A framework for probabilistic seismic risk assessment of NG distribution networks

Lifelines are essential infrastructures for human activities and the economic developm ent of a region. Lifelines vulnerability reduction is an actual question, particularly with reference to NaTech events, like earthquakes. In this regard, worldwide past seismic experiences revealed heavy damages to NG distribution networks. It is therefore essential to perform seismic risk assessment of NG buried pipelines systems with the aim to identify potential criticalities and avoid significant consequences. For such reasons, this work illustrates the proposal of a probabilistic framework for seismic risk assessment of NG lifelines. The proposed procedure is subsequently applied to a specific case study in Italy to highlight its feasibility

Biochemical and immunochemical similarities among mammalian bilitranslocase and a plant flavonoid translocator

Flavonoids are a large class of plant secondary metabolites, belonging to polyphenol family, which possess pharmacological and nutritional properties. Their synthesis takes place only in plants, while mammals can acquire them only with diet. It has been demonstrated that flavonoid uptake occurs in rat also by the activity of bilitranslocase, a carrier that is involved in anion transport in liver cell, vascular endothelium and gastric mucosa. A sequence of bilitranslocase interacting with flavonoid moieties is already known and characterized. Antibody raised against such protein epitope were shown to exhibit cross-reactivity against plant membrane proteins in tissues involved in flavonoid transport and accumulation, such as teguments of carnation petals and skin of grape berries. Further immunolocalization studies allowed to demonstrate the presence of cross-reacting protein not only at the level of tegumental tissues, but also associated to sieve elements and seed teguments in grape berries

LIPOXYGENASE ACTIVITY AND HYDROPEROXIDE FORMATION IN COFFEE (Coffea arabica L.) CHERRIES CULTIVATED BY DIFFERENT AGRONOMIC TECHNIQUES

It is widely accepted that biotic stress induces acidic lipoxygenase (LOX) activity mainly due to the effects of methyl jasmonate [1]. The activation of LOX pathway leads to the production of a large amount of oxidation products, which may affect the coffee organoleptic profile [2]. Hitherto the presence of LOX in coffee (Coffea arabica L.) plants is still scarcely examined [3], therefore the aim of this study was to investigate the possible involvement of this enzyme in oxidative stress of cherries from plants cultivated in organic and conventional manners. Primary and secondary oxidation products were evaluated in three separate cherry fractions: pulp, parchment seed and green coffee. Hydroperoxides, measured by HPLC, were highest in parchment seed and lowest in green coffee, but no significant difference was found between the samples obtained from the two cultivations. However, the pulp fraction from organic cultivated plants had a larger amount of primary peroxidation products, when compared to that from conventionally cultivated plants. The presence of LOX was then examined in all cherry fractions by immunochemical analysis. Only the pulp fraction showed an 84 kDa protein that exhibited a cross-reactivity against a pea LOX antibody. Subsequently, the presence of LOX was confirmed in heavy (28000 g) and light (100000 g) membrane pulp fractions by an enzymatic assay, immunochemical method and hydroperoxide formation. In both organic and conventional cultivation systems, the light membrane showed the highest LOX activity, measured as conjugated diene formation, at pH 8.0. Instead, the heavy membrane fraction highlighted a different pH optimum, which was found to be 5.5 in pulps from organic cultivations and 8.0 in the conventional ones, respectively. All the activities reported were inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, well known LOX inhibitors. The NDGA-sensitive hydroperoxide production was simultaneously confirmed by RP-HPLC analysis. Oxidative stress was also detected by evaluating the antioxidant activity in all fractions. A lipophilic extraction, performed using dioxane:water (95:5, v/v), revealed that parchment seeds possessed the lowest antioxidant capacity. On the other hand, a significant difference in the antioxidative capacity was detected in parchment seeds from organic and conventionally cultivated plants. Taken together, these results show, for the first time, the presence of LOX in coffee cherries, whose activity was associated to membrane fractions. Furthermore, the cultivation techniques seem to induce the activation of different isoenzymes. In particular, the organic ones were subjected to an oxidative stress in coffee fruits leading to the expression of an acidic LOX activity, detectable in the pulp fraction. [1] Buzi A., et al. (2004). J. Phytopathol., 152: 34-42. [2] Kohlmann M., et al. (1999) Eur. J. Biochem. 260:885-895. [3] Rojas M.L., et al. (1993). Physiol. Mol. Plant Path., 43: 209-219

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures

Background Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. Results Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. Conclusions Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence

CHARACTERIZATION OF LIPASE ACTIVITY IN GREEN COFFEE BEANS DURING STORAGE AND GERMINATION

Lipase, green coffee, germination, storage, lipid Coffee seeds possess an intermediate storage pattern, showing a non-quiescent behaviour characterized by various metabolic reactions occurring during storage. In particular, lipase is the main enzyme involved in the mobilization of triacylglycerols, providing energy and a source of carbon skeleton during early stages of germination. During storage, the triacylglycerols might be involved in the generation of undesirable compounds (known as \u201coff-flavours\u201d), lowering both the viability of coffee seeds and the cup quality. In this work, the soluble protein fraction from coffee seeds and plantlets was extracted by acetone and then was utilised to assay lipase activity. Green coffee beans, harvested in Colombia, were stored at room temperature for 2-3 months, in order to verify the influence of prolonged storage on lipase activity. For germination experiments, the seeds were imbibed for 7 days at 30 \ub0C and transferred in perlite at 28 \ub0C and 90% R.H. for further 3 weeks. Lipase activity was detected by a colorimetric method based on specific degradation of a chromogenic substrate, at pH 8.2. Green coffee seeds exhibited an appreciable lipase activity that was slightly increased during storage. Such an activity was inhibited by tetrahydrolipstatin (THL) in a concentration-dependent manner, while it was slightly stimulated by both EGTA and EDTA. During the germination, after 10, 14, 17 and 21 days, lipase activity showed an initial increase that was followed by a gradual decrease. The effect of the presence or absence of the parchment (seed coat), during the first stages of germination, has also been investigated

