A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C18 analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at ?Ex = 340 nm and ?Em = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 apparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format

The meeting on ADP-ribosylation reactions, PARP2017, was held in Budapest(17-19.5.2017). The event is held every two years in Europe, Alternating with the USA session, held at Cold Spring Harbor Laboratories. ADP-ribosylation is a reversible post-translational modification that has been studied in animals, plants, and bacteria, either plant and human pathogenic species. ADP-ribose transferases (ARTs) are enzymes adding poly-ADP-ribose (PARylation) and mono-ADP-ribose (MARylation) to proteins. In recent years, a new wave of interest is occurring on the topics on PARP/ART proteins. ADP-ribosylation is a reversible post-translational modification (PTM) that was thought to modify very few amino acids, mainly glutamate residues, with mono-ADP-ribose (MAR) or poly-ADP-ribose (PAR). Bacteria toxins, as exception, were known to modify other residues, as an exception. For this activity, ADP-ribosyltransferases (ARTs) have been defined writers. ART enzymes are collectively named MAR- or PAR- writers. ADP-ribosylation exerts allosteric effects on enzymes, thereby controlling their catalytic activity, or can modify amino acids that are no longer available for other types of PTM, such as phosphorylation, or methylation. Moreover, the MAR and PAR regions can be read by multiple protein motifs mediating protein-protein interactions.

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.

The transfer of well-studied native and chimeric pattern recognition receptors (PRRs) to susceptible plants are strategies to improve host resistance. In most cases, the ectodomain determines PRR recognition specificity, while the endodomain determines output quality and intensity. Here we report the generation and characterization of a chimeric receptor, carrying the ectodomain of the Arabidopsis thaliana EF-Tu receptor (EFR), and the endodomain of the tomato Cf-9 resistance protein. The chimera was expressed both transiently and stably in tobacco, and triggered a robust hypersensitive response upon elf18 treatment. Co-immunoprecipitation and virus-induced gene silencing studies showed that the chimera constitutively interacts with the SUPPRESSOR OF BIR1-1 (SOBIR1) co-receptor, and requires both SOBIR1 and kinase-active BRI1-ASSOCIATED KINASE-1 (BAK1) for its function. Transgenic plants expressing EFR-Cf-9 were more resistant to the hemibiotrophic bacterial pathogen Pseudomonas amygdali pv tabaci, indicating that this chimeric receptor is a valuable tool for both investigating the molecular mechanisms responsible for the activation of defense response by PRRs and R proteins, and for biotechnological use to improve crop disease resistance.

A method for isolating potential probiotic lactobacilli directly fromtraditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as subdominant populations.

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.

The microbial contamination in food packaging has been a major concern that paved the way to search for novel, natural anti-microbial agents, such as modified ?-mangostin. In the present study, twelve synthetic analogs were obtained through semi-synthetic modification of ?-mangostin by Ritter reaction, reduction by palladium-carbon (Pd-C), alkylation, and acetylation. The evaluation of the anti-microbial potential of the synthetic analogs showed higher bactericidal activity than the parent molecule. The anti-microbial studies proved that I E showed high anti-bacterial activity whereas I I showed the highest anti-fungal activity. Due to their microbicidal potential, modified ?-mangostin derivatives could be utilized as active anti-microbial agents in materials for the biomedical and food industry.

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.

The review recapitulate the current knowledge on the roles and importance of non-coding RNAs (ncRNAs) and small RNAs in bacteria. Several cases of regulating and signaling RNAs are presented either in Gram positive and Gram negative bacteria, with a focus on bacterial RNAs involved in pathogenesis and stress responses. Examples of small RNAs and their mechanisms of action, transcriptional and post-transcriptional regulation, induction of new genes, increased stability of their targets or their destination to the degradation pathway.

Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp, Comamonas spp., in response to stress conditions, i.e. under high carbon and low nitrogen (24:1) sources. PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Bioreactors are provided with biosensors to monitor physio-chemical parameters, such as temperature probe and pH sensor (linked to pumps to add NaOH or HCl), stirring speed, air flux regulation or micro-bubble dispersion by sparging (BIOSTAT Q Multi-Fermentor Bioreactor System with dissolved oxygen probe), to provide dissolved oxygen, needed for aerobic growth. Turbidity (OD600) and glucose consumption need to be 9 measured, at 0, 4, 8, 12, 24, 48, 72 and 96 h. PHA production need to be evaluated too, since after PHA accumulation bacterial cells are collected for PHA extraction. In order to make the process sustainable and economically convenient, two factors need to be optimised: high optical density of cells, and continuous presence of 5-10% sugars. The bacteria need to reach an OD higher than 25- possibly near 50, the obtain a good ratio of cells/volume, exploiting the maximum volume of bioreactor, without dispersing sugar in a high volume. PHA is detected by staining cells with Nile Blue, and evaluating the fluorescence intensity. Here we propose to apply three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by spectrophotometry or other sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining (with washing step), and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

Food pathogens frequently cause food-borne diseases. There is a need to rapidly identify the source of the bacteria in order to contain their spread and the epidemics. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. In this review we present an update on molecular methods alternative to nucleic acid based detection for species identification. Biosensor based methods rely on recognition of antigen targets or receptors by antibodies, aptamers or high affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high//affinity and high/specificity recognition molecule. Various different detection methods are discussed, from label-free sensors and immunosensors to fluorescence-based ones. Each method shows advantages and disadvantages in terms of equipment, sensitivity, simplicity and cost-effectiveness. Finally, Lab-on-a-chip (LOC) devices, are introduced briefly, with the potential to be fast, sensitive, and useful for on-site bacteria detection in food processing laboratories to check potential contamination by sample monitoring combined with a rapid pre-enrichment step.

Carpoglyphus lactis is a stored product mite infesting saccharide rich stored commodities including dried fruits, wine, beer, milk products, jams and honey. The association with microorganisms can improve the survival of mites on dried fruit.Methods and Results: The microbial communities associated to C .lactis were studied in specimens originating from the packages of dried apricot, plums and figs and compared to the laboratory strain. Clone libraries of 16S rRNA and ITS genes were constructed and analyzed at OTU97. The population growth on dried yeasts was 12 to 25 times higher on rearing diet than on dried fruits. The food boli formed by bacteria and plan remnants were common on control and dried apricot diet. The treatment and pretreatment of rearing diet by antibiotics provided a clue about the absence of antagonistic bacteria which might slow down the C. lactis population growth.Conclusions: The sequences of high similarity to Leuconostoc pseudomesenteroides, Elizabethkingia anopheles, Ewingella americana, Erwinia billingiae, Bacillus cereus and Serratia rubidaea were prevailing in the 16S rRNA library from mites fed on dried fruits. The ITS library showed smaller differences among laboratory strains on rearing diet and field strains, with the exception of mites on dried plums. The C. lactis had no antagonistic bacterial strains which were suppressed by antibiotics.Significance and Impact of the Study: The shifts of the microbial community in the gut of C. lactis were induced by the diet changes. The identified yeast and bacteria are suggested as the main food source of stored product mites on dried fruits.

Coffee is an important crop that assures a sustainable economy to tropical regions farmers. Coffee beans are produced from coffee berries by removing the outer fruit layers through dry or wet processes. Quality of coffee depends on optimized protocols of cultivation, ripe berries collection, fermentation or other processes to remove the mucilage, and, finally, moisture reduction. A big concern for coffee production is currently represented by climate change, that threatens the survival of Coffea arabica cultivation worldwide and imposes different strategies to react to this issue. Storage and shipment represent two steps where bean quality needs to be preserved by preventing fungal contamination that may impact the final product. In this review we describe the challenges afforded by the coffee industry to guarantee quality from production to roasting and brewing. An overview on novel technologies, such as the application of starter strains and the exploitation of industrial enzymes in accelerating the process of flavour development in coffee beans, is given. Moreover, the results of studies on microbial populations and the differences found in fungi, yeasts and bacteria composition among the investigations, are summarized. In particular, this review describes new attempts to contain the development of mycotoxigenic fungi, through the application of antagonistic microorganisms such as S. cerevisiae. The new wave of specialty coffees, i.e. those with a cupping score higher than 85/100, is also presented. It is shown how, through careful coffee production methods and controlled fermentation processes producers may increase their income by producing specialty coffees with high standards of quality and high added value for the sector area of coffee experience.

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.

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.

There is a need to identify the presence of bacterial pathogens in foods earlier than it is currently carried out. At present, pre-enrichment culture followed by selective agar plating are standard microbiological methods applied to detect Listeria monocytogenes and other bacterial pathogens with zero tolerance in food products. PCR and biosensor methods may shorten the time required to individuate contamination by L. monocytogenes in a pre-enrichment culture. In this study, a protein chip immunosensor with a sensitivity of 100 bacteria/ml was developed for Listeria spp. detection. The method provides a 10 fold increase in sensitivity compared to published work on bacteria immunosensing coupled to fluorescence detection. When 40 ml of Listeria spp. pre-enrichment culture were processed by immunoseparation, the limit of detection (LOD) was lowered to 2.5 cfu/ml. The results were confirmed by a qPCR method whose sensitivity was of 0.1 cfu/ml. The method based on immunosensors combined with immunomagnetic separation (IMS) on pre-enrichment cultures may shorten the time required for species identification. We also showed the potential for multiplexing of the protein chip method demonstrating the selective detection of Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli, and Campylobacter spp. A future development is the integration into a system for rapid screening of pathogens contaminating food productions.

In the recent years, a huge number of human transcripts have been found in the human genome thatdo not encode for proteins, which have been named non-coding RNAs (npcRNAs) containing secondarystructures or short regions highly conserved within mammalian sequences.Long RNAs (antisense RNA, structured RNA, and long interspersed ncRNAs) and small RNAs (miRNAs, siRNAs, snoRNAs) have shown to exert many roles: functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, controlling protein ubiquitination and degradation, regulating epigenetic marks and affecting chromosome structure. One group of npcRNAs thatis well-characterized, at the biochemical level, is represented by miRNAs. This group comprises alarge class of small npcRNAs (~22-nucleotide RNAs) acting through base pairing to partially complementary sites in the 3'untranslated regions (3'UTR) of the targeted messenger RNA.Circular RNAs, competing endogenous RNAs (ceRNAs) acting as RNA sponges, natural antisense RNAs (NAT), enhancer RNAs (eRNAs), and RNA decoys are further expanding the wide array of functionalities exerted by ncRNAs. In the last case, as example, Growth Arrest Suppressor 5 (GAS5) forms a structured RNA tha is a decoy for the glucocorticoid receptor (GR), mimicking the DNA structure of the GR element (GRE). Long noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Many of the mechanisms that modify the 3' UTRs, or that affect differential splicing, make use of RNA regulation, antisense RNAs, and may involve RNP complexes. Many human ncRNAs have been characterized in terms of function or expression profiles. HOTAIR, described in detail in the review by Ge Shan, is a structured RNA that assemble several proteins to form an epigenetic regulation complex: it assembles Polycomb Repressive Complex (PRC) proteins and determines the silencing of specific genes. Terminal differentiation induced ncRNA (TINCR) destabilises ALU elements in mRNAs through the RNA binding protein Staufen 1 binding to polypurine tract.Thus, proteins involved in the functioning of ncRNAs are highly varied: RNA binding proteins, ribonucleoprotein complexes, alternative splicing proteins, alternative polyadenylation proteins, chromatin remodeling complexes, and gene activation and repression complexes (PRC) and enzymes positioning or eliminating histone marks. Then, it is clear that the changes determined in several diseases and in cancer are caused non only by mutated genes but also by epigenetic deregulation and byalternative spliced genes and alternative polyA tails that evade microRNA recognition. Concerning miRs, the varied presence of Argonaute family of proteins and the link with diseases are well detailed in more than one review in this special issue.

