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The impact of tea constituents on the insulin-signaling pathway as well as their antidiabetic activity are still debated questions. Previous studies suggested that some tea components act as Protein Tyrosine Phosphatase 1B (PTP1B) inhibitors. However, their nature and mechanism of action remain to be clarified. This study aims to evaluate the effects of both tea extracts and some of their constituents on two main negative regulators of the insulin-signaling pathway, Low-Molecular-Weight Protein Tyrosine Phosphatase (LMW-PTP) and PTP1B.The effects of cold and hot tea extracts on the enzyme activity were evaluated through in vitro assays. Active components were identified using gas chromatography-mass spectrometry (GC-MS) analysis. Finally, the impact of both whole tea extracts and specific active tea components on the insulin-signaling pathway was evaluated in liver and muscle cells.We found that both cold and hot tea extracts inhibit LMW-PTP and PTP1B, even if with a different mechanism of action. We identified galloyl moiety-bearing catechins as the tea components responsible for this inhibition. Specifically, kinetic and docking analyses revealed that epigallocatechin gallate (EGCG) is a mixed-type non-competitive inhibitor of PTP1B, showing an ICAltogether, our data suggest that tea components are able to regulate both protein levels and activation status of the insulin receptor by modulating the activity of PTP1B.

Capsicum plant species are globally cultivated in warm and temperate regions, being important for agro-economic, biological and cultural aspects. While their worldwide spread and their ability of cross-pollination to easily hybridize play an important role in the formation of numerous species and varieties but also create confusion for their classification. For this reason, the categorization of species and varieties is complex and several methods have been used to evaluate pepper plant origin and evolution. Therefore, the objectives of this study were to compare a wild pepper (Capsicum chacoense) with other two domesticated cultivars belonging to different species such as Capsicum annuum and C. baccatum and draw conclusions about their origins using different approaches. For this purpose three methodologies have been used and compared: the comparison of their fruits volatile organic compounds (VOCs) emissions , their capsaicin and dihydrocapsaicin content and the leaves proteomic profiles. The VOCs analysis has been conducted by a time-of-flight mass spectrometry (ToF-MS) with an innovative approach to better identify all the compounds detected, in particular using two different ionization agents (H3O+ and NO+) to better identify all the compounds detected. The VOCs and pungency analyses were then used to build back propagation neural networks (BPNN) and a Random Tree classifier to conduct a multivariate analysis and evaluate the most species-specific volatiles. The outcomes appeared to be a most accurate approach with respect to the traditional varieties descriptors used for peppers discrimination. The BPNN led to the identification of several putative volatiles as good candidates for the recognition of these species or significant nodes in a decision learning tool. Finally, protein profiles have been obtained by SDS-PAGE analysis on the leaves to perform a fast proteomic comparison among the species. The protein profiles showed the C. baccatum and C. chacoense were more similar to the domesticated pepper C. annuum.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.

The involvement of transactivation response (TAR) DNA-binding protein 43 (TDP-43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. After 12 yr it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full-length protein, which has limited so far the characterization of its structure, function, molecular interactors, and pathobiology. Using a novel protocol we have achieved the purification of the full-length TDP-43, with both a short pectate lyase B tag and a glutathione S-transferase tag, which consisted in its expression in bacteria, solubilization from inclusion bodies, purification under denaturing conditions, refolding, and a final size exclusion chromatography (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and its stability. The protein is pure, as determined with electrophoresis, Western blotting, and mass spectrometry; properly refolded, as revealed by circular dichroism and fluorescence spectroscopies; functional, because it binds to DNA and protein partners; and stable to degradation and aggregation in a physiologic solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer.-Vivoli Vega, M., Nigro, A., Luti, S., Capitini, C., Fani, G., Gonnelli, L., Boscaro, F., Chiti, F. Isolation and characterization of soluble human full-length TDP-43 associated with neurodegeneration.

Functional and nutritional properties of baked goods can be enhanced by the use of sourdough fermentation, which is defined as a mixture of water and flour fermented by lactobacilli (LAB) and yeasts. Previous data highlighted the ability of sourdoughs obtained with selected LAB strains and commercial flour to produce bioactive peptides equipped with antioxidant and anti-inflammatory properties. More recently, it has been proven that choosing the most suitable combination of selected LAB and high-functional-value flours greatly increase the amount of low-molecular-weight antioxidant compounds responsible for improving the nutritional value of the products. This work aimed to isolate bioactive peptides both from sourdoughs and baked breads obtained with pigmented wheat and barley and a selected combination of LABs. Soluble water extracts were obtained, and low-molecular-weight peptides were isolated. Antioxidant activity was detected by assaying the intracellular ROS production in stressed cultured macrophages, treated with peptides. Moreover, anti-inflammatory activity, highlighted by NFkB pathway inhibition and by COX2 reduction in stressed cells, was demonstrated for peptides obtained from breads. The results allowed the conclusion that the combination of flours and LAB used in the present manuscript led to the production of bakery products with beneficial effects on oxidative and inflammatory status.

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) biovar 3 involved all global interest since 2008. We have found that in Psa3 genome, similarly to other P. syringae, there are three putative genes, lscα, lscβ and lscγ, coding for levansucrases. These enzymes, breaking the sucrose moiety and releasing glucose can synthetize the fructose polymer levan, a hexopolysaccharide that is well known to be part of the survival strategies of many different bacteria. Considering lscα non-coding because of a premature stop codon, in the present work we cloned and expressed the two putatively functional levansucrases of Psa3, lscβ and lscγ, in E. coli and characterized their biochemical properties such as optimum of pH, temperature and ionic strength. Interestingly, we found completely different behaviour for both sucrose splitting activity and levan synthesis between the two proteins; lscγ polymerizes levan quickly at pH 5.0 while lscβ has great sucrose hydrolysis activity at pH 7.0. Moreover, we demonstrated that at least in vitro conditions, they are differentially expressed suggesting two distinct roles in the physiology of the bacterium.

The importance of training in regulating body mass and performance is well known. Physical training induces metabolic changes in the organism, leading to the activation of adaptive mechanisms aimed at establishing a new dynamic equilibrium. However, exercise can have both positive and negative effects on inflammatory and redox statuses. In recent years, attention has focused on the regulation of energy homeostasis and most studies have reported the involvement of peripheral signals in influencing energy and even inflammatory homeostasis due to overtraining syndrome. Among these, leptin, adiponectin, ghrelin, interleukin-6 (IL6), interleukin-1β (IL1β) and tumour necrosis factor a (TNFa) were reported to influence energy and even inflammatory homeostasis. However, most studies were performed on sedentary individuals undergoing an aerobic training program. Therefore, the purpose of this review was to focus on high-performance exercise studies performed in athletes to correlate peripheral mediators and key inflammation markers with physiological and pathological conditions in different sports such as basketball, soccer, swimming and cycling.

The use of sourdough fermentation and whole grain flours in baked goods manufacturing are known to enhance their functional and nutritional features. In this context, it is necessary to select the most suitable lactic acid bacteria strains and flour combination to achieve this goal. A characterization of 70 lactobacilli strains based on pro-technological and nutritional properties was carried out. The screening allowed the selection of 10 strains that were used to ferment sourdoughs made with two varieties of common wheat, the conventional red-grained cv Aubusson, a blue-grained variety rich in anthocyanins cv Skorpion, and a hull-less barley variety, cv Rondo. From each fermented sourdough, a water soluble extract was obtained and evaluated for its antioxidant activity performed on cultured cells (RAW 264.7 murine macrophage) by assaying Reactive Oxygen Species (ROS) content. Sourdoughs made with pigmented wheat and barley, had an antioxidant activity greater than that recovered in those made with conventional wheat flour, in spite they have been inoculated with the same LAB strains. Results highlighted the interdependence between flour and the inoculated lactic acid bacteria that has to be taken into account for the development of healthy breads exploiting high functional value cereals through biotechnological processes.

Twenty years of research on cerato-platanin family proteins (CPPs) have led to some clear conclusions: CPPs are exclusively present in the fungal kingdom and possess an outstanding capacity to stimulate the immune system of plants. Recent discoveries have highlighted remarkable structural and functional similarities between CPPs and expansins, a class of non-enzymatic proteins found in both plants and microbes possessing loosening ability on the cell wall structure. Nevertheless, the determination of a biological role for CPPs in fungi is becoming a complicated puzzle to solve, since experimental data are often divergent and point to functional diversification. A general consensus appears however possible: CPPs from pathogenic and beneficial fungi may be considered as microbe-associated molecular patterns (MAMPs) and likely play a dual role, exerting functions in the fungal cell wall and/or in plant colonization. In this review, which celebrates 20 y of research on CPPs, we trace the history of these proteins and highlight experimental evidence and still unsolved issues.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by local follicular metabolic processes and in part a plasma transudate. Since the causes of impaired fertility may be due to a metabolic imbalance, metabolomics is useful to identify low molecular weight metabolites. Oxidative stress is involved in human infertility and the use of metabolomics can be crucial to identify which other metabolites besides reactive oxygen species are involved in oxidative stress correlated to infertility. To obtain new information on the study of signaling molecules in FF, the knowledge of the lipid content will be important to improve information on the understanding of follicular development. The objective of this study is to identify (a) a metabolic profile and a lipid profile of FF in women undergoing in vitro fertilization and (b) to correlate the previous information obtained regarding adiponectin and oxidative stress with the metabolic and lipid profile obtained in the present study. As result, we found an increase in oxidative stress due to both an increase of androgens and an accumulation of lipids in the follicular environment and we suggest that this might be one of the causes of reduced fertility.

Sourdough fermentation influences several properties of leavened baked goods also because Lactic acid bacteria (LAB) and yeasts produce bioactive peptides with a positive effect on human health. In an early study, three Lactobacilli strains (L. farciminis H3 and A11 and L. sanfranciscensis I4) possessing different proteolytic activities were used to produce sourdoughs containing peptides equipped with anti-inflammatory and/or antioxidant properties. This work was aimed to assess whether these properties could be retained after cooking. The selected LABs were used to produce breads from which low molecular weight (LMW-) peptides were extracted. The results provide solid proofs of keeping both antioxidant and anti-inflammatory activities of peptides from cooked products. Sequences of LMW-peptides either from doughs and breads were determined by mass spectrometry: differences have been noticed in amino acidic composition and in sequences, however, all the strains produce peptides equipped with antioxidant and anti-inflammatory activities.

Summary Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers., Graphical Abstract, Highlights • ETR cells show high miR-23b-3p that reduces SLC6A14 and amino acids upload • ETR cells promote autophagy and aspartate and glutamate import via SLC1A2 • Aspartate and glutamate fuel anabolic and catabolic pathways in ETR breast cancers • Targeting amino acid metabolic reprogramming is effective in ETR cells, Bacci et al. find that endocrine-resistant ER+ breast cancers are characterized by enhanced miR-23b-3p, autophagy activation, and import of aspartate and glutamate that fuel catabolic and anabolic pathways, which are essential for their aggressive features. The molecular players involved in this metabolic scenario are of clinical significance and have prognostic and predictive value.