Phospholipase activities in green coffee beans (Coffea arabica L.) harvested in different countries

Triacylglycerols (TAGs) are accumulated in specialised organelles called \u201coil bodies\u201d, which are enclosed in a phospholipid monolayer embedded with some unique proteins. Upon germination, such membranes are modified to allow the availability of TAGs as an energy source during early stages of seedling growth in oilseeds. This process occurs by the sequential and/or collective action of many hydrolytic enzymes, such as phospholipases, lipoxygenases and lipases that are associated to oil body membranes. In contrast, during seed storage, oilseed lipids may undergo lipolytic degradation processes leading to a wide range of metabolites potentially harmful for seed viability. In particular, green coffee endosperm consist of approx. 99% of the mature seed mass and contains many polyunsaturated fatty acids whose degradation leads to volatile compound formation through the oxylipin pathway. In spite of this, the enzymes involved in TAGs degradation (particularly lipases) are poorly studied. Therefore, the aim of this work was to evaluate the involvement of phospholipase activity in oil body membrane degradation during storage of green coffee (Coffea arabica L.) and to determine the correlations between storage lipid mobilization and maintenance of seed viability in beans harvested in different countries (Ethiopia, India, Kenya and Tanzania). Green coffee beans were frozen and powdered in liquid nitrogen and oil bodies were extracted with cold acetone. Phospholipase A1 and A2 activities were assayed in crude extracts by a fluorimetric method, using different probes. Such activities were just partially stimulated by free Ca2+, in contrast with what reported by others. Furthermore, PLA2 activity was assayed in a wide range of pH, evidencing two peaks of pH optimum. These results suggests that green coffee bean presents at least two isoforms of PLA2. Phospholipase profiles (PLA2 and total) were correlated with the provenience of the beans, showing a higher activity in those harvested in Ethiopia, while the lower was associated to beans from India

The permeability transition in plant mitochondria: The missing link

The synthesis of ATP in mitochondria is dependent on a low permeability of the inner membrane. Nevertheless, mitochondria can undergo an increased permeability to solutes, named permeability transition (PT) that is mediated by a pore (PTP). PTP opening requires matrix Ca2+ and leads to mitochondrial swelling and release of intramembrane space proteins (e.g. cytochrome c). This feature has been initially observed in mammalian mitochondria and tentatively attributed to some components present either in the outer or inner membrane. Recent works on mammalian mitochondria point to mitochondrial ATP synthase dimers as physical basis for PT, a finding that has been substantiated in yeast and Drosophila mitochondria. In plant mitochondria, swelling and release of proteins have been linked to programmed cell death, but in isolated mitochondria PT has been observed in only a few cases and in plant cell cultures only indirect evidence is available. The possibility that mitochondrial ATP synthase dimers could function as PTP also in plants is discussed here on the basis of the current evidence. Finally, a hypothetical explanation for the origin of PTP is provided in the framework of molecular exaptation

Properties of the permeability transition of pea stem mitochondria

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by MgADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel

Agronomic performance of 21 new disease resistant winegrape varieties grown in northeast Italy

The goal of the field trial was to evaluate the agronomic performance of 21 (10 red and 11 white) winegrape varieties obtained from recent breeding programmes for disease resistance developed in Hungary, Germany, and Italy. The tested red varieties were as follows: ‘Cabernet Carbon’, ‘Cabernet Eidos’, ‘Cabernet Volos’, ‘Julius’, ‘Merlot Khorus’, ‘Merlot Kanthus’, ‘Monarch’, ‘Prior’, UD. 31.103, ‘Vinera’. The tested white varieties were as follows: ‘Aromera’, ‘Bronner’, ‘Fleurtai’, ‘Johanniter’, ‘Muscaris’, ‘Souvignier Gris’, ‘Sauvignon Kretos’, ‘Sauvignon Nepis’, ‘Sauvignon Rytos’, ‘Solaris’, ‘Soreli’. ‘Merlot’ (red) and ‘Glera’ (white) were included as control. The experimental vineyard was established in Castelfranco Veneto on the plain, in 2014. Spray treatments were applied against downy and powdery mildew, by using only copper and sulphur. Grape production, grape quality, and phenology were recorded over a six-year-period, while disease resistance (downy mildew, powdery mildew, black rot and anthracnose) was detected only during a few years. The most significant findings were: a) all varieties showed a good level of downy mildew resistance, especially ‘Cabernet Carbon’, ‘Monarch’, ‘Prior’, UD 31.103, ‘Muscaris’, ‘Solaris’, ‘Souvignier Gris’, ‘Bronner’, ‘Fleurtai’, ‘Aromera’; b) no powdery mildew attacks were detected in any variety; c) ‘Monarch’, ‘Muscaris’, ‘Solaris’ and ‘Souvignier Gris’ also showed a high level of resistance towards black rot and anthracnose; d) red grape varieties had an earlier bud burst as compared to ‘Merlot’, and, concerning ripening, some varieties were earlier than ‘Merlot’, other ones were later; e) white varieties had a later bud burst but an earlier ripening time as compared to ‘Glera’; f) grape production and quality changed significantly depending on the varieties, being titratable acidity higher than 6.4 g L-1 tartaric acid and pH lower than 3.5; also the year affected in a significant way those parameters as well as the interaction between the genotype and the year. In conclusion, the tested varieties behaved positively in terms of environmental sustainability

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma