Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disordersthat develop as a result of a defective immune response toward intestinal bacteria. Intestinaldysbiosis is associated with the onset of IBD and has been reported to persist even in patientsin deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiotacomposition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of adiet enriched in polyphenols administered to mild but established chronic intestinal inflammation.The Bronze-enriched chow administered for two weeks was not able to produce any macroscopiceffect on the IBD symptoms, although, at molecular level there was a significant induction ofanti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A andIFN production. Analysis of the microbial composition revealed that two weeks of Bronze enricheddiet was sufficient to perturb the microbial composition of Winnie and control mice, suggestingthat polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species.In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remissionaffecting the intestinal dysbiosis.

Paramagnetic iron oxide nanoparticles have been synthetized and covered by a silica shell for a dual function: SiO2 capping improves the stability of the nanoparticles and at the same time promotes the bonding between the paramagnetic nanoparticles@SiO2 (MNPs@SiO2) and biogenic amines. The constituents of the paramagnetic nanoparticles have been identified to be magnetite and maghemite by Infrared and Raman spectroscopy; these optical investigations allow also to confirm the key role of the capping layer in the interaction with the amines. The magnetic adducts with the biogenic amines can be removed simply and rapidly through the application of weak magnetic fields. The observation of the quickness and ease of the biogenic amines elimination have prompted us to check the application of this new approach to real commercial wine samples containing these toxic fermentation products: their complete removal has been observed by absorption spectra, thus confirming the potentiality of this novel approach in agroindustrial area and agribusiness.

Polyphenols are natural compounds capable of interfering with the inflammatory pathways of several in vitro model systems. In this study, we developed a stable and effective strategy to administer polyphenols to treat in vivo models of acute intestinal inflammation. The in vitro suppressive properties of several polyphenols were first tested and compared for dendritic cells (DCs) production of inflammatory cytokines. A combination of the polyphenols, quercetin and piperine, were then encapsulated into reconstituted oil bodies (OBs) in order to increase their stability. Our results showed that administration of low dose reconstituted polyphenol OBs inhibited LPS-mediated inflammatory cytokine secretion, including IL-6, IL-23, and IL-12, while increasing IL-10 and IL-1R? production. Mice treated with the polyphenol-containing reconstituted OBs (ROBs) were partially protected from dextran sodium sulfate (DSS)-induced colitis and associated weight loss, while mortality and inflammatory scores revealed an overall anti-inflammatory effect that was likely mediated by impaired DC immune responses. Our study indicates that the administration of reconstituted quercetin and piperine-containing OBs may represent an effective and potent anti-inflammatory strategy to treat acute intestinal inflammation. © 2014 Cavalcanti et al.

ADP ribosylation is a post-translational modification of proteins shared by animals, plants and bacteria, and catalysed by mono- or poly-ADP- ribosyl transferases (ART). In plants, little is known about the targets of the various ART enzymes. The DAWDLE (DDL), a Forkhead-associated (FHA) domain protein, has been identified as a target of PARP2, with multiple PARylation sites, which was enhanced upon treatment of bacterial flagellin. Forkhead-associated (FHA) domains can bind and recognise ADP ribose units (Readers). DDL positively regulates plant defense to both adapted and non-adapted pathogens. DDL PARylation is required for its function in plant immunity. In addition to the endogenous ADP-ribosylation, plant pathogens contain effectors with mono ART activity (HopU1, HopF2, AvrRpmi1), while HopM1 interacts and induce degradation of an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) involved in vesicle trafficking. In parp triple mutants, Parylation was found increased, pointing to PARP-like domain proteins, Radical Cell Death 1 (RCD-1) and Similar to RCD One (SRO). Two LRR Receptors, FLAGELLIN SENSING 2 (FLS2) and EF-Tu RECEPTOR (EFR) are activated by flagellin peptide and by EF-Tu protein. Otherwise, CLADOSPORIUM FULVUM-9(Cf-9) R protein senses Cf-9. EFR/Cf-9 chimeric receptor is able to trigger in tobacco (Nicotiana tabacum) an HR in response to elf18C conferring resistance to different strains of P. syringae. We study FLS2/Cf-9 and EFR/Cf-9 fused with GFP, testing for immune response in tobacco plants expressing the avirulence gene Avr-9 and infiltrated with Agrobacterium carrying the native Cf-9 gene. SOBIR1 forms a constitutive complex with FLS2/Cf-9 and EFR/Cf-9, and BAK1/SERK3 plays a crucial role in plant immune response (PCD). Responsive plants show leaves with a Hypersensitive Response (HR). We are studying the requirement of signaling partners in the activation of immune response, in the presence of PARP inhibitors. In parallel, we will establish plants transformed with GFP-Macrodomain to detect subcellular localization of mono- and poly- ADP-ribosylated protein targets.

Brassicaceae are promising oil feedstock for cultivation in centralsouthern Italy. Therefore, a two-year investigation on Brassica carinata A. Braun (cv. CT 204) was carried out in three sites of Apulia region [Site 1, Alberobello - Murgia foreland; Site 2, Troia (Foggia) - Daunian sub-Apennines; Site 3, Monteroni (Lecce) - Area of Salento], and in one site of Basilicata region (Site 4, Hill of Matera). The aim was to identify site-specific management practices [by comparing minimum vs conventional tillage, low sowing density vs high sowing density; different levels of nitrogen (N) supply and organic fertilisers] in the four different marginal areas, to achieve optimum yield performance for biodiesel prospective production. The crop showed a good adaptability in the study sites, and the highest N level positively influenced the yield performance in Sites 1, 2 and 3. Moreover, the reduction of mechanical operations (minimum tillage) did not negatively influence crop production and seed oil content. The highest density of sowing tested determined the best crop performance in Site 3, particularly showing the maximum seed oil content with the lowest N supply. Finally, in Site 4 the compost mixed with mineral N fertiliser as well as the sewage sludge from urban wastewater determined productive results comparable to those obtained with mineral fertiliser, evidencing that organic fertilisers could (partially or completely) substitute the mineral one for this crop in the study site. On the whole, seed yield and oil content showed a potential for biodiesel production of Brassica carinata cultivated with site-specific agronomic techniques in four different marginal areas of Southern Italy, suggesting it can be likely achieved the crop environmental adaptation.

ADP ribosylation reactions are relevant either in animals and plants challenged by pathogens and in their immune response. Pathogens intracellularly or through introduction of effectors alter the signalling pathways and the protein hubs at various levels. SirTM-type are ADP-ribosylating sirtuins present in pathogenic organisms such as Staphylococcus aureus and Streptococcus pyogenes (Rack et al., 2015), in strains of Clostridiaceae, Enterococcaceae, Spirochaetaceae , in the fungi Aspergillus, Candida, Entamoeba, and in Fusarium and Phytophthora plant pathogens.Legionella pneumophila modulates cellular and innate metazoan processes (as in humans, drosophila, and C. elegans), interfering with TLR2 signalling, and immunity. L. pneumophila Sde protein is a mono-ADP-ribosyltransferase protein that converts ubiquitin to ADP-ribosylated Ub (ADPR-Ub), processes ADPR-Ub to phosphoribosyl-Ub (PR-Ub), and conjugates PR-Ub to a serine residue in substrates, transferring ubiquitin to endoplasmic reticulum-associated Rab-family GTPases (i.e. Rab33b). In this way, bacteria and fungi promote inactivation of vesicular trafficking, autophagy and phagocytosis.Also plant pathogens contain effectors with ADP-ribosylating activity (HopU1, HopF2, AvrRpmi1); HopM1 interacts and induce degradation of an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) involved in vesicle trafficking. In plants, PARP-like domain proteins are Radical Cell Death 1 (RCD-1) and Similar to RCD One (SRO). PARylation of protein targets occurs during Pattern Triggered immunity: one ADP-ribosylated protein is the Forkhead Associated (FHA) protein Dawdle (DDL), a transcriptional activator. RCD1-type proteins are phosphorylated, and interact with Mut9-like kinases (MLKs). An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression, suggesting that MLKs also affect transcriptional regulation of SA signaling. The effector HaRxL106 from Hyaloperonospora arabidopsidis suppresses transcriptional activation of salicylic acid (SA)-induced defense genes, probably by converting RCD1 into a transcriptional co-repressor.

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36 h in YES medium, and 72 h in hazelnut agar medium. Significantly, the amount of anatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease. potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular. ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts. (C) 2009 Elsevier B.V. All rights reserved.

Resveratrol, a naturally occurring phytoalexin, has long been known to play an important regulatory role in key functions in cell physiology. This multifunctional role of resveratrol is explained by its ability to interact with several targets of various cell pathways. In the recent past, synthetic chemical modifications have been made in an attempt to enhance the biological effects of resveratrol, including its anti-cancer properties. In this study, we investigated the molecular mechanisms of action of novel trans-restricted analogues of resveratrol in which the C-C double bond of the natural derivative has been replaced by diaryl-substituted imidazole analogues. In ovarian cancer models, the results of in vitro screening revealed that the resveratrol analogues exhibited enhanced anti-proliferative properties compared with resveratrol. We found that the resveratrol analogues also significantly inhibited Akt and MAPK signalling and reduced the migration of IL-6 and EGF-treated cells. Finally, in ascite-derived cancer cells, we demonstrated that the resveratrol analogues reduced the expression of epithelial mesenchymal transition (EMT) markers. Collectively, these findings indicate the enhanced anti-cancer properties of the resveratrol analogues.

Introduction: Polyphenols of wine have been extensively studied in relation to their health promoting properties. Red wine polyphenols consisted of different class of compounds belonging to flavonoid pathway, such as the resveratrol, quercetin, and anthocyanins which have long been considered to reduce the incidence of mortality and morbidity from cardiovascular diseases (CVD). Endothelial and monocyte activation is a pivotal event in atherosclerotic CVD, in this study was examined the anti-inflammatory effects of Italian red wines polyphenols in human vascular cells, such as endothelial and monocytoid cells. Since the wine polyhenols depend on different factors such as grape cultivar, and wine-making practices, aim of this study was to examine the anti-atherogenic effects of polyphenolic extracts from Italian red wines obtained by two Apulian grape cultivar: Primitivo and Negroamaro. Methods: Six Apulian red wines produced by the cv Negramaro and Primitivo grapes from the 2007-2008 vintage were analyzed. For each wine, triplicate lipophilic fraction analyses were carried out as per Giovinazzo et al., 2005. HPLC wine anthocyanin analysis was performed by direct injection of wine samples as reported by De Villiers et al. 2004. Human Umbilical Vein Endothelial Cells (HUVEC) and human monocytoid cells (U937) were pre-treated with chemically synthesised polyphenols, or with Primitivo and Negroamaro polyphenolic extracts and the corresponding de-alcoholised wines, at 0,2-2 % (v/v), before stimulation with 20 nmol/L phorbol myristate acetate (PMA) or 2 mg/mL lypopolysaccaride (LPS) for 24 h. Then, HUVEC were tested for the expression of Vascular Cell Adhesion Molecule(VCAM)-1 by enzyme immunoassays (EIA), and U937 supernatants were tested for the release of matrix metalloproteinases (MMP)-9 by gelatine zymography. Results: All chemically synthesized polyphenol tested reduced, in a concentration dependent manner, the stimulated expression of VCAM-1 in LPS triggered HUVEC. In the same conditions, lipophilic fraction extracted from both Primitivo and Negroamaro wines as well as the corresponding de-alcoholised wines exhibited an even higher inhibition of VCAM-1 expression (up to 50-70 fold higher than those expected on the base of individual polyphenol content). Similarly, either Primitivo and Negroamaro polyphenolic extracts reduced the PMA stimulated release of MMP-9 by PMA triggered U937.Conclusions: In this study different class of polyphenols from Primitivo and Negramaro red wines were characterised. These fractions, significantly inhibited the monocyte-derived extracellular matrix proteases release as well as the endothelial expression of athero-adhesion molecules. These inhibitory effects could explain, at least in part, the anti-inflammatory and cardio-protective properties of red wine polyphenols.

Dietary phytochemicals found in vegetables and fruits consist of a wide variety of biologically active compounds with anti-carcinogenic activity. The aim of this study was to evaluate the antigrowth activity of carnosol, a dietary diterpene, as a single agent or in combination with other dietary phytochemicals or chemotherapeutic drugs against a panel of tumor cell lines. Carnosol decreased cell viability in human breast, ovarian, and intestinal tumor cell lines, and inhibited cancer cell adhesion on fibronectin and growth of cancer cells in suspension. Carnosol also inhibited EGF-induced epithelial mesenchymal transition in ovarian cancer cells. The combination treatment with other dietary phytochemicals increased the anti-proliferative activity of carnosol. The combination with curcumin resulted in a synergistic reduction of vitality in SKOV-3 and MDA-231 cells and potently inhibited viability of primary cancer cells isolated from the pleural fluid or ascites of patients with metastatic cancers. These results provide additional evidence about the anticancer role of carnosol and its potential in blocking the growth of tumor cells. © 2014 the Partner Organisations.

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards novel classes of potentially healthy flavonoids. Using a structural gene (encoding a grape stilbene synthase), we were able to produce transgenic tomatoes accumulating new stilbene-related phytochemicals. Afterwards, we performed a breeding programme taking advantage of the ability of some transcription factors (MYB12, Delila and Rosea) to strongly activate the expression of several phenylpropanoid biosynthetic genes and provide high rates of metabolic flux for different classes of polyphenols. Biochemical analyses showed that these new tomato lines were able to accumulate high levels of specific classes of polyphenols such as stilbenes, flavonols and anthocyanins. We demonstrated that, due to the presence of the novel phytochemicals, the transgenic tomato fruits displayed a significantly higher antioxidant profile. Our data show that a combination of biosynthetic and regulatory genes together with the availability of natural tomato varieties could provide novel insights into genetic and biochemical regulation of the flavonoid pathway in this worldwide important vegetable.

Microwave treatments have been used in drying of almonds and pistachios, and in hazelnuts roasting. Radiofrequency (RF) energy and microwave are promising technologies to be applied in the AgroFood sector and at industry level. RF energy has been used as a means to rapidly heat nuts (almonds, walnuts, hazelnuts) to control post-harvest insects, in a rapid process able to maintain the quality and sensorial properties of nuts. Several food productions, such as dried fruits, ham, sausages, and cheese, are subject to mite infestation and contamination with bacteria and fungi. The excessive presence of mites induces a change in sensory properties and lowers the quality of stored foods. In addition, in the drying process, RF and microwave treatments reduce rapidly the moisture content and contribute to inhibit the activity of enzymes involved in lipid peroxidation, ensuring not only an extension of shelf-life, but also preserving the sensory properties and quality of the food productions during storage.

Dendritic cells (DCs) are the most potent antigen-presenting cells able to trigger the adaptive immune response to specific antigens. When non-self-antigens are captured, DCs switch from an "immature" to a "mature" state to fulfill their function. Among the several surface proteins involved in DCs maturation, the role of aquaporins (AQPs) is still poorly understood. Here we investigated the expression profile of Aqps in murine bone marrow derived dendritic cells (BMDCs). Among the Aqps analyzed, Aqp9 was the most expressed by DCs. Its expression level was significantly upregulated 6 h following LPS exposure. Chemical inhibition of Aqp9 led to a decreased inflammatory cytokines secretion. BMDCs from AQP9-KO mice release lower amount of inflammatory cytokines and chemokines and increased release of IL-10. Despite the reduced release of inflammatory cytokines, Aqp9-KO mice were not protected from DSS induced colitis. All together, our data indicate that AQP9 blockade can be an efficient strategy to reduce DCs inflammatory response but it is not sufficient to protect from acute inflammatory insults such as DSS induced colitis.

The development of energy crops can provide environmental benefits and may represent an opportunity to improve agriculture in areas considered at low productivity. In this work, we studied the energy potential of two species (Brassica carinata A. Braun and Cynara cardunculus L.) and their seed oil productivity under different growth conditions. Furthermore, the biodiesel from the oil extracted from the seeds of these species was produced and analysed in term of utilisation as fuels in compression ignition engines. In particular, the spray penetration and shape ratio were measured in a constant-volume chamber and compared with the results obtained with a standard diesel fuel. These results were obtained using a standard common rail injection system at different injection pressure, injection duration, and constant-volume chamber pressure.

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. In particular polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their ignificant antioxidant activity, important both for plant physiology and human nutrition. With the aim of generating plants and fruits with increased antioxidant capability and a wider health benefits, the tomato flavonoid route was modified through the over-expression of the grape stilbene synthase cDNA under a constitutive promoter (Giovinazzo et al., 2005) and a tissue specific, TomLoxB, promoter (D'Introno et al., 2009). The expression of the StS gene in tomato tissues resulted in the synthesis of new compounds, identified as trans-resveratrol and trans-resveratrol-glucopyranoside with a different tissue specificity, both depending on the promoter and the availability of precursors in ripening tissues (Nicoletti et al., 2007). The level synthesis of compounds belonging to different class of polyphenols (i.e. chlorogenic acid, naringenin, rutin, and quercetin), were monitored in both transgenic and wild type fruits tissues at different stages of maturity. Furthermore, we wanted to analyse whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues. The results indicated that the stilbenes and the flavonoids found in transgenic plants were mainly detected as conjugates. This could increase the bioavailability of these compounds. Hence, the structure of the stilbenes synthesized in the transformed tomato plants is of considerable interest from a nutritional point of view. Moreover, the synthesis of resveratrol affected the levels of other antioxidants. In particular, ascorbate and glutathione pool increased in the transformed tissues, proportionally to the amount of the resveratrol. Noteworthy, the transformed fruits also showed increased activities of different enzymes involved in redox homeostasis, (such as ASC - redox enzymes and catalase) which finally resulted in a lower levels of lipid peroxidation. The alteration induced by the presence of resveratrol on cell metabolism will be discussed.

A protein chip containing 11 different proteases was used to investigate the expression of protease inhibitors induced by A. carbonarius infection of potato tubers. Kunitz-type protease inhibitors B1 (KPI-B1) recombinant proteins with trypsin/chymotrypsin protease selectivity were used as internal control and inter-assay control in protease chips targeting KPI proteins. KPI-B1 was spotted on each chip as a reference to compare fluorescence intensities between different hybridization experiments. In order to validate the use of KPI-B1 as control, we studied the performance of recombinant KPI-B1 protein. Interactions between KPI-B1 and proteases in the absence and in the presence of phenylmethylsulfonyl fluoride (PMSF) indicated that a free substrate binding pocket in protease is required for binding with the recombinant KPI-B1 inhibitor. Atomic Force Microscopy (AFM) was employed to analyse structures of protein complexes formed by KPI-B1. KPI-B1/antibody complexes have diameters of ca. 450 nm and heights of ca. 8 nm, while trypsin/KPI-B1/antibody complexes have relatively small diameters (ca. 300 nm) but very great heights (ca. 50 nm). On the basis of AFM data, trypsin-KPI-B1 complexes, instead of KPI-B1 alone, could be a better internal control for protein chip to calibrate fluorescence signals obtained from different hybridization experiments.

Dietary polyphenols are associated with a wide range of health benefits, protecting against chronic diseases and promoting healthy aging. Dietary polyphenols offer a complementary approach to the treatment of inflammatory bowel diseases (IBDs), a group of common chronic intestinal inflammation syndromes for which there is no cure. Tomato is widely consumed but its content of polyphenols is low. We developed a tomato variety, Bronze, enriched in three distinct classes of polyphenols: flavonols, anthocyanins, and stilbenoids. Using Bronze tomatoes as a dietary supplement as well as Indigo (high anthocyanins and flavonols), ResTom (high stilbenoids) and wild-type tomatoes, we examined the effects of the different polyphenols on the host gut microbiota, inflammatory responses, and the symptoms of chronic IBD, in a mouse model. Bronze tomatoes significantly impacted the symptoms of IBD. A similar result was observed using diets supplemented with red grape skin containing flavonols, anthocyanins, and stilbenoids, suggesting that effective protection is provided by different classes of polyphenols acting synergistically.

This study have been considered the effects of the three type of biodiesel (brassica, cardoon and coffee) on the performance, tailpipe emissions and combustion characteristics of a single cylinder direct injection compression ignition engine operated in four different speeds (1200, 1700, 2200, 2700 rpm) and three different engine loads (15, 30 and 45%). The differences in combustion and performance parameters and exhaust emissions of engine fueled by these fuels have been compared. The free fatty acid profile of the biodiesels has been considered. The properties of the biodiesels according to the ASTM D6751 have been analyzed. Highest degree of unsaturation is achieved for biodiesel produced from brassica (94.64%) compared to biofuels derived from cardoon and coffee, 79.81% and 57.65%, respectively. The high value of the erucic acid (C22:1) in the brassica biodiesel (48.7 wt%) is the reason of higher unsaturation degree than the other biodiesels. The components of the free fatty acid profile of the considered biodiesels mostly include long chain free fatty acids (C18 and higher). The physical properties of the biodiesel fuel is influenced by the fatty acid profile. The CN of the brassica, cardoon and coffee is 56.44, 56.11 and 57.44, respectively. The surface tension of the brassica, cardoon and coffee biodiesel fuel is 42.05, 40.99 and 37.62 mN/m. The oxygen content of the brassica, cardoon and coffee is 13.44, 10.91 and 7.77%, accordingly. Dynamic viscosity of the brassica, cardoon and coffee was 6, 5.7 and 9.5 cSt, respectively. The ignition delay of the brassica, cardoon and coffee biodiesel diesel fuel blends at 15% engine load is 9.52, 11.05, 5.07% and at 30% engine load is 12.88, 13.85, 15.78% lower than diesel fuel on average, respectively. The additional oxygen have decreased the CO and THC emissions. The highest reduction of the THC emission than standard diesel fuel was 41.19%. The maximum BTE (brake thermal efficiency) obtained for different biodiesel diesel blends fuels was lower than that of standard diesel fuel. The CA50 of the biodiesel diesel fuels was lower than diesel fuel due to their lower ignition delay.

Four cultivars of chickpea (C. arietinum) were used in this study, two Desi (ICC 4958 and ICC 6098) cultivars and two Kabuli (Beja-1 and Amdoun-1) each one with a water stress tolerant and sensitive variety, inoculated with a Mesorhizobium strain. The effects of drought stress on plant growth and the response of genes related to metabolism were analysed by means of qRT-PCR at various time points of treatments. Nodules and root tissue was used for RNA extraction. We measured the expression of key genes involved by qRT-PCR on samples from stressed and control roots and nodules of a different cultivar using available Taqman assays. In addition phenotype characterisation was performed, such as Relative water content (RWC), quantification of biomass, evaluation ''in situ' of' acetylene reduction activity at the end of the vegetative stage, and nitrogenase activity. The expression analysis was done after 7 days of drought stress and after 10 days of drought stress. We studied genes implicated in the metabolism (glutamine synthase and AAT) or transport of proteins (peptide amino acid transporter like protein PAAT and proline rich protein PPP), sucrose synthase, malate dehydrogenase, sucrose phosphate synthase, sugar transporter) and Sucrose transporter, the antioxidant genes ascorbate peroxidase, Glutathione-S-transferase, Leghemoglobin, catalase, and two lipoxygenase genes.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that par- ticipates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic forcemicroscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of -cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Fruits and vegetables are rich in plant polyphenols, whose consumption is encouraged in healthy dietary regimes due to their antioxidants and anti-inflammatory effects. These organic molecules exhibit numerous properties including phylochelation; the ability to complex metal ions, including highly reactive iron. Among polyphenols, we focused our attention on quercetin that previously demonstrated its ability to reduce dendritic cells (DCs) inflammatory cytokine secretion and antigen presentation following LPS exposure. Dendritic cell inflammatory response is also associated with modulation of several iron metabolism related genes.

In plants, the Leucine Rich Receptor proteins (NS-LRR and LRR-RK) are at the basis of ETI and HR response to pathogen elicitors. It was shown that swapping the domains that trigger Effector Triggered Immunity or Hypersensitive Response made possible the modulation of plant response to pathogen attack.Most pathogenic and nonpathogenic/symbiotic bacteria use type III secretion system to inject into host cells, effector proteins promoting virulence or symbiosis (Chisholm et al., 2006; Xin et al., 2012).The second evasion strategy is exemplified by the Pseudomonads AprA protein, which digests monomeric flagellin that hampers plants and vertebrates recognition. Finally, the last evasion mechanism is based on MAMP modification. Indeed, pathogen PRR recognition triggers defence mechanisms that create a pressure of selection on bacteria. Some successful microbes such as plant pathogens Ralstonia solanacearum or Xanthomonas campestri pv campestris B186 (XccB186) have evolved to evade TLR5 or FLS2 recognition by mutating their flagellin-encoding genes. Since these bacteria require motility for colonization and persistence in their hosts, sequences contain not only changes that permit PRR evasion, but also compensatory mutations that restore flagellin polymerization.Considering the important role of PRR in innate immunity and the possibility of a PRR transfer to crop that confers broad-spectrum resistance to several bacterial pathogens, we sought to enhance plant recognition capabilities to express the TLR5 in Arabidopsis thaliana.

Curcumin is a natural hydrophobic polyphenol found in the powdered rhizomes of Curcuma longa. Due to its capacity to interfere with many signalling pathways, it has been shown that curcumin has potential beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic properties. However, the use of curcumin is fairly restricted because of its poor water solubility, low bioavailability, inadequate tissue absorption and degradation at alkaline pH. In the present contribution, we first verified the anti-proliferative effects of natural curcuminoids towards two different cell lines derived from an ovarian and a breast adenocarcinoma cancer. Later, curcuminoids were successfully encapsulated into reconstituted oil bodies. Once encapsulated into the triacylglycerol cores of the reconstituted oil bodies, curcumin, the most hydrophobic and active of the three curcuminoids, was better stabilized in comparison with albumin stabilization. Oil body encapsulated curcuminoids showed the same effects on cancer cell viability as the free drug, confirming the great potential of natural oil bodies as micro/nano-capsules in drug delivery applications.

Conformational switching induced in ethane-bridged bisporphyrins was used as a sensitive transduction method for revealing the presence of urea dissolved in water via nonenzymatic approach. Bisporphyrins were deposited on solid quartz slides by means of the spin-coating method. Molecular conformations of Zn and Ni monometalated bis-porphyrins were influenced by water solvated urea molecules and their fluorescence emission was modulated by the urea concentration. Absorption, fluorescence and Raman spectroscopies allowed the identification of supramolecular processes, which are responsible for host-guest interaction between the active layers and urea molecules. A high selectivity of the sensing mechanism was highlighted upon testing the spectroscopic responses of bis-porphyrin films to citrulline and glutamine used as interfering agents. Additionally, potential applicability was demonstrated by quantifying the urea concentration in real physiological samples proposing this new approach as a valuable alternative analytical procedure to the traditionally used enzymatic methods.

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards novel classes of potentially healthy flavonoids. Using a structural flavonoid gene (encoding a grape stilbene synthase), we were able to produce transgenic tomatoes accumulating new stilbene-related phytochemicals. Afterwards, we performed a breeding programme taking advantage of the ability of some transcription factors (MYB12, Delila and Rosea) to strongly activate the expression of several phenylpropanoid biosynthetic genes and provide high rates of metabolic flux for different classes of polyphenols. Biochemical analyses showed that these new tomato lines were able to accumulate high levels of specific classes of polyphenols such as stilbenes, flavonols and anthocyanins. We demonstrated that, due to the presence of the novel phytochemicals, the transgenic tomato fruits displayed a significantly higher antioxidant profile. Our data show that a combination of biosynthetic and regulatory genes together with the availability of natural tomato varieties could provide novel insights into genetic and biochemical regulation of the flavonoid pathway in this worldwide important vegetable.

Polyphenols represent a great variety of compounds occurring in fruits, vegetables and plant-derived products. Dietary polyphenols have been found displaying several biological properties, such as anti-inflammatory, antioxidant and anti-aging activities, cardiovascular and neuro-protection, and reduction of the risk of intestinal diseases. The bio-efficacy of polyphenols is tightly linked to their bioavailability, to structural complexity and composition of food matrix in which they are present. Since most of the polyphenols are naturally stored in food matrices as glycosylated and/or variously decorated forms, they need an intestinal bio-conversion in more absorbable forms. Recent findings are highlighting the polyphenols-gut microbiota interplay in the health benefits linked to these compounds. Furthermore, the prebiotic-like activities of polyphenols on microbiota and their potential use in preventive/therapeutic strategies for gastrointestinal disorders are recently emerging.

Halloysite nanotubes (HNTs) are efficient nano-containers capable of entrapping a range of active agents within the inner lumen, followed by their retention and slow release. Halloysite is a green environmentally friendly object available in commercial quantities. The lumen of the halloysite tube accomodates globular protein diameters, allowing their entrapment within the inner lumen of the halloysite while retaining their activity for use in biocatalysis. In this work a combination of high resolution imaging technique such as TEM, SEM and SFM have been employed to elucidate the structure. We have investigated their visco-elastic properties by force-indentation measurements and performed cytotoxicity tests utilizing neoplastic cell lines (breast and cervical cancer cells). Furthermore their uptake has been confirmed by Confocal Microscopy after their functionalisation with fluorescence molecules. The results indicate that halloysite nanotubes have been readly uptaken by neoplastic cells and exhibit a high level of biocompatibility.

The spectroscopic behavior of mono-zinc ethane-bridged bis-porphyrin (ZnH2Po2) was studied as a solidfilm upon deposition from a chloroform solution by means of the spin-coating method. The angular speedused during the deposition strongly influences the conformational arrangement of bis-porphyrin. A preferentialarrangement as the anti- (opened) conformer was obtained when high speeds were used. Theobtained thin film was successfully employed to detect ammonia and acetone as the relevant analytes inaqueous solutions. This was a result of the supramolecular interaction between each of these two compoundsused and the bis-porphyrin active layer, inducing the corresponding conformational switchingeasily detectable by UV-vis absorption spectroscopy. The spectral variations of ZnH2Po2 were evident inthe range of concentrations between 1 ppm and 20 ppm for both the analytes. This is particular importantin light of the fact that ammonia and acetone concentrations are changed within this limit throughoutthe menstrual women's cycle, reaching their maximum during the fertility days. Hence, ZnH2Po2 canbe effectively applied as a potential active layer for medical devices to monitor the hormonal variationsduring the women's menstrual cycle.Additionally, the reversible sensing mechanism with reusable up to 8 cycles, makes this ZnH2Po2 basedactive layer practically efficient

Astrocytes have a key role in the pathogenesis of several diseases, including multiple sclerosis, and are proposed as a possible target for immunotherapy. Our earlier study reported that astrocytes treated with IFN-² modified their biomechanical properties possibly due to changes in the expression of the proteins involved in cytoskeleton organization and other important physiological processes. To gain insight into the mechanism underlying IFN-² action during inflammation, we stimulated astrocytes with LPS, a bacterial wall component used as a model for both in vitro and in vivo immunological stimulation of microglia and astrocytes. We showed that IFN-² reverses the effects of LPS on the proteome of astrocytes. To better examine this result, we performed a proteomic analysis of astrocytes treated with LPS or LPS plus IFN-². Treatment with LPS caused increases both in a series of proteins mainly involved in cytoskeletal changes and in protein degradation, as well as protective enzymes like superoxide dismutase. IFN-² reverses LPS effects on astrocytes proteome, supporting its protective role during inflammatory insults

The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism.Results. KPI-A genes from the genomes of S. tuberosum cv. Istrinskii and the wild species S. palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software.The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011. Our results concerning the number, location, and orientation of KPI genes of groups ? and ? were found to differ considerably from the published data.Conclusions. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time

Small molecule modulators of the Endoplasmic Reticulum glycoprotein folding quality control (ERQC) machinery have broad-spectrum antiviral activity against a number of enveloped viruses and have the potential to rescue secretion of misfolded but active glycoproteins in rare diseases. In vivo assays of candidate inhibitors in mammals are expensive and cannot be afforded at the preliminary stages of drug development programs. The strong conservation of the ERQC machinery across eukaryotes makes transgenic plants an attractive system for low-cost, easy and fast proof-of-concept screening of candidate ERQC inhibitors. The Arabidopsis thaliana immune response is mediated by glycoproteins, the folding of which is controlled by ERQC. We have used the plant response to bacterial peptides as a means of assaying an ERQC inhibitor in vivo. We show that the treatment of the plant with the iminosugar NB-DNJ, which is a known ER alpha-glucosidase inhibitor in mammals, influences the immune response of the plant to the bacterial peptide elf18 but not to the flagellin-derived flg22 peptide. In the NB-DNJ-treated plant, the responses to elf18 and flg22 treatments closely follow the ones observed for the ER alpha-glucosidase II impaired plant, At psl5-1. We propose Arabidopsis thaliana as a promising platform for the development of low-cost proof-of-concept in vivo ERQC modulation.

L'articolo illustra gli obiettivi ed i risultati più salienti relativi al PSR Regione Puglia progetto SaVeGraINPuglia.

Phenolic coumpounds present in grape berries are extracted from the skin, seeds and flesh during the winemaking process. These substances have a potentially positive effect, on human health, thus giving to red wine "bioactive properties" that an contribute to decrease the incidence of atherosclerosis, cancer, neurodegenerative diseases. Grape variety, quality, climate, geographical origin and phythopatologies can affect the quality and quantity of phenolic compounds that accumulate in the cells. Viticulture and vinification practice, which varies in different countries, determine the concentration of phenolic compounds in wine. During the vinemaking process numerous parameters, such as temperature, presence of seeds and berry skins and addition of enzymes, have been reported to affect the extraction of phenolic compounds during grape must fermentation. Conversely, poor knowledge is available on the effects of different yeast strains on the final concentration of polyphenols in red wine. Indeed, yeasts enzymatic activity are likely to influence the extraction of phenolic substances from grape tissues. With the aim to evaluate the existence of a potential correlation between polyphenols content, antioxidant capacity of wine and yeast starter cultures, three selected autochthonous strains of Saccharomyces cerevisiae, were used to inoculate large scale must fermentation. At the end of the process, the experimental wines obtained were evaluated for the content of different class of polyphenols and antioxidant capacity. The statistical analysis of obtained results revealed a significant influence of the autochthonous strain on the concentration of several class of polyphenols and total antioxidant capacity in the produced wines. The different strain influence on the analysed parameters assumes a technological significance and it strongly contribute in determining the final quality of the wine by influencing "functional parameters" important for the preservation of human health.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two ?-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first ?-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

The importance of hemoproteins for life lies largely in their iron-mediated chemical properties. In the human body, there are about 4 g of iron, a precious resource preserved by sophisticated recycling mechanisms. Iron is also important for pathogen growth, so it is not surprising that immune cells developed mechanisms to reduce iron availability in cases of inflammation. In healthy conditions, macrophages degrade hemoproteins and export iron, while if inflammation develops, they retain cytoplasmic iron to reduce extracellular iron concentrations. Iron-rich macrophages possess a stronger inflammatory ability, which explains the chronic inflammatory response observed in states of iron overload. Inflammatory bowel syndromes are often characterized by intestinal blood loss and consequent anemia, but iron-supplementation therapies may exacerbate the inflammatory response. In chronically transfused patients iron overload is frequently observed; the iron can become toxic and in excess, even fatal if not treated with iron-chelating drugs.

Plants frequently live in environments characterized by the presence of simultaneous and different stresses. The intricate and finely tuned molecular mechanisms activated by plants in response to abiotic and biotic environmental factors are not well understood, and less is known about the integrative signals and convergence points activated by plants in response to multiple (a)biotic stresses. Phytohormones play a key role in plant development and response to (a)biotic stresses. Among these, one of the most important signaling molecules is an oxylipin, the plant hormone jasmonic acid. Oxylipins are derived from oxygenation of polyunsaturated fatty acids. Jasmonic acid and its volatile derivative methyl jasmonate have been considered for a long time to be the bioactive forms due to their physiological effects and abundance in the plant. However, more recent studies showed unambiguously that they are only precursors of the active forms represented by some amino acid conjugates. Upon developmental or environmental stimuli, jasmonates are synthesized and accumulate transiently. Upon perception, jasmonate signal transduction process is finely tuned by a complex mechanism comprising specific repressor proteins which in turn control a number of transcription factors regulating the expression of jasmonate responsive genes. We discuss the latest discoveries about the role of jasmonates in plants resistance mechanism against biotic and abiotic stresses. Finally, the deep interplay of different phytohormones in stresses signaling will be also discussed.

The plant phenol trans-resveratrol, which is mainly found in grape, displays a wide range of biological effects. A cell suspension culture was developed from calli of grape leaves of Vitis vinifera cv. Negramaro in order to study the bioproduction of resveratrol. The effects of a number of secondary plant metabolism elicitors, namely chitosan, methyl jasmonate, jasmonic acid, coronatine, and 12-oxo-phytodienoic acid, were tested on this cell suspension culture. The identification and quantification of stilbenes was achieved with high performance liquid chromatography, with both spectrophotometric and mass spectrometric detection. Of the tested elicitors, methyl jasmonate was the most effective in inducing the biosynthesis of approximately 4 mg g-1 dry weight (about 60 mg L-1) of resveratrol. Conversely, 12-oxo-phytodienoic acid, jasmonic acid, and coronatine were able to trigger the synthesis of approximately 20 mg g-1 dry weight (200-210 mg L-1) of viniferins. Taken together, our results show for the first time different modulatory effects of closely-related jasmonates on stilbene biosynthesis.

Legumes such as Medicago truncatula and pea (Pisum sativum) have been selected as plant species to study the response to drought, salt, wounding, and fungal attack, and induction of double stresses. The transcriptional activation of several classes of genes, such as RNAs (microRNAs, siRNAs, tasiRNAs, antisense RNAs), and mRNAs coding for key signaling enzymes (hormone signaling: Jasmonic acid, Abscisic acid, Salicylic acid biosynthesis genes and signal regulating genes) will be studied. Libraries of clones have been constructed to perform Next Generation Sequencing.On one side, a Systems Biology approach is required to the understanding of stress signalling networks. In a comparative study, cloning of orthologous of resistance genes in other legume species will be required to monitor their activation and/or fine tuning.

Oleosin, caleosin and steroleosin, are normally expressed indeveloping seed cells and are targeted to oil bodies. In the present work,the cDNA of each gene, tagged with fluorescent proteins, was transientlyexpressed into tobacco protoplasts, and the fluorescent patterns observedby confocal laser scanning microscopy. Our results indicated cleardifferences in the endocellular localisation of the three proteins. Oleosinand caleosin both share a common structure consisting of a centralhydrophobic domain flanked by two hydrophilic domains and were correctlytargeted to LDs, whereas steroleosin, characterised by an N-terminal oilbody anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplasts fractionation on sucrose gradients indicated that both oleosinand caleosin-GFP peaked at different fractions than where steroleosin-GFPor the ER marker BiP, were recovered. Chemical analysis confirmed thepresence of triacylglycerols in one of the fractions where oleosin-GFP wasrecovered. Finally, only oleosin- and caleosin-GFP were able toreconstitute artificial oil bodies in the presence of triacylglycerols andphospholipids. Taken together our results pointed for the first time thatleaf LDs can be separated by the ER and both oleosin or caleosin areselectively targeted due to the existence of selective mechanismscontrolling protein association with these organelles.

Flavonoids represent a wide group of plant secondary metabolites implicated in many physiological roles, from the attraction of pollinators to the protection against biotic or abiotic stresses. Flavonoids are synthetized in a number of horticultural crops that are important components of our daily diet. In the last decades, the consumption of vegetables rich in antioxidants has been strongly promoted from the perspective of prevention/protection against chronic diseases. Therefore, due to their nutritional importance, several attempts have been made to enhance flavonoid levels in species of agronomic interest. In this review, we focus on the flavonoid biodiversity among the major horticultural species, which is responsible of differences among closely related species and influences the qualitative/quantitative composition. We also review the role of flavonoids in the nutritional quality of plant products, contributing to their organoleptic and nutritional properties, and the main strategies of biofortification to increase their content.

Resveratrol, a plant phenolic compound, is accumulated in grape and red wine, but it is not widely distributed in other food crops. Our previous works showed that resveratrol biosynthesis can be induced in tomato plant (Lycopersicum esculentum Mill.) via the heterologous expression of a grape (Vitis vinifera L.) stilbene synthase cDNA under the control of a constitutive (CaMV 35S) or a fruit specific (TomLoxB) promoter. Resveratrol synthesising fruits showed an increase in the levels of ascorbate and glutathione, the soluble antioxidants of primary metabolism and the total antioxidant activity. These results paralleled with a higher capability of transgenic fruit to counteract the pro-inflammatory effects of phorbol ester in mocyte-macrophages, via the inhibition of induced cyclooxygenase-2, and indicated the potential of our approach to improve the nutritional value of tomato. Since the levels of resveratrol detected in transgenic fruit were limited to a maximum of 50 ug/g fresh weight, we started a breeding programme to increase the content of this metabolite.We took advantage of the ability of an Arabidopsis myb-type transcription factor (AtMYB12) to strongly activate the expression of several phenylpropanoid biosynthetic genes that act upstream of stilbene synthase, suggesting that AtMYB12 can provide high rates of metabolic flux for resveratrol biosynthesis. To further increase the level of resveratrol, we have introduced in the breeding programme a spontaneous flavanone 3-hydroxylase tomato mutant. The presence of this mutation reduces the amount flavonols and should therefore enhance substrate availability to stilbene synthase. Preliminary data show that a combination of biosynthetic and regulatory genes together with the availability of natural tomato varieties is a powerful strategy for plant metabolic engineering.

European hazelnut is an important nut crop in Italy, where about 121,750 tons of in-shell nuts are produced every year. Roasting is the most important practice for hazelnut preservation and commonly is carried out in commercial electrical ovens at 120-160°C for 10-20 min. This needful practice is time and energy expensive, so the development of new processing methods is required to reduce processing costs and to obtain top quality roasted nuts. The aim of this study was to develop a simple microwave treatment for hazelnuts peeling and roasting.With this aim, some physical (colour, temperature, moisture) and chemical (taste, lipoxygenase activity, fatty acids, vitamins, sensory attributes) features of inshell nuts and kernels of three Italian hazelnut varieties (Tonda di Giffoni, Tonda Romana and Nocchione) after conventional oven or microwave roasting were evaluated.Results showed that microwave roasting of kernels for 450 s gave a higher peeling score than the conventional oven treatment. This paralleled with better colour and taste scores for microwaved roasted kernels. Furthermore, a 360-450 s microwave roasting was able to inactivate almost completely lipoxygenases, avoiding adverse effects on fatty acids hydroperoxides and PUFA content. A shorter microwave treatment (360 s) was enough to obtain good peeling and sensory scores of inshell hazelnuts.Taken together our results indicated that microwave technology can be successfully applied to both kernels and inshell hazelnuts to obtain suitable peeling and high quality roasted nuts.

Improved crop varieties are needed to sustain the food supply, to fight climate changes, water scarcity, temperature increase and a high variability of rainfalls. Variability of drought and increase in soil salinity have negative effects on plant growth and abiotic stresses seriously threaten sustainable agricultural production. To overcome the influence of abiotic stresses, new tolerant plant varieties and breeding techniques using assisted selection are sought. A deep understanding of the mechanisms that respond to stress and sustain stress resistance is required. Here is presented an overview of several mechanisms that interact in the stress response. Localised synthesis of plant hormones, second messengers and local effectors of abiotic stress response and survival, the signaling pathways regulated by plant hormones are today better understood. Metabolic networks in drought stress responses, long distance signaling, cross-talk between plant organs finalised to tissue-specific expression of abiotic stress relieving genes have been at the centre of most recent studies.

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effectiveproduction system for bioactives and other high value ingredients.

Plants show a remarkable developmental plasticity to adapt their growth to changing environmental conditions. Thus, understanding how non-coding RNAs work, locally andsystemically, may reveal novel mechanisms involved in growth control and differentiation. A new EU collaborative project may identify and assign a role to RNA species in legumes responding to biotic and abiotic stresses.

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis inflower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrolproducing(35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrolproducing(LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of thestilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and inone of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SSor LoxS tomato flowers, at an even higher extent (about 8e10 times) in the former line. We furtherinvestigated whether stilbene synthase expression may have resulted in impaired naringenin accumulationduring flower development. In the 35SS flowers, naringenin was significantly impaired by about50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increasedin transgenic flowers, thereby excluding the diminished production of flavonols as a reason forparthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resultedchanges to pollen structure. Microscopic observations revealed the presence of few and abnormal flakelikepollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric andferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion ofthese compounds, therefore suggesting an impairment in structural compounds as a reason for pollenablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major roledisplayed by resveratrol synthesis on parthenocarpy in tomato fruits.

Multiple stresses are becoming common challenges in modern agriculture due to environmental changes. A large set of phytochemicals collectively known as oxylipins play a key role in responses to several stresses. Understanding the fine-tuned plant responses to multiple and simultaneous stresses could open new perspectives for developing more tolerant varieties. We carried out the molecular and biochemical profiling of genes, proteins and active compounds involved in oxylipin metabolism in response to single/combined salt and wounding stresses on Medicago truncatula. Two new members belonging to the CYP74 gene family were identified. Gene expression profiling of each of the six CYP74 members indicated a tissue- and time-specific expression pattern for each member in response to single/combined salt and wounding stresses. Notably, hormonal profiling pointed to an attenuated systemic response upon combined salt and leaf wounding stresses. Combined, these results confirm the important role of jasmonates in legume adaptation to abiotic stresses and point to the existence of a complex molecular cross-talk among signals generated by multiple stresses.

The carrot (Daucus carota L.) is an important vegetable source of bioactive compounds in the human diet. In the Apulia region (Southern Italy), local farmers have domesticated colored landraces of carrots over the years, strictly related to local cults and traditions. Amongst these, an important landrace is the carrot of Saint Ippazio or the Tiggiano carrot. In the present study, we evaluated the content of carotenoids, anthocyanins, phenolic acids, sugars, organic acids, and antioxidant activity in Tiggiano carrots. Our results indicated that yellow-purple carrots have the highest levels of bioactive compounds, together with the highest antioxidant capacity compared to the yellow and cultivated orange varieties. These data point out the nutritional value of purple Tiggiano carrots and may contribute to the valorization of this typical landrace.

Oil bodies (OBs) are specialised organelles ubiquitously detected in plant oil seeds, which serve as lipid storage compartments. OBs consist of a hydrophobic core of triacylglycerol (TAGs), surrounded by a monolayer of phospholipids (PLs) embedded with some specific proteins with a size ranging from 0.5 to 2 mu m. In this work, we report an easy method to reconstitute OBs starting from their constituents and to encapsulate lipophilic molecules, i.e. the fluorescent fluorescein isothiocyanate (FITC) and carboxyfluorescein (CF), into reconstituted OBs. This methods allowed us to produce OBs 4- to 10-fold smaller (50-200 nm) than the native one and to obtain a good recovery (about 40%) of both the fluorescent compounds used in the present work. The properties of reconstituted OBs were investigated by a combination of Brewster angle microscopy, scanning force microscopy, zeta-potential techniques. OBs were stable and formed ordered monolayers when patterned on hydrophobic substrates whereas they showed a higher tendency to aggregate into larger, coalescing OBs when were deposited onto hydrophilic substrates or at the air/water interface. Furthermore, we verified the uptake of FITC-loaded OBs by the MCF-7 breast cancer cell line. Our results indicated that OBs could be envisaged as novel carriers to deliver hydrophobic bioactive compounds

Plants have evolved complex signalling pathways to cope with different biotic stresses. Complex interactions among these pathways permit a tight control between development and stress response. Oxylipin metabolism represents one of the main defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX), to form fatty acid hydroperoxides. These fatty acids hydroperoxides are finally converted into an array of bioactive compounds, collectively known as oxylipins, by the action of several enzymes. Some of these, namely allene oxide synthase (AOS), hydroperoxide lyase (HPL) and divinyl ether synthase (DES) form a specialised cytochrome P450s subfamily, known as CYP74. CYP74 enzymes are specialised for the metabolism of hydroperoxides and, differently from other P450 enzymes, do not bind molecular oxygen but use already oxygenated fatty acids as oxygen donors and as source for reducing equivalents. AOS, together with allene oxide cyclase (AOC), catalyse the formation of 12-oxophytodienoic acid (OPDA) or dinor-OPDA, which are the first intermediates in jasmonic acid biosynthetic pathway. Alternatively, hydroperoxides can be cleaved into some other signalling molecules, such as volatile aldehydes and oxo-acids by HPL or converted into divinyl ethers by DES. At the end of these enzymatic reactions, an array of different oxylipins, which includes jasmonates, aldehydes, ketols, divinyl ethers, each showing specific biological functions, are produced. Levels of oxylipins are low in normal physiological conditions but increase rapidly in response to mechanical wounding, herbivore and pathogen attack and other environmental or developmental inputs.In the present review we will focus on recent advances related to oxylipins biosynthesis and their contribution to local and systemic defence mechanisms of plants.

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs-QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs-QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs-QP-exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs-QP administration suppresses antigen-specific T-cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti-inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

Group A Kunitz-type protease inhibitors (KPIA) are involved in protecting potato plants frommicroorganisms and pests. While the nucleotide sequence is known for many KPIA genes of various potatocultivars (Solanum tuberosum subsp. tuberosum) and a few genes of tomato (Solanum lycopersicum), there are no data on their allelic diversity in other species of the genus Solanum. KPIA fragments were cloned, amplified, sequenced, and analyzed from plants of the subgenera Potatoe sect. Petota (five genes from S. tuberosum ssp. andigenum and two genes from S. stoloniferum) and Solanum (five genes from S. nugrum), and their con sensus sequences were established. An identity of 97-100% was observed among these sequences and the KPIA sequences of the sections Petota (cultivated potato Solanum tuberosum ssp. tuberosum) and Etuberosum (S. palustre) The interspecific variation of KPIA did not exceed its intraspecific variation for all but one species (S. lycopersicum). The distribution of highly variable and conserved sequences in the mature protein coding region was the same in all of the above species. The same primers failed to amplify the homologous genes from Solanum dulcamara, S. lycopersicum, and Mandragora officinarum. Phylogenetic analysis of the KPIA sequences showed that S. lycopersicum clustered separately from all of the other species examined, that S. nigrum clustered together with species of the sections Etuberosum and Petota, and that these species produced no speciesspecific clusters. Although S. nigrum is resistant to all known races of the oomycete Phytophthora infestans, which causes one of the most economically important diseases of Solanaceae, the aminoacid sequences encoded by S. nigrum KPIA differed slightly, if at all, from their counterparts of cultivated potato, which is susceptible to P. infestans infection.

Red wine is a treasured source of polyphenols, such as stilbenes, flavonols and hydroxycinnamic acids, depending on different factors, such as grape cultivar and wine-making practices. The red wine polyphenols (RWPs) have long been associated to reduced mortality and morbidity from cardiovascular diseases and cancer. Since matrix metalloproteinase (MMP) activity is involved in extracellular matrix degradation, a crucial step both in inflammation and cell migration, aim of this study was to examine the effects of specific classes of polyphenols from Primitivo and Negroamaro grapes and wines produced in the Apulian region (Southern Italy), on the release and activity of MMP-9 in human monocytoid cells.U937 monocitoid cells were pre-treated with increasing concentrations of polyphenolic extracts (PE) of Primitivo and Negramaro grape and wine, before stimulation with 30 nM phorbol myristate acetate (PMA) for 24 h. The release and activity of MMP-9 in culture medium was tested by ELISA and zymography, respectively. Both Primitivo and Negroamaro PE exhibited a concentration-dependent inhibition of MMP-9 release and activity, without any reduction in cell viability. The inhibitory effect was higher than that produced by individual chemically synthesized polyphenols.. In this study, the PE from Primitivo and Negramaro grapes and red wines were characterized, and shown to significantly and synergistically inhibit the monocyte-derived release and activity of extracellular matrix proteases. These inhibitory effects could explain, at least in part, the anti-inflammatory and anti-cancerogenic properties of RWP.

There is a need for sustainable fertilizers because common mineral fertilizers are increasingly costly and often induce water and air pollution. For instance, seagrass compost could be used as fertilizer in the coastal areas of the Mediterranean, thus also solving the issue of beached resi- dues. Here, we studied organic fertilization by application of seagrass-based compost in a tomato and lettuce crop succes- sion. Composts were made of posidonia and yard wastes at 1/4 w/w fresh weight ratio. Compost was applied at two concentrations: 10 and 20 Mg ha-1 fresh weight. Fresh leaves of posidonia were also tested directly as organic mulch in plots treated with conventional fertilizers. A control treatment in- volved mineral fertilization without mulching soil. Herbicide applications were performed in non-mulched plots immedi- ately before transplanting of tomato and lettuce for controlling the weeds, while mulched treatments did not receive any herbicide application. Results show that 20 Mg ha-1 of posidonia-based compost is equivalent to the inorganic fertil- ization because the nutritional status and yield of tomato and lettuce were statistically similar. No phytotoxicity symptoms were observed in any of the tomato and lettuce plants mulched with posidonia. The same growth rate was observed for toma- to and lettuce growing under conventional management prac- tices: mineral fertilizer and herbicide applications. Overall, our findings show that seagrass-based compost is a promising, sustainable fertilizer for tomato and lettuce.

Stored food and house dust arthropods include many species of mites and beetles affecting human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and B4. We manufactured a protein chip with immobilized KPI-B1 and B4 and showed trypsin/chymotrypsin-binding specificity indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens.

Using four different chromatographic steps, b-galactosidase was purified from the ripe fruit of sweetcherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunusavium b-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of fourdifferent active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and bgalactosidase-activity staining. The active polypeptides were individually excised from the gel andsubjected to SDS-PAGE. Each of the four native enzymes showing b-galactosidase activity was composedof two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjectedto N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry b-galactosidaseshowed a 43% identity with the 44 kDa subunit of persimmon and apple b-galactosidases and the 48 kDasubunit of carambola galactosidase I. The sweet cherry b-galactosidase exhibited a strict specificitytowards p-nitrophenyl b-D-galactopyranoside, a pH optimum of 4.0 and Km and Vmax values of 0.42 mMand 4.12 mmol min
1 mg
1 of protein respectively with this substrate. The enzyme was also activetowards complex glycans. Taken together the results of this study prompted a role for this class ofenzymes on sweet cherry fruit ripening and softening.

A lateral flow (LF) device combined with Quantum dots (QDs) technology was developed for rapiddetection of a specific mycobacterial flavoprotein reductase (FprA). In order to develop the LF assay based on a double-antibody sandwich format, two monoclonal antibodies recognizing different epitopes located in separated FprA domains, were identified. The first monoclonal antibody was immobilized onto the detection zone of a porous nitrocellulose membrane, whereas an other monoclonal antibody was conjugated to QDs nanoparticles as a detection system. Using these monoclonal antibodies we recorded a good fluorescence signal, whose intensity was directly proportional to the concentration of FprA protein. The use of antibodies conjugated with fluorescent semiconductor QDs via biotin-streptavidin bridge, allowed the detection of FprA protein at concentration as low as 12.5 pg/?l in less than 10 min. The reported technology could be useful for the diagnostic investigation of Mycobacterium tuberculosis and other human pathogens in clinical specimens.

Oil bodies (OBs) are micelle-like structures with an outer phospholipid monolayer embedding some specific proteins (oleosins) and surrounding a hydrophobic core of triacylglycerols (TAGs). Oleosins are alkaline hairpin-like proteins that are anchored into the OBs structure with their hydrophilic domains covering the surface. We performed surface pressure (?) and Brewster Angle Microscopy investigations of reconstituted OBs (ROBs) and of trypsin digested ROBs. The obtained ? vs time isotherms clearly show the formation of a surface layer. Upon ROBs suspension injection into the subphase, a clear-cut ? enhancement is recorded, followed by a long plateau region for ROBs suspensions more concentrated than 12.5. ?g/ml. The BAM analysis highlighted the presence of a dark background, ascribable to a 2D layer due to free components rearrangement and brilliant circular 3D domains, due to unaltered ROBs or small aggregates of ROBs. Increasing ROBs concentration, large domains appeared. We hypothesize that the presence of an excess of free TAGs in the 2D layer is crucial for the generation of such domains. We verified the generation of such typical structures, studying the behavior of a ROBs suspension (concentration of 12.5. ?g/ml) with two different approaches: after injection under a concentrated TAGs floating layers and after digestion with trypsin. These two procedures resulted in similar effects since proteinase digestion is like to induce the same morphology of a TAGs excess. © 2014 Elsevier B.V.

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. Polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their significant antioxidant activity, important both for plant physiology and human nutrition. With the aim to verify whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues, in the present work we took advantage of the availability of two different transgenic tomato lines in which a grape stilbene synthase gene was expressed under a constitutive or a tissue specific promoter, respectively. The induction of resveratrol synthesis in tomato affected the redox status of transformed tomato fruit. In particular, ascorbate and glutathione pool increased, significantly and proportionally to the amount of the resveratrol synthesised in transformed tissues. Noteworthy, the increase in ascorbate and glutathione pool paralleled with a significant increase in the activities of the main enzymes involved in redox homeostasis, i.e. catalase, ascorbate peroxidase as well the enzymes responsive for ascorbate recycling. Furthermore, lipoxygenase activity and levels of polyunsaturated fatty acids hydroperoxides, were reduced in fruit tissues from transgenic tomato lines. The synthesis of resveratrol, together with the global redox status had an impact on the total antioxidant activity of transgenic fruits. Again the total antioxidant capability increased proportionally to the amount of synthesised resveratrol with the most remarkable increase recorded from the lipophilic-resveratrol containing-fraction of transgenic fruits. Overall, our results pointed a higher nutritional value of resveratrol synthesizing tomato fruits.

The plant polyphenol trans-resveratrol (3, 5, 4?-trihydroxystilbene) mainly found in grape, peanut and otherfew plants, displays a wide range of biological effects.Numerous in vitro studies have described various biologicaleffects of resveratrol. In order to provide more informationregarding absorption, metabolism, and bioavailability ofresveratrol, various research approaches have been performed,including in vitro, ex vivo, and in vivo models. Inrecent years, the induction of resveratrol synthesis in plantswhich normally do not accumulate such polyphenol, hasbeen successfully achieved by molecular engineering.Inthis context, the ectopic production of resveratrol has beenreported to have positive effects both on plant resistance tobiotic stress and the enhancement of the nutritional value ofseveral widely consumed fruits and vegetables. The metabolicengineering of plants offers the opportunity to changethe content of specific phytonutrients in plant - derivedfoods. This review focuses on the latest findings regardingon resveratrol bioproduction and its effects on the preventionof the major pathological conditions in man.

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3? (GSK-3?). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance. © 2012 The Royal Society of Chemistry.

Carcinoma progression is associated with the loss of epithelial features, and the acquisitionof a mesenchymal phenotype by tumour cells. Herein we show that exposure of MCF-7cells to epidermal growth factor (EGF) resulted in morphological alterations characteristicof epithelial-to-mesenchymal transition (EMT). EGF treatment resulted in increased motilityalong with an up-regulation of transcription factors Slug, Zeb1, Zeb2, and mesenchymalmarkers Vimentin and N-cadherin.Treatment of MCF-7 cells with a combined stimulation of EGF and resveratrol, a naturallyoccurring stilbene with antitumor properties, failed to alter cell morphology, motility andoverexpression of EMT markers induced by EGF. Using specific chemical inhibitors, wedemonstrated that EGF-induced EMT is mediated by extracellular signal-regulated kinase1/2 (ERK 1/2) signalling pathway and that resveratrol is able to repress EGF-induced ERKactivation.In summary, these data provide new evidence of the inhibitory effect of resveratrol onEGF-induced EMT cell transformation.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that in response to microbial infections generate long-lasting adaptive immune response. Following microbial uptake, DCs undergo a cascade of cellular differentiation that ultimately leads to "mature" DCs. Mature DCs produce a variety of inflammatory cytokines, including tumor necrosis factor-alpha (TNFbeta) a key cytokine for the inflammatory cascade. In numerous studies, polyphenols, including quercetin, demonstrated their ability to suppress TNFalpha secretion and protect from the onset of chronic inflammatory disorders. We show that murine bone marrow derived DCs express Slpi following quercetin exposure. Slpi is known to suppress LPS mediated NFkappaB activation, thus, it was hypothesized that its expression could be the key step for polyphenol induced inflammatory suppression. Slpi-KO DCs poorly respond to quercetin administration failing to reduce TNFalpha secretion in response to quercetin exposure. Supernatant from quercetin exposed DCs could also reduce LPS-mediated TNFalpha secretion by unrelated DCs, but this property is lost using an anti-Slpi antibody. =, oral administration of quercetin is able to induce Slpi expression. Human biopsies from inflamed tract of the intestine reveal the presence of numerous SLPI+ cells and the expression level could be further increased by quercetin administration. We propose that quercetin induces Slpi expression that in turn reduces the inflammatory response. Our data encourages the development of nutritional strategies to improve the efficiency of current therapies for intestinal chronic inflammatory syndrome and reduce the risks of colorectal cancer development.