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.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O-157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

Oxylipin family of signals represents one of the mechanisms employed by plants to communicate and respond to wounding, herbivores, and to biotic and abiotic stresses. This family comprises fatty acid hydroperoxides, hydroxy-, keto- or oxo- fatty acids, volatile aldehydes, divinyl ethers and Jasmonic Acid (JA). Most of them are volatile compounds participating in several physiological processes, defence mechanism, stress adaptation and communication with other plants and microorganisms. Studies on the comparison of jasmonates, OPDA, and Abscisic Acid (ABA) content and of gene expression variation in chickpea roots from a drought tolerant and a responsive variety, have confirmed preliminary studies made on drought and salt stress on different chickpea varieties, showing that involvement and up-regulation of specific LOX, AOS and HPL isoforms is required for stress tolerance. In this context, various levels of regulation of jasmonate signaling and JA biosynthesis pathway are discussed, sustained by observations made in roots and nodules of salt stressed chickpea varieties. Finally, an additional level of regulation of JA by epigenetics and microRNAs, with the involvement of ABA and NO responsive elements in promoters of transcription factor genes, is briefly introduced.Here we report about new insights on the role of the differential activation of JA biosynthesis during abiotic stress in roots of varieties differently responding to drought and salt stress, and on the importance of earlier and stronger JA induction as a trait conferring better drought tolerant in legume varieties able to cope with water stress. Real-time PCR may be useful to evaluate the timing and expression levels of specific gene isoforms in tolerant varieties, thus supporting breeding programmes for the identification of hybrids with improved JA synthesis, able to activate oxylipin specific pathways in a sustained and prolonged time course after stress perception.

Legumes play an increasing role in mixed cropping systems, crop rotations and as target crops for food, livestock feeding, and in human diets. Among grain legumes, the most important protein-rich crops are peas (Pisum sativum). The availability of early varieties favours the production of grain pea in shorter periods, after the end of rainy seasons and before the long periods of drought before summer. A field trial using an early pea variety was set up in march and lasted until the middle of June, when we measured the soil content in nitrogen and organic matter, to assess the enrichment rate in this period. The data presented in the study support the improvement of carbon and nitrogen input into the soil through a rotation of crops in the same year, to achieve an increase of resource use efficiency, and to reduce agriculture environmental impact with ad hoc climate mitigation solutions.

Food pathogens contaminate food products that allow their growth on the shelf and also under refrigerated conditions. Therefore, it is of utmost importance to lower the limit of detection (LOD) of the method used and to obtain the results within hours to few days. Biosensor methods exploit the available technologies to individuate and provide an approximate quantification of the bacteria present in a sample. The main bottleneck of these methods depend on the aspecific binding to the surfaces and on a change in sensitivity when bacteria are in a complex food matrix in respect to bacteria in a liquid food sample. In this review we introduce Surface Plasmon Resonance (SPR), new advancements in SPR techniques, and Electrochemical Impedance Spectroscopy (EIS), as fluorescence-free biosensing technologies for detection of L. monocytogenes in foods. The application of the two methods has made possible L. monocytogenes detection with LOD of 1 log CFU/ml. Further advancement are envisaged through the combination of biosensor methods with immunoseparation of bacteria from larger volumes, application of lab-on-chip technologies, and EIS sensing methods for multiplex pathogen detection. Validation efforts are being conducted to demonstrate the robustness of detection, reproducibility and variability in multi-site installations.

Raw meat, milk, seeds and vegetables are the source of bacteria, that are transferred through cross-contamination during food preparation. The standard microbiology method in food pathogens detection is the colony counting on agar plate, a procedure that requires several days for revealing the presence of a pathogen. This review will give importance to methods for detection of bacteria in food samples incubated in a pre-enrichment broth for times ranging from 24 to 48 hr, in order to accumulate a sufficient number of bacteria in the exponential growth phase peak. The highest sensitivity in species identification of bacteria has been achieved with molecular methods through amplification of DNA. In addition to the quantitative evaluation of the amplified DNA, DNA biosensors have been developed which are based on the hybridisation of target DNA with highly selective probes bound to a surface. The detection limit of DNA methods range between 10 to 100 colony forming units/ml of sample. Up to now, many biosensor-based methods are labour intensive and cost effective methods, which in addition are difficult to be implemented for in-field application.An ideal biosensor should detect target molecules directly without the use of labelled ligands or multiple washing steps. There are several bottlenecks to be solved in order to perform an efficient analysis. The first is the use of proper surfaces, the second is to efficiently and uniformly print the capture probe (antibody, binding proteins, aptamers) and to have a constant probe density in order to obtain the detection of targets with high reproducibility. The third is to obtain a high sensibility even at a low concentration of the targets. A number of methods are used for the rapid detection of biomolecules in solution. These include immunoassays, chromatographic methods, magnetic and biological biosensor methods (using screen-printed biosensors with immobilized enzymes), and antibody based detection methods. In this review we present an overview on these biosensors , on the imporvements in detection that are comparable to the methods based on DNA amplification (100 colony forming units/ml of sample.).

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

A great improvement in food safety and quality controls worldwide has been achieved through the development of biosensing platforms. Foodborne pathogens continue to cause serious outbreaks due to the ingestion of contaminated food. The development of new, sensitive, portable, high-throughput, and automated platforms is a primary objective to allow detection of pathogens and their toxins in foods. Listeria monocytogenes is one common foodborne pathogen. Major outbreaks of listeriosis have been caused by a variety of foods, including milk, soft cheeses, meat, fermented sausages, poultry, seafood and vegetable products. Due to its high sensitivity and easy setup, electrochemical impedance spectroscopy (EIS) has been extensively applied for biosensor fabrication and in particular in the field of microbiology as a mean to detect and quantify foodborne bacteria. Here we describe a miniaturized, portable EIS platform consisting of a microfluidic device with EIS sensors for the detection of L. monocytogenes in milk samples, connected to a portable impedance analyzer for on-field application in clinical and food diagnostics but also for biosecurity purposes. To achieve this goal microelectrodes were functionalized with antibodies specific for L. monocytogenes. The binding and detection of L. monocytogenes was achieved in the range 2.2 x 103 cfu/ml to 1 x 102 with a Limit of Detection (LoD) of 5.5 cfu/ml.

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

It is becoming apparent that spatio-temporally controlled patterns of DNA methylation and histonemodifications ensure that each cell in the body gets differentiated into its required cell fate during normaldevelopment. It is a well-coordinated and tightly synchronized unidirectional process that involves stepwisegene silencing associated with developmental stages, as well as the activation of tissue-specificprogramming of genes. However, dysregulations in epigenetic modifications can severely disturb normaldevelopmental pathways, thus leading to cancer development and progression. Furthermore, increasing listsof research publications are examining tumor suppressor and oncogenic miRNAs, which are epigeneticallymodified in different cancers, as well as upregulated long non-coding RNAs acting as miRNA sponges.

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.

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.

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.

Actually, the knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae xSaccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from “Primitivo” grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing a rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

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.

The importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown. NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart. Findings: we screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation. Conclusion: our data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

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.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized in cytoplasmic granules in bacteria, such as Cupriavidus necator (Ralstonia eutropha), and several other bacteria. PHAs accumulation occurs in response to stress conditions, i.e. under high carbon and low nitrogen (24:1 ratio). In this study, E. coli was genetically modified for PHA production in biofermentors. PHA was synthesized in bacteria transformed with the operon phbA/phbB/phbC. The bacteria were fed using a basal medium supplemented with three different plant by-products, potato tuber skin hydrolysate, corn hydrolysate, and banana juice supplement. The growth in biofermentor was monitored through the evaluation of consumption of sugars and quantification of PHA synthesis. A microarray scanner was used to read fluorescence intensity of Nile Blue stained bacteria. PHA production by E. coli fed on a banana juice supplement outperformed all the other fermentation media, with highest amount of PHA per dry cell weight.Keywords: Biodegradable polymers; Bacteria; E. coli; Hydrolysate