Honey is a food known for its medical properties. In this work, we have studied the impact of different types of honey on insulin signalling pathway. We found that honey extracts inhibit the enzyme PTP1B, one of the main negative regulators of insulin receptor signalling. HPLC-MS analysis allowed us to confirm the presence of several natural PTP1B inhibitors in the honey extracts analysed. Statistical analysis methods show a correlation between specific 1H-NMR resonance frequencies/HPLC peaks and the inhibitory power of the samples. This finding will allow the prediction of the biological properties of honey samples applying relative simple analytical methods. Finally, we demonstrated that the treatment of HepG2 cells with honey extracts enhances the expression of insulin receptor, and stimulates glucose uptake. For the first time, our results demonstrate that bioactive components of honey could improve glycaemic control by both inhibiting PTP1B and stimulating the expression of insulin receptor in liver cells.

In this work, a non-metallicolous and a metallicolous population of S. paradoxa were exposed to copper excess and fungal elicitation, and investigated for phytohormone production and cytological alterations. Under the stress applied separately and in combination, S. paradoxa plants varied phytohormone concentration in a population-specific way, suggesting a different signalling in response to biotic and abiotic stimuli according to the environment of origin. Generally, the stress responses consisted in increased levels of salicylic acid, auxin, and gibberellin in the non-metallicolous population, and of jasmonic and abscisic acid in the metallicolous one. Interestingly, the metallicolous population increased the level of such phytohormones following exposure to the fungal elicitor only in the presence of copper. This alternative hormonal signalling could derive from the incompatibility between the ordinary ROS-mediated response to pathogens and the acquired mechanisms that prevent oxidative stress in the population from the metal-rich soil. Furthermore, stress-induced autophagic phenomena were more evident in the non-metallicolous plants than in the metallicolous ones, suggesting that the adaptation to the metalliferous environment has also affected autophagy intensity and signalling in response to copper excess and fungal elicitation.

Background: Most studies on oxidative stress markers and antioxidant levels have been conducted in male athletes, although female participation in sport has increased rapidly in the past few decades. In particular, it could be important to assess oxidative stress markers in relation to the training load because the anaerobic path becomes predominant in high-intensity actions. Methods: Ten female professional basketball players, performing five 2 h-lasting training sessions per week, and 10 sedentary control women were investigated. Capillary blood and saliva samples were collected in the morning before the training session. The antioxidant capacity and the levels of reactive oxygen metabolites on plasma were determined measuring Reactive Oxygen Metabolite and Biological Antioxidant Potential (d-ROMs and the BAP Test). Salivary cortisol was detected by using commercial enzyme-linked immunosorbent assay kit. Results: The antioxidant capacity (BAP value) was significantly higher in elite basketball players (21.2%, p &lt, 0.05). Conversely, cortisol (51%, 0.009) and the levels of oxidative species (d-ROM, 21.9%, 0.05) showed a significant decrease in elite athletes.

We investigated how the adaptation to metalliferous environments can influence the plant response to biotic stress. In a metallicolous and a non-metallicolous population of Silene paradoxa the induction of oxidative stress and the production of callose and volatiles were evaluated in the presence of copper and of the PAMP fungal protein cerato-platanin, separately and in combination. Our results showed incompatibility between the ordinary ROS-mediated response to fungal attack and the acquired mechanisms of preventing oxidative stress in the tolerant population. A similar situation was also demonstrated by the sensitive population growing in the presence of copper but, in this case, with a lack of certain responses, such as callose production. In addition, in terms of the joint behaviour of emitted volatiles, multivariate statistics showed that not only did the populations respond differently to the presence of copper or biotic stress, but also that the biotic and abiotic stresses interacted in different ways in the two populations. Our results demonstrated that the same incompatibility of hyperaccumulators in ROS-mediated biotic stress signals also seemed to be exhibited by the excluder metallophyte, but without the advantage of being able to rely on the elemental defence for plant protection from natural enemies.

Background L-Homocysteine (Hcy) is a non-proteinogenic α-amino acid synthesized from dietary methionine. In healthy humans, high Hcy levels are a risk factor for cardiovascular diseases, stroke and type 2 diabetes. A recent study reports that Hcy reacts with Cys10 of transthyretin (TTR), generating a stable covalent adduct. However, to date the effect of S-homocysteinylation on TTR conformational stability remains unknown. Methods The effect of Hcy on the conformational properties of wt- and L55P-TTR were analysed using a set of biophysical techniques. The cytotoxicity of S-homocysteinylated L55P-TTR was also evaluated in the HL-1 cardiomyocyte cell line, while the effects of the assemblies on kinematic and dynamics properties of cardiac muscle cells were analysed in cardiomyocyte syncytia. Results We found that Hcy stabilizes tetrameric wt-TTR, while it destabilizes the tetrameric structure of the L55P mutant, promoting the accumulation of self-assembly-prone monomeric species. Conclusions Our study demonstrated that S-homocysteinylation of the L55P-TTR mutant impairs protein stability, favouring the appearance of toxic monomers. Interestingly, S-homocysteinylation affected only mutant, not wt-TTR. Moreover, we also show that assemblies of S-homocysteinylated L55P-TTR impair cardiomyocytes functional parameters. General significance Our study offers new insights on the negative impact of S-homocysteinylation on L55P-TTR stability, whose aggregation is considered the causative agent of a form of early-onset familial amyloid polyneuropathy and cardiomyopathy. Our results suggest that high homocysteine levels are a further risk factor for TTR cardiomyopathy in patients harbouring the L55P-TTR mutation.

This work was performed to evaluate if metal adaptation can affect the response to biotic stress in higher plants. Three populations of Silene paradoxa, from a noncontaminated soil, a serpentine soil and a copper mine soil respectively, were cultivated in the presence/absence of nickel or copper and then were exposed to pathogen-associated molecular patterns (PAMP). In particular, the non-catalytic fungal protein cerato-platanin, secreted by the parasitic Ascomycete Ceratocystis platani, was used, because of its well documented ability to act as a PAMP, and the production of phytoalexins was assayed. Cerato-platanin exposition determined phytoalexin production in a population- and treatment-dependent way. Particularly, an over-production of phytoalexins was recorded for the copper mine population grown in the presence of copper, suggesting that, in particular cases, the adaptation to metalliferous environments can effectively affect plant response to biotic stress. Nevertheless, this supposition cannot be generalized to all the types of metalliferous environments and of metals studied; however, this work can be considered one of the first example of positive interaction between abiotic and biotic stimuli. © 2013 Elsevier B.V., SCOPUS: ar.j, info:eu-repo/semantics/published

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-populin (Pop1) produced by Ceratocystis populicola; a protein orthologous of cerato-platanin (CP), the core member of the CP family. These two proteins interact with host and non-host plants. In plane leaves they induce synthesis of phytoalexins, disruption of intercellular and intracellular leaf tissue, cell plasmolysis, programmed cell death, over-expression of defence-related genes, H2O2 and NO production, activation of MAPK cascade and plant resistance. All these features point to CP and Pop1 as defence inducers, though Pop1 shows a reduced efficiency. Pop1/CP similarity is 73 %. CD spectroscopy highlights some secondary structure differences between Pop1 and CP. Indeed, the region between the first two cysteines (C20–C57), that in CP includes the β2-strand and it is involved in GlcNAc (N-acetyl-d-glucosamine) interaction, in Pop1 is predicted to be fully disordered.

Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered E. coli SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.

Cerato-platanin (CP) is the founder of a fungal protein family consisting in non-catalytic secreted proteins, which work as virulence factors and/or as elicitors of defense responses and systemic resistance, thus acting as PAMPs (pathogen-associated molecular patterns). Moreover, CP has been defined an expansin-like protein showing the ability to weaken cellulose aggregates, like the canonical plant expansins do. Here, we deepen the knowledge on CP PAMP activity by the use of a multi-disciplinary approach: proteomic analysis, VOC (volatile organic compound) measurements, and gas exchange determination. The treatment of Arabidopsis with CP induces a differential profile either in protein expression or in VOC emission, as well changes in photosynthetic activity. In agreement with its role of defense activator, CP treatment induces down-expression of enzymes related to primary metabolism, such as RuBisCO, triosephosphate isomerase, and ATP-synthase, and reduces the photosynthesis rate. Conversely, CP increases expression of defense-related proteins and emission of some VOCs. Interestingly, CP exposure triggered the increase in enzymes involved in GSH metabolism and redox homeostasis (glutathione S-transferase, thioredoxin, Cys-peroxiredoxin, catalase) and in enzymes related to the "glucosinolate-myrosinase" system, which are the premise for synthesis of defence compounds, such as camalexin and some VOCs, respectively. The presented results are in agreement with the accepted role of CP as a PAMP and greatly increase the knowledge of plant primary defences induced by a purified fungal elicitor.

Based on sequence homology, several fungal Cys-rich secreted proteins have been grouped in the cerato-platanin (CP) family, which comprises at least 40 proteins involved mainly in eliciting defense-related responses. The core member of this family is cerato-platanin, a moderately hydrophobic protein with a double ?-? barrel fold. CP and the recently identified orthologous cerato-populin (Pop1) are involved in host-fungus interaction, and can be considered non-catalytic fungal PAMPs. CP is more active in inducing defense when in an aggregated conformation than in its native form, but little is known about other CP-orthologous proteins. Here, we cloned, expressed, and purified recombinant Pop1, which was used to characterize the protein aggregates. Our results suggest that the unfolded, self-assembled Pop1 is more active in inducing defense, and that the unfolding process can be induced by interaction with hydrophobic inanimate surfaces such as Teflon, treated mica, and gold sheets. In vivo, we found that both CP and Pop1 interact with the hydrophobic cuticle of leaves. Therefore, we propose that the interaction of these proteins with host cuticle waxes could induce unfolding and consequently trigger their PAMP-like activity.

Moderate wine consumption is associated with human health benefits (reduction of cardiovascular risk and neurodegenerative diseases, decrease of onset of certain cancers) attributed to a series of bioactive compounds, mainly polyphenols, with antioxidant power capable of counteracting the negative action of free radicals. Polyphenols are naturally present in the grapes, but an additional amount originates during winemaking. The aim of this work was to assess the ability of four commercial and two indigenous Saccharomyces cerevisiae strains to produce bioactive compounds (tyrosol, hydroxytyrosol, tryptophol, melatonin and glutathione) during alcoholic fermentation. In order to exclude the fraction of antioxidant compounds naturally occurring in grapes, the strains were inoculated in a synthetic must. At the end of fermentation the bioactive compounds were analysed by High-Performance Liquid Chromatography, while antioxidant activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Moreover, freeze-dried samples, originating from the experimental wines, were used to perform ex-vivo assays on cultured cells (RAW 264.7 murine macrophages) with the aim to evaluate their antioxidant and anti-inflammatory activities. The results indicated that the production of the considered bioactive compounds is a strain-specific property; therefore, the different yeast strains utilized during fermentation have different capabilities to modify the antioxidant and anti-inflammatory properties of the wine.

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-populin (Pop1) produced by Ceratocystis populicola; a protein orthologous of cerato-platanin (CP), the core member of the CP family. These two proteins interact with host and non-host plants. In plane leaves they induce synthesis of phytoalexins, disruption of intercellular and intracellular leaf tissue, cell plasmolysis, programmed cell death, over-expression of defence-related genes, H2O2 and NO production, activation of MAPK cascade and plant resistance. All these features point to CP and Pop1 as defence inducers, though Pop1 shows a reduced efficiency. Pop1/CP similarity is 73%. CD spectroscopy highlights some secondary structure differences between Pop1 and CP. Indeed, the region between the first two cysteines (C20-C57), that in CP includes the β2-strand and it is involved in GlcNAc (N-acetyl-D-glucosamine) interaction, in Pop1 is predicted to be fully disordered.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψβ-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.