Lipid droplets (LDs) are ubiquitous organelles in plant cells, but their physiological roles are largely unknown. To gain insight into the function of LDs in plants, we have characterized the Arabidopsis homologues of SEIPIN proteins, which are crucial factors for LD biogenesis in yeast and animals. SEIPIN1 is expressed almost exclusively in embryos, while SEIPIN2 and SEIPIN3 have broader expression profiles with maximal levels in embryos and pollen, where LDs accumulate most abundantly. Genetic analysis demonstrates that all three SEIPINs contribute to proper LD biogenesis in embryos, whereas in pollen, only SEIPIN2 and SEIPIN3 play a significant role. The double seipin2 seipin3 and triple seipin mutants accumulate extremely enlarged LDs in seeds and pollen, which hinders their subsequent mobilization during germination. Interestingly, electron microscopy analysis reveals the presence of nuclear LDs attached to type I nucleoplasmic reticulum in triple seipin mutant embryos, supporting that SEIPINs are essential for maintaining the correct polarity of LD budding at the nuclear envelope, restricting it to the outer membrane. In pollen, the perturbations in LD biogenesis and turnover are coupled to reduced germination in vitro and with lower fertilization efficiency in vivo. In seeds, germination per se is not affected in seipin2 seipin3 and triple seipin mutants but there is a striking increase in seed dormancy levels. Our findings reveal the relevance of SEIPIN-dependent LD biogenesis in pollen transmission and in adjusting the timing of seed germination, two key adaptive traits of great importance in agriculture.

Anthocyanins, extracted from grape skin (Vitis vinifera), were dissolved in ultrapure aqueous subphaseand an oligophenylenevinylene derivative was spread on the subphase surface. Such oligomer waschosen as anionic counterpart of hydrosoluble anthocyanins in order to perform a LangmuireSchäferfilm of the dyad. Interface interactions between oligophenylenevinylene derivative and anthocyaninswere studied by Brewster angle microscopy and reflection spectroscopy. Additionally, the oligomerexhibits a chemical structure able to ensure an enhancement of the stability under UVevisible irradiationsof the film of the dyad without any variation of the natural pigment absorption in the visible range.The visible spectra of cast anthocyanins film and LangmuireSchäfer oligomer/anthocyanins film afterexposure to 254 nm irradiation showed a remarkable increase of the film stability, probably due to thescreening effect of the oligomer. Preliminary test of a LangmuireSchäfer film of oligophenylenevinylenederivative/anthocyanins as a herbicide sensor showed that sensing is completely reversible, stable andrepeatable.

Anthocyanins, extracted from grape skin (Vitis vinifera), were dissolved in ultrapure aqueous subphase and an oligophenylenevinylene derivative was spread on the subphase surface. Such oligomer was chosen as anionic counterpart of hydrosoluble anthocyanins in order to perform a LangmuireSchäfer film of the dyad. Interface interactions between oligophenylenevinylene derivative and anthocyanins were studied by Brewster angle microscopy and reflection spectroscopy. Additionally, the oligomer exhibits a chemical structure able to ensure an enhancement of the stability under UVevisible irradiations of the film of the dyad without any variation of the natural pigment absorption in the visible range. The visible spectra of cast anthocyanins film and LangmuireSchäfer oligomer/anthocyanins film after exposure to 254 nm irradiation showed a remarkable increase of the film stability, probably due to the screening effect of the oligomer. Preliminary test of a LangmuireSchäfer film of oligophenylenevinylene derivative/anthocyanins as a herbicide sensor showed that sensing is completely reversible, stable and repeatable.

Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

An immunosensor for highly sensitive detection of Salmonella spp has been developed using Surface Plasmon Resonance (SPR) as transducing technique. S.enteritidis spp, of various serotypes are major pathogens present in foodstuff and one of the causes of enteric diseases in humans. Most of the severe foodborne diseases are caused by nontyphoidal Salmonella serovars, with nearly 1.4 million cases of illnesses. The conventional methods of detecting foodborne pathogens require time-consuming preparation procedures including pre-enrichment, selective enrichment and colony isolation, and biochemical testing before the organism can be identified. To overcome these problems, label-free biosensors for this pathogen are highly desirable. SPR technique is a widely used optical technique for biosensing purposes. The reason of such interest is that it allows real-time monitoring of chemical and bio-chemical interactions occurring at the interface between a thin gold film and a dielectric interface, without the need for labeling the reagents. In this work, a high performance SPR device provided with a miniaturised microfluidics system, has been realized for the proposed application. A self-assembled layer of protein A was realized onto a typical SPR transductor via 11-MUA cross linker to achieve uniform, stable, and sterically accessible antibodies coating. The subsequent binding of polyclonal anti-Salmonella spp antibodies (Pab) Pabs have been characterized by Elisa assay to determine the specificity inter species and cross reactions against other pathogens such as E. coli and P. aeruginosa. The presented data showed that SPR allowed to attain a good and remarkable detection of bacteria, with improvement of sensibility. Exploitation of the proposed application for food safety is envisaged.

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined smallangle X-ray scattering (SAXS) and crystal structures of themain ERQC enzyme, ER alpha-glucosidase II (alpha-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the + 1 and + 2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of D-mannose at the + 1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the alpha-GluII + 1 and + 2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.

The lipoxygenase (LOX) catalysed peroxidation of polyunsaturated fatty acids (PUFA) is a key starting point indefence mechanisms common to plant, animal and at least some microorganisms. In human, the peroxidation ofarachidonic acid finally leads to the biosynthesis of important defence effectors such as leukotrienes and lipoxins.In plant, the most common substrates of lipoxygenases are represented by linoleic (C18:2) and linolenic (C18:3)acids. Differently from auto-oxidation reactions which produce a huge number of hydroperoxide derivatives, theoxidation reaction catalysed by LOXs is positional and stereo-specific. Indeed, only 9- or 13-hydroperoxides (witha prevalence of the S steroisomers) are produced from these substrates. PUFA hydroperoxides are per se signalmolecules or can be used as substrates for a number of enzymatic reactions carried out by other enzymes of theLOX pathway. At the end, an array of volatile and non volatile compounds, collectively known as phyto-oxylipins areproduced upon specific stress signals, by the contribution of a multitude of enzymes localised in different subcellularcompartments. Thanks to the excellent work of several groups of scientists around the world, our knowledge on thecontribution of the oxylipin pathway on plant defence mechanisms dramatically increased in recent years.In the present contribution, we'll focus our attention on the hydroperoxide lyase branch of the plant oxylipinspathway, responsible for the synthesis of volatile aldehydes, alcohols and other related compounds which areimportant constituents of fruit aromas and the green leaf volatiles.

Inflammatory bowel disease (Crohn's disease (CD) and ulcerative colitis (UC)) is a multifactorial disease resulting from immune dysregulation in the gut. The underlying colitis is characterized by high levels of inflammatory cytokines, including TNF alpha. Biological intervention for IBD patients using anti-TNF alpha antibodies is often an effective therapeutic solution. However, TNF alpha neutralization fails to induce remission in a subgroup of IBD patients, primarily in UC patients. There is a dearth of suitable animal models representing TNF alpha non-responders. Here we have combined one of the best UC models currently available, namely Winnie and the TNF alpha KO mouse to generate a TNF alpha-deficient Winnie to study early onset colitis. The induced TNF alpha deficiency with underlying colitis does not influence general health (viability and body weight) or clinical parameters (colon weight, colon length and histological colitis) when compared with the Winnie genotype alone. The molecular characterization resulted in identification of Il1 beta as the major elevated cytokine during early phases of colitis. Further, in vitro functional assay using bone marrow-derived dendritic cells confirmed IL-1 beta as the major cytokine released in the absence of TNF alpha. This study has generated a successful model of colitis that remains TNF alpha non-responsive and has demonstrated that IL-1 beta expression is a major pathway for theprogression of colitis in this system. These data also suggest that IL-1 beta can be a potential target for clinical intervention of UC patients who fail to respond to TNF alpha neutralization.

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.