Huge advances there have been made since the first discovery of large sets of non-coding RNA transcripts as functional elements involved in the regulation of transcription and translation in which the FANTOM team performed the functional annotation of the mouse genome. John Mattick proposed that transcribed RNAs form a hidden layer of complexity amplifying the evolution potential of the genetic code, with fine-tuning role and tight control of transcripts and tissue specific regulation of transcription: microRNAs and small RNAs have been the paradigm of these functions. The binding of miRNA to its target mRNA results in inhibition of the translation of the target mRNA to a functional protein or degradation of target mRNAs.In addition, a complex network of competing endogenous RNAs (ceRNAs), acting as sponges, and able to sequester microRNAs or RNA binding proteins , interfering with the activity of miRNAs, have been described. Thanks to these findings, nowadays new databases have been set up, for long ncRNAs, ceRNAs, for tiny tRNA fragments originating small RNAs (tRFs) ; and various classes of transcribed RNAs .MicroRNAs recognise seed complementary sequences often based on the 3' UTRs of mRNAs. Therefore, single nucleotide polymorphisms in microRNA sequence or splice variants in mRNAs producing alternative 3' UTRs contribute to individual differences in the control of translation of specific transcripts. Combined analysis of protein-coding and small non-coding RNAs is crucial for a better understanding and more accurate prediction of the complex dynamics of post-transcriptional mRNA regulation. Alternative polyadenylation (APA) as well as single-nucleotide polymorphisms (SNPs) affect miRNA targeting of transcripts from different individuals and tissues.

In 1963, the team led by Pierre Chambon described the synthesis of poly(ADP-ribose) (PAR). ADP-ribosylation is a reversible post-translational modification used by PARP-domain proteins sharing a NAD+ binding pocket, that modify protein targets. It consists in mono ADP-ribose (MAR) or multiple ADP-ribose subunits (PAR). PARylation was then studied either in plants (Miwa et al. 1975), as well as in animals (Sugimura 1973, Miwa et al. 1973, Hayaishi and Ueda 1977). However, only starting from 1995 plant PARPs were studied at molecular and biochemical level (Lepiniec et al., 1995, Berglund et al., 1996, Babiychuk et al., 1998). Recently, two groups have analysed the PARP superfamily of proteins (Citarelli et al., 2010, Perina et al., 2014) whilst the Kangasjarvi team updated the phylogenetic tree focusing particularly on PARP domain containing SRO (Similar to RCD One) like proteins, such as Radical-Induced Cell Death1 (RCD1) (Jaspers et al., 2010).A tight collaboration between Japanese and Italian teams working on PARP and PAR was started since 1985 by Hisanori Suzuki, professor of Chemical Biology at Verona University. Following jointed efforts, in 1997 an Italy-Japan bilateral seminar on ADP-ribosylation was organised in Kyoto, with chairman Kunihiro Ueda, a pioneer of ADP-ribose studies. This special issue wants to acknowledge these efforts, that made possible not only to advance the studies on PARPs but also to tighten personal friendships. This topic on PARP superfamily and proteins binding or degrading PAR intercepts a new wave of interest and revival on the topics on PAR.

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.

House dust mites produce antibacterial proteins suppressing bacterial growth. The 14.5-kDa bacteriolytic protein (UniProtKB Q8MWR6) has been known in Dermatophagoides pteronyssinus. We have applied polymerase chain reaction and reverse transcription-PCR to detect a homologous gene sequence coding for a Q8MWR6-related protein in Dermatophagoides farinae (Hughes) using genomic DNA and total RNA, respectively. The resulting PCR product of expected size, 243 bp, was obtained from both Dermatophagoides spp., while no amplification was achieved from stored product mite samples. Sequence of the gene fragment from D. farinae showed 83% similarity to the previously described one in D. pteronyssinus. Successful amplification of the expected product from cDNA generated with oligo-dT primer implies that the NlpC/P60-like protein in Dermatophagoides mites is of eukaryotic or mite origin.

Poly-hydroxy-alcanoates (PHAs) are biodegradable and biocompatible polymers synthesized and accumulated in intracellular compartments in several bacterial species. Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp., Comamonas spp., in response to stress conditions, i.e., under high carbon and low nitrogen (24:1 ratio). PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Recombinant E. coli systems were studied to produce PHB using metabolic engineering. In biofermentors, the critical points are the excess of fermentable sugars and the ratio of nutrients versus cell optical density. In order to allow production in biofermentors in automated system, sensors are envisaged to evaluate critical parameters such as sugar consumption, bacteria concentration and level of synthesis of PHA. The need of fermentors and operation control has compelled for application of three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by enzyme linked sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining, and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

There is an arm race between plants and their pathogens, by fungi, and bacteria, as well as between plants and insects. Plant proteases are hydrolytic enzymes, grouped on the basis of the catalytic amino acid, as serine, cysteine, aspartic acid, or metal dependent activity. Plant-fungi interactions, as well as plants with other invaders, have been elucidated in recent years, showing an evolutionary adaptation of hosts and invaders to produce proteases and evolve new protease inhibitors. Interactions between protease inhibitors and the target proteases provide information on the ways organisms interact and defend themselves from pathogens, recognizing symbionts from parasite organisms. A comparative analysis of protease inhibitors in plants with sequenced genomes have been recently performed. In the analysis of PIs, protease biochemical assays, protein-protein interaction studies and protease chips were used to analyze constitutive and inducible inhibitors under different conditions. Recently, activity-based protease profiling (ABPP) was used to differentiate enzymes tissue specificity, and roles in various physiological and pathological states. Specificities of PIs toward different protease (serine and cysteine proteases) can allow to selectively and differentially bind and detect various proteases. In this review we summarize the most recent knowledge on plant pathogens and the mechanisms they evolved to circumvent plant defences among which pathogen effectors, proteases and proteases inhibitors. Finally, we introduce the recent findings on pathogen bodyguards, proteins interfering with plant defence mechanisms or decoys, mimicking Transcription Activator Like Effectors (TALE). It is envisaged that further advances in understanding the function of pathogen effectors will provide new ways to improve plant immunity and mechanisms of defence against their pests.

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.

Plants activate an immune response in defense against microbial pathogens. The first layer of immunity consists in the recognition of microbial fingerprints, called Pathogen Associated Molecular Pattern (PAMP), by a set of Pattern Recognition Receptors (PRR). In addition, the degradation products from fungi, bacteria and plant cells are recognised as Damage Associated Molecular Pattern (DAMP). The first layer of plant defence is based on Pattern Recognition Receptors (PRR) on the membrane. These receptors, either receptor kinases or receptor-like proteins (RLPs), associating with cytoplasmic kinases, recognize the presence of PAMPs, thus activating a local response named PAMP-triggered immunity (PTI), that is not strong but effective towards many pathogen species. Here we discuss and focus on Elongation Factor Tu Receptors (EFR) and flagellin sensing (FLS) receptors. In leucine-rich repeat (LRR) receptor proteins, the hydrophobic LLR domains are exposed on external membranes, providing the protein-protein interaction modules.Plants evolved this protein-protein interaction domain several times during the development of mechanisms to defend themselves from viruses, virulence factors, enzymes and effectors of bacterial and fungal pathogens. Pathogens in addition evolved proteins and enzymes that are injected in the plant cell to counterfight plant immune signaling pathways. These effectors are recognised by plant receptors sensing their presence of their cognate avirulence genes. These receptors originated from recombination during evolution and only occur in some specific tomato genotypes, instead of the widely occurring PPRs. Effector Triggered Immunity (ETI) allows a plant response to effector proteins that is more strong, but is race specific. It leads to local necrosis and apoptosis, and to the establishment of the hypersensitive response (HR). For biotrophic or hemibiotrophic pathogens, necrosis is an effective way to limit their spread, while for necrotrophic pathogens this is not efficient and sufficient way to limit their spread, since depends on the timing of infection and on the plant development phase. Pathogenic fungi strategy relies on the formation of specialised structures, or haustoria, that facilitate the nutrient uptake form plant cells. In this review we summarize the most recent knowledge on plant pathogens and the mechanisms they evolved to circumvent plant defences among which pathogen effectors, protein decoys inactivating plant defence signals. Effectors are recognised through their binding to plant proteins by means of plant receptors, that activate the Effector Triggered Immunity (ETI). In particular, we focus on the Solanaceae, discussing general mechanisms and specific pathways that confer resistance to various pathogens.There is an arm race between plants and fungal and bacterial pathogens that has led to new protein variants and protein decoys (pseudokinases, inhibitors and sponges blocking glucanases, and Transcr

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.

