efeito do tratamento complementado pelo extrato da planta Mikania glomerata, na intoxicação experimental por Bothropoides jararaca. Foram usados ratos Wistar,divididos em três grupos: C - controle, VB - veneno botrópico + soro antiofídico e VBM - veneno botrópico + soro antiofídico + Mikania glomerata. As citocinas foram quantificadas, no soro e no homogenato desses animais, pelo teste ELISA, em três momentos (M1 - 30 minutos, M2 - seis horas e M3 - 24 horas após a inoculação do veneno). Os resultados obtidos evidenciaram que a intoxicação por veneno botrópico estimula principalmente a produção de IL-6 no soro e TNF-α, IL-1β, IL-6 no homogenato da pata de animais experimentalmente intoxicados. O tratamento complementar, com o extrato da planta Mikania glomerata, teve influência principalmente na produção de IL-6, IL-10 e IFN-γ no soro e IL-6, IL-1β e IFN-γ no homogenato. Porém, são necessários novos estudos com o extrato de Mikania glomerata para que se possa entender a ação dessa planta sobre a intoxicação botrópica, bem como verificar qual a melhor via para administrá-lo. This experiment aimed to quantify the pro-inflammatory cytokine levels, including TNF-α, interleukin-1β (IL-1β) and IL-6 as well as the anti-inflammatory ones such as IL-10 and INF-γ. It was also proposed to compare the effect of the conventional treatment to a treatment in which was added the Mikania glomerata plant in the experimental intoxication using Bothropoides jararaca venom. It was used Wistar rats that were randomly divided into 3 groups: C - control; VB - Bothrops venom + antivenom serum; and VBM - Bothrops venom + antivenom serum + Mikania glomerata. Cytokines were quantified in the serum and paw homogenate using ELISA test in three different moments (M1- 30 minutes, M2- 6 hours and M3- 24 hours after venom injection). The intoxication by Bothropoides jararaca venoms mainly stimulated the production of IL-6 in the serum and TNF-α, IL-1β, IL-6 in paw homogenate of animals experimentally intoxicated. Adjunctive treatment with the extract of the Mikania glomerata plant mainly influenced the production of IL-6, IL-10 and IFN-γ in the serum and IL-6, IL1β and IFN-γ in paw homogenate. Further research is necessary with the extract of Mikania glomerata in order to understand the action of this plant on the Bothropoides poisoning and also to verify the best way to manage it. Unesp Faculdade de Medicina Veterinária e Zootecnia Unoeste Faculdade de Ciências Agrárias Unesp Instituto de Biociência de Botucatu Unesp Faculdade de Medicina Veterinária e Zootecnia Unesp Instituto de Biociência de Botucatu

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y+l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.

Background:\ud A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).\ud \ud Methods:\ud Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.\ud \ud Results:\ud Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.\ud \ud Conclusions:\ud The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

Cassava is one of the main foods in Brazil, playing a key role in the diet of several communities. In the Central-Southern region of Brazil, several pests are associated with cassava, among which the cassava mealybug stands out due to its high potential for damage. The aim of the present study was to evaluate the population dynamics and the effect of pruning and different cassava management on the developmental stages of mealybug, Phenacoccus. manihoti. The experiment was conducted in Santa Monica, PR, with ‘Cauiá’ cultivar, involving a randomized complete block design with four replications and six treatments: 1) No pruning: intact plants, 2) Pruning soil: pruned close to the soil without plant material, 3) Pruning at 15 cm: conventional pruning at 15 cm of the soil with vegetative material, 4) Pruning + branches: conventional pruning, with the middle portion of the plant in the plot and removal of the apical region of the plant, 5) Pruning + apical: conventional pruning, with the removal of the middle portion of the plant and retaining the apical parts of the plant, and 6) Pruning - branches - apical: conventional pruning, with the removal of the middle and apical portions of the plant. After 7-days of pruning, the number of eggs, nymphs, and adults were counted, and the population variation was monitored at weekly interval for a month. For the insect count, four sites were randomly chosen in a zigzag manner within each plot and four plants were evaluated, totaling 16 plants per plot. Based on the counts, the percentage of plants with eggs, nymphs, and adults was calculated. The data of the variables were transformed by ?(x+5), subjected to analysis of variance and Scott Knot test (5%). The percentage of plants with egg mass and adults of P. manihoti was highest in plots that had no pruning. The pruning close to the soil combined with the removal of vegetative material resulted in plants with fewest eggs of P. manihoti. Removal of the apical region portion of the cassava plant reduced the number of P. manihoti nymphs and adults.

Pineapple is commonly planted in monoculture systems. It is a long-cycle crop that takes time to monetize, which hinders its cropping by small farmers. The objective of this work was to evaluate the production and quality of pineapple with short-cycle crops, at the beginning of the growing period, as an alternative for family farming in the state of Tocantins. The experimental design was randomized complete block with three treatments and four replications. The pineapple was intercropped with rice and cowpea. The treatments consisted of T1: pineapple + rice; T2: pineapple + cowpea and T3: pineapple in monoculture. The evaluated variables of the pineapple fruit were pH, soluble solids, titratable acidity, yield, fruit mass, fruit length with crown, fruit length without crown and fruit circumference. For the rice and cowpea, the yield and the Area Equivalence Index (AEI) were determined. The cropping system did not influence the pineapple fruits quality. The pineapple yielded less. Cowpea yielded more when intercropped with pineapple. The AEI of the pineapple + rice intercropping was 2.07, being feasible for increasing the use of the area by 100%. The AEI of the pineapple + cowpea intercropping was 2.48, being feasible as it increased the use of the area by 148%. The results obtained showed that it is possible to intercrop pineapple with rice or cowpea at the beginning of pineapple cultivation, and it can be a viable alternative for family farming.

It has been previously shown that rust (Uromyces pisi) infection can be reduced in pea (Pisum sativum) by exogenous applications of systemic acquired resistance elicitors such as BTH and BABA. This protection is known to be related with the induction of the phenolic pathway but the particular metabolites involved have not been determined yet. In this work, we tackled the changes induced in phytoalexin content by BTH and BABA treatments in the context of the resistance responses to pea rust. Detailed analysis through high-performance liquid chromatography (HPLC) showed qualitative and quantitative differences in the content, as well as in the distribution of phytoalexins. Thus, following BTH treatment, we observed an increase in scopoletin, pisatin and medicarpin contents in all, excreted, soluble and cell wall-bound fraction. This suggests fungal growth impairment by both direct toxic effect as well as plant cell wall reinforcement. The response mediated by BTH was genotype-dependent, since coumarin accumulation was observed only in the resistant genotype whereas treatment by BABA primed phytoalexin accumulation in both genotypes equally. Exogenous application to the leaves of scopoletin, medicarpin and pisatin lead to a reduction of the different fungal growth stages, confirming a role for these phytoalexins in BTH- and BABA-induced resistance against U. pisi hampering pre- and postpenetration fungal stages., The authors are greatly indebted to the Spanish AGL2014-52871-R and Spanish-Italian HI2007-0233 projects for financial support. Dr. E. Barilli was granted by JAE-Doc program, co- financed by the European Social Foundation

Toxocariasis, caused most commonly by Toxocara canis, is an important cosmopolitan zoonosis. Paratenic hosts have been employed to provide knowledge regard to the transmission of toxocariasis. Transmammary transmission in murine experimentally infected was observed based on the recovery of larvae from the tissue. The aim of this study was to evaluate the possibility of transmammary transmission of Toxocara canis in rabbits by detecting larvae directly in milk. Seventeen sexually mature virgin white New Zealand female rabbits were divided into two groups. Twelve animals were orally inoculated with 1,000 T. canis embryonated eggs (infected group), and five animals remained uninfected (control group). One month following the infection, the females were mated. Manual collection of 500 ?L of milk from each rabbit was performed on days +7, +14 and +21 of lactation for three consecutive lactations. The recovery of larvae was determined via a centrifuge-sedimentation technique using ether and formalin solutions. ELISA test was run to confirm the production of anti-T. canis antibodies (IgG) by infected rabbits. The presence of larvae was observed in milk samples from 5 (41.7%) of the 12 infected rabbits. The total number of recovered larvae was 20, ranging from 1 to 4 larvae per lactation/rabbit. Larvae were recovered exclusively on days 7 and 14 of lactation. Recovery was verified in different lactations. No significant difference was observed with respect to the number of larvae either in the same lactation period or in different lactation periods. Anti-T. canis antibodies were detected in all infected rabbits. In conclusion, the presence of larvae in rabbit milk samples suggests the possibility of galactogenic transmission of T. canis in paratenic hosts. Moreover, the technique employed in this study allows for the recovery of larvae directly from milk.

Pea powdery mildew incited by Erysiphe pisi represents a major constraint for pea crop worldwide. Only three resistance genes (er1, er2 and Er3) have been identified in pea germplasm so far. Cellular and molecular aspects involved in their interaction with the pathogen are still unknown. In the present study, we selected three well-characterised powdery mildew pea accessions carrying each of these resistance genes JI2302 (er1), JI2480 (er2) and IFPI3260 (Er3) for a combined cDNA array and histological analyses. Transcripts of twenty pea genes, including three housekeeping genes, were generated at 24, 48 and 72 h after fungal inoculation. The 17 genes analysed encoded for phenylpropanoids, structurally related genes, disease relative response genes, or homologues genes to characterised pathogenesis-related family (PR) genes from barley known to be up-regulated after powdery mildew infection. A total of 16 genes out of the 20 studied were differentially expressed between genotypes and/or E. pisi infection, as well as between the time points considered. The transcript inductions observed are expressed as reinforcement of cell walls, activation of pathogenesis-related proteins and the activation of the phenylpropanoid pathway. Leaves of genotype JI2302 (er1) showed mainly Pschitin and Chi2 (encoding for endochitinases) accumulation after E. pisi inoculation, as well as genes encoding for pea defensins. Leaves of IFPI3260 (Er3) showed the overall highest expression of DRR230a, DRR230b and DRR230c (encoding pea defensins) and Prx7 (encoding an elicitor-inducible peroxidase) after pathogen inoculation. Genotype JI2480 (er2) showed Pschitin and Chi2 accumulation after E. pisi inoculation, as well as reduced activation of pea defensins, compared to er1 and Er3 genotypes, after pathogen attack. Up-regulation of genes involved in these mechanisms combined with high constitutive expression determines a more effective defence against E. pisi. © 2014 Springer Science+Business Media Dordrecht., Financial support by AGL2011-22524 Project co-financed by FEDER is acknowledged. E. Barilli was financed by a JAEdoc Grant.

Resumen pt: As fossas septicas biodigestoras constituem alternativa para a producao de adubo orgânico proveniente dos residuos sanitarios. Tem-se um adubo de excelen...

Ascochyta blight caused by Didymella pinodes (formerly Mycosphaerella pinodes) is one of the most important fungal diseases of pea (Pisum sativum) worldwide that can also infect the model legume Medicago truncatula. The objective of this study was to identify quantitative trait loci (QTLs) controlling resistance to D. pinodes in M. truncatula. Response to D. pinodes was studied under controlled conditions in seedlings of a population derived from the cross J6 × F83005.5, two M. truncatula lines that are, respectively, resistant and susceptible to D. pinodes. A combined map using two different recombinant inbred line populations was then used to identify the genomic regions bearing putative QTLs and to improve the position of the QTLs. A single QTL associated with resistance to D. pinodes was detected on linkage group 2, explaining up to 13 % of the total phenotypic variation for relative disease severity against the pathogen. Two simple sequence repeat markers, MTE80 and mtic890 (3 cM apart) were the ones most significantly associated with the QTL. These markers are located in bacterial artifical chromosomes AC119409 and AC125474, respectively, both of them overlapping on M. truncatula chromosome 2. The integration of QTL analysis and genomics in M. truncatula will contribute to the development of new markers and facilitate the identification of candidate genes for Ascochyta blight resistance. © 2013 Springer Science+Business Media Dordrecht., Financial support by FP6-2002-FOOD-1-506223 European Union Integrated Project and by Spanish project AGL2011-22524 is acknowledged. EM and EB were given grants by Juan de la Cierva and I3P programmes, respectively, co-financed by FEDER.

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on T. gondii tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable polymerase chain reaction (PCR) positivity. This protocol was then used to analyse milk samples from sheep of three different farms in Southern Italy, including real time PCR for DNA quantification and PCR-restriction fragment length polymorphism for genotyping. The pre-treatment protocol using ethylenediaminetetraacetic acid and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, real time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of T. gondii transmission through consumption of raw milk and its unpasteurised derivatives.

Changing agricultural practices from conventional to conservation tillage generally leads to increased weed populations and herbicide use. To gain information about the possible use of lucerne (Medicago sativa L.) cover crop as an alternative and sustainable weed-control strategy for winter wheat (Triticum aestivum L.), an experiment was performed at Thiverval-Grignon, France, from 2008 to 2010. We compared conventional and reduced tillage as well as the presence and absence of living mulch (i.e. lucerne) on weeds and wheat production. Percentage soil coverage and aboveground biomass of wheat, lucerne and weeds were measured at the end of grain filling. Weed communities were analysed in terms of composition and diversity. During both seasons, wheat biomass did not significantly decrease in reduced-till trials compared with conventional ones (7.0 and 7.2 t ha–1, respectively, in 2008–09; 6.9 and 7.1 t ha–1 in 2009–10). Regardless of soil management, the percentage soil coverage by wheat significantly decreased when it was intercropped, although wheat biomass was not significantly reduced compared with the sole crop. To minimise cash-crop losses, we studied the competition between wheat, lucerne and weeds, testing various herbicide strategies. Early control of lucerne allowed better balance between weed control and wheat development. In addition, weed communities varied among treatments in terms of abundance and composition, being reduced but more varied in plots associated with lucerne. A functional group analysis showed that grasses benefited from reduced-till conditions, whereas problematic weeds such as annuals with creeping and climbing morphologies were substantially reduced. In addition, annual and perennial broad-leaf species with rosette morphology were also significantly decreased when lucerne was used as living mulch. Wheat production in reduced-till conditions intercropped with lucerne living mulch may be useful for integrated weed management, reducing the need for herbicides.

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPβ transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD.

l-Carnitine, in addition to playing a fundamental role in the β-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l-carnitine, GM-CSF–induced differentiation massively increased the saturable Na+-dependent uptake of l-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l-carnitine transport is mediated by a high-affinity component (Km ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low-affinity component (Km > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.

Didymella pinodes is the principal causal agent of ascochyta blight, one of the most important fungal diseases of pea (Pisum sativum) worldwide. Understanding its host specificity has crucial implications in epidemiology and management; however, this has not been clearly delineated yet. In this study we attempt to clarify the host range of D. pinodes and to compare it with that of other close Didymella spp. D. pinodes was very virulent on pea accessions, although differences in virulence were identified among isolates. On the contrary, studied isolates of D. fabae, D. rabiei, and D. lentil showed a reduced ability to infect pea not causing macroscopically visible symptoms on any of the pea accessions tested. D. pinodes isolates were also infective to some extend on almost all species tested including species such as Hedysarum coronarium, Lathyrus sativus, Lupinus albus, Medicago spp., Trifolium spp., Trigonella foenum-graecum, and Vicia articulata which were not mentioned before as hosts of D. pinodes. On the contrary, D. lentil and D. rabiei were more specific, infecting only lentil and chickpea, respectively. D. fabae was intermediate, infecting mainly faba bean, but also slightly other species such as Glycine max, Phaseolus vulgaris, Trifolium spp., Vicia sativa, and V. articulata. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to confirm identity of the isolates studies and to determine phylogenetic relationship among the Didymella species, revealing the presence of two clearly distinct clades. Clade one was represented by two supported subclusters including D. fabae isolates as well as D. rabiei with D. lentil isolates. Clade two was the largest and included all the D. pinodes isolates as well as Phoma medicaginis var. pinodella. Genetic distance between D. pinodes and the other Didymella spp. isolates was not correlated with overall differences in pathogenicity. Based on evidences presented here, D. pinodes is not specialized on pea and its host range is larger than that of D. fabae, D. lentil, and D. rabiei. This has relevant implications in epidemiology and control as these species might act as alternative hosts for D. pinodes., This work was supported by Spanish project AGL2014-52871-R. EB was granted by a contract founded by the Spanish JAEdoc program and MC by the Spanish Juan de la Cierva program.

Ninety pig carcasses and twenty one food contact surfaces (FCSs) were tested for Salmonella in a slaughterhouse processing ca. 380 pigs/h between 2014-2015. Sampling was performed during seven sessions. Four carcass sites of 100 cm2 each (back, belly, jowl externally, and the diaphragmatic area internally) were swabbed after evisceration. Meat conveyors and dressing tables were tested swabbing areas of 200 to 400 cm2. After pre-enrichment in buffered peptone water, samples were tested by Salmonella MDS® assay and the presumptive positives were confirmed by the ISO 6579 method. Salmonella isolates were serotyped following the Kauffman- White-Le Minor scheme and genotyped by XbaI pulsed field gel electrophoresis. Salmonella was isolated from 16/90 [17.8%; confidence interval (CI) 95%=11.2-26.9] carcasses and 4/21 (19.0%; CI 95%=7.7-40.0) FCSs. Four serovars were identified on carcasses. S. enterica 4,[5],12:i:- was the most prevalent (43.75%), followed by S. Rissen (31.25%), S. Derby (12.5%) and S. Bovismorbificans (12.5%). Two serovars were found on FCSs, namely S. Derby (75%) and S. Livingstone (25%). During one sampling session, a failure in carcass dehairing occurred and caused significantly higher prevalence of carcass contamination (60%) than in the remaining sessions. Moreover, in the same session, Salmonella prevalence was marginally significantly higher on FCSs than in the remaining sampling days, suggesting that dehairing affects contamination not only on carcasses, but also on the working surfaces.

Systemic acquired resistance (SAR) to Uromyces pisi in pea was studied by using a proteomic approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea genotypes displaying different phenotypes (susceptible and partial resistance to the fungus), and in response to parasite infection under the effect of two inducers of SAR, BTH and BABA. Multivariate statistical analysis identified 126 differential protein spots under the experimental conditions (genotypes/treatments). All of these 126 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 50 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, biosynthesis, binding and defense response, whose behavior pattern was different in relation to susceptibility/resistance of the genotypes studied and to the BTH/BABA induction to pathogen response. Results obtained in this work suggested that plants could reduce their photosynthesis and other energy metabolism and enhance the production of defense-related proteins to cope the stress. On the other side, we postulated that resistance induced by the chemicals operates via different mechanisms: BABA inducer could act via phenolic biosynthesis pathway, whereas resistance provided by BTH inducer seems to be mediated by defense and stress-related proteins. The results are discussed in terms of response to rust under the effect of inducers. © 2012 Elsevier B.V., The authors would like to thank the Spanish AGL2011-22524 and Andalusian P07-AGR-02883 projects for financial support.

Rust is a serious disease of pea whose casual agent is not always understood. In this paper we studied reaction of pea accessions to seven rust species infecting closely related legumes, finding that indeed pea can be infected mainly by . Uromyces pisi, followed by . Uromyces viciae-fabae. Other rust species like . Uromyces striatus, . Uromyces ciceris-arietini, . Uromyces anthyllidis and . Uromyces vignae can also infect and reproduce on pea, although in a minor extent. All . U. pisi isolates tested were very virulent on pea accessions, but isolates UpPt-03 and UpKeS-05 (from Palmar de Troya, Spain and Kafr-El-Sheik, Egypt respectively) were significantly most infective. In addition to this we studied in detail the host range of . U. pisi by inoculating multiple accessions of various legumes with urediospores of seven isolates of . U. pisi from different geographical origins. Both experiments were performed under controlled conditions. Based on the evidence presented here the host range of . U. pisi is greater than previously recorded, including genotypes belonging to . Cicer arietinum, . Vicia articulada, . Vicia ervilia and . Vicia faba, which were not mentioned before. © 2012 Elsevier Ltd., Financial support by the Spanish ProjectsAGL2008-01239 and P07-AGR02883 is acknowledged.