MicroRNAs are aberrantly expressed in many cancers and can exert tumor suppressive or oncogenic functions. Since oncomirs promote growth of cancer cells and support survival during chemotherapy, thus microRNA silencing therapies could be a valuable approach to be associated with anticancer drugs and chemotherapy treatments. miR-155 microRNA was found overexpressed in different types of cancer, such as leukemias (PML, B-cell lymphomas), lung cancer and glioblastoma. GABA-A receptor downregulation was found correlated with glioma grading, with decreasing levels associated to higher grade of malignancies. A relationship between knock down of miR-155 and re-expression of GABRA 1 protein in vivo was recently individuated. This finding has implication on the effectiveness of RNA silencing approaches against miR-155 with the scope to control proliferation and signalling pathways regulated by GABA-A Receptor. Applying microRNAs for treatment of brain tumours poses several problems, and fields to be solved are mainly the passage of the brain-blood-barrier (BBB) and the targeted delivery to specific cell types. GBM cells bud off microvesicles that deliver cytoplasmic contents to nearby cells. Thus, the exploitation of these mechanisms to deliver antagomir therapeutics targeting microvescicles in the brain could take the lead in the near future in the treatment of brain cancers in substitution of invasive surgical intervention.

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.

Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 hours of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 ml, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 ?l. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions.

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.

The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation.

NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organism homeostasis. NAD+ is a coenzyme in redox reactions, a donor of adenosine diphosphate-ribose (ADPr) moieties in ADP-ribosylation reactions, a substrate for sirtuins, a group of histone deacetylase enzymes that use NAD+ to remove acetyl groups from proteins; NAD+ id also a precursor of cyclic ADP-ribose, a second messenger in Ca++ release and signaling, and of diadenosine tetraphosphate (Ap4A) and oligoadenylates (oligo2'-5'A), two immune response activating compounds. In the biological systems considered in this review, NAD+ is mostly consumed in ADP-ribose (ADPr) transfer reactions. In this review the roles of these chemical products are discussed in biological systems, such as in animals, plants, fungi and bacteria. In the review, two types of ADP-ribosylating enzymes are introduced as well as the pathways to restore the NAD+ pools in these systems.

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short chain length (scl) or medium chain length (mcl) polymers: class I, class III, and class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while class II phaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization,catalysis mechanism, substrate recognition and affinity, product formation, and product egressroute. The assays based on amino acid substitution by mutagenesis have been useful to validatethe hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates,and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimizedchemical properties.

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.

high-risk HPV subtypes are driving forces for human cancer development: HPV-16 and HPV-18 are responsible for most HPV-caused cancers.OBJECTIVE: This review describes the present knowledge on HR-HPV genomes coding potential for viral miRNAs.METHOD: HPV subtypes miRNA database, VIRmiRtar, has been constructed applying bioinformatics and a computational method, ViralMir, exploiting structural features, presence of hairpins, and validation by comparison with RNA sequencing datasets.RESULTS: Several miRNA candidates have been localised in the genomes of high-risk HPV subtypes. Among these, HPV-16 miR-1, miR-2 and miR-3. The database contains a list of host candidate gene targets that may be responsible for the oncogenesis in the various cellular environments.CONCLUSION: miRNA silencing therapies, based on specific cellular uptake of miRNA mimics and antagomiRs, directed towards HPV encoded miRNAs and/or microRNAs deregulated in the host cells, could be a valuable approach to support pharmaceutical interventions in the treatment of HPV dependent cancers.

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.

La Lattoria è una azienda zootecnica del Salento per la produzione di latte bovino, che ha investito in impianti automatizzati per il veicolamento alla mungitura delle bovine e la raccolta robotizzata del latte. In questo modo, il latte è di alta qualità, paucimicrobico, e, grazie ad una alimentazione varia e controllata, ben equilibrato in sostanza grassa.L'azienda produce uno yogurt cremoso, non sgrassato, disponibile al pubblico tramite un distributore automatico refrigerato, da cui i clienti si riforniscono nelle 24 h, oltre che allo spaccio aziendale e in alcuni punti vendita a Lecce. Il benessere degli animali e un buon livello di pulizia forniscono una materia prima di elevato gusto, per la produzione di yogurt. La collaborazione con ISPA-CNR è in corso per evidenziare punti di forza e criticità e ampliare i segmenti di mercato (ceppi starter, tipi di frutta, alimenti funzionali).