Persulfidation of mitoKv7.4 channels contributes to the cardioprotective effects of the H2S-donor Erucin against ischemia/reperfusion injury.

BACKGROUND: Hydrogen sulfide (H2S) is a gasotransmitter deeply involved in cardiovascular homeostasis and implicated in the myocardial protection against ischemia/reperfusion. The post-translational persulfidation of cysteine residues has been identified as the mechanism through which H2S regulates a plethora of biological targets. Erucin (ERU) is an isothiocyanate produced upon hydrolysis of the glucosinolate glucoerucin, presents in edible plants of Brassicaceae family, such as Eruca sativa Mill., and it has emerged as a slow and long-lasting H2S-donor. AIM: In this study the cardioprotective profile of ERU has been investigated and the action mechanism explored, focusing on the possible role of the recently identified mitochondrial Kv7.4 (mitoKv7.4) potassium channels. RESULTS: Interestingly, ERU showed to release H2S and concentration-dependently protected H9c2 cells against H2O2-induced oxidative damage. Moreover, in in vivo model of myocardial infarct ERU showed protective effects, reducing the extension of ischemic area, the levels of troponin I and increasing the amount of total AnxA1, as well as co-related inflammatory outcomes. Conversely, the pre-treatment with XE991, a blocker of Kv7.4 channels, abolished them. In isolated cardiac mitochondria ERU exhibited the typical profile of a mitochondrial potassium channels opener, in particular, this isothiocyanate produced a mild depolarization of mitochondrial membrane potential, a reduction of calcium accumulation into the matrix and finally a flow of potassium ions. Finally, mitoKv7.4 channels were persulfidated in ERU-treated mitochondria. CONCLUSIONS: ERU modulates the cardiac mitoKv7.4 channels and this mechanism may be relevant for cardioprotective effects.

Selective chemiluminescent TURN-ON quantitative bioassay and imaging of intracellular hydrogen peroxide in human living cells.

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 µM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

Effect of Aureobasidium pullulans strains against Botrytis cinerea on kiwifruit during storage and on fruit nutritional composition.

Kiwifruit, wounded at the equator or by pedicel removal, to simulate the stem end wound, were treated with Aureobasidium pullulans (L1 and L8 strains) and subsequently inoculated with conidia of Botrytis cinerea. Fruits were stored at -1 °C in normal refrigeration (NR) or in controlled atmosphere (CA) (2% O2; 4.5% CO2). After 4 months, both antagonists significantly reduced the disease in all experiments, L1 better than L8. In NR, their efficacy was higher than 80%. In CA, the disease reduction was lower: between 30% (L1) and 60% (L8). The ability of both strains to compete with the pathogen for nutrients was tested in kiwifruit juice (0.5%) by in vitro experiments. Antagonists significantly reduced pathogen conidia germination in water and in juice. An HPLC analysis was performed to define the amino acid composition of kiwifruit juice upon L1 and L8 treatment. L1 and L8 greatly increased the concentration of both glutamic and aspartic acids and stimulated the production of new amino acids, although at low concentrations. Each amino acid displayed an antifungal effect against mycelium growth of B. cinerea. Finally, L1 and L8, cold tolerant and active strains in CA, can be effectively applied to control the stem end rot of kiwifruit in long storage.

Antifungal effect of volatile organic compounds produced by Bacillus amyloliquefaciens CPA-8 against fruit pathogen decays of cherry.

The present work focuses on the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus amyloliquefaciens CPA-8 against Monilinia laxa, M. fructicola and Botrytis cinera, three postharvest fruit pathogens of sweet cherry fruit. VOCs were evaluated with a double petri dish assay against mycelial and colony growth of target pathogens. For this purpose, CPA-8 was grown on different media and cultured for 24 and 48 h at 30 °C before assays. Data showed that mycelial growth inhibition was higher when CPA-8 was grown on Tryptone Soya Agar (TSA) while no differences were generally observed when CPA-8 was cultured for either, 24 and 48 h. Moreover, no effects were observed on colony growth. The main volatile compounds emitted by CPA-8 were identified by solid-phase microextraction (SPME)-gas chromatography as 1,3 pentadiene, acetoin (3-hydroxy-2-butanone) and thiophene. Pure compounds were also tested in vitro on mycelial growth inhibition and their EC50 values against the three pathogens were estimated. Thiophene was the most effective VOC, showing more than 82% suppression of mycelial growth at the highest concentration (1.35 µL/mL headspace) and EC50 values ranging from 0.06 to 6.67 µL/mL headspace. Finally, the effectiveness of thiophene and CPA-8 VOCs was evaluated against artificially inoculated cherry fruits. Among the target pathogens, M. fructicola was clearly controlled by CPA-8 with less than 25% of rotten fruits compared to the control (65% disease incidence) and for all pathogens, less than 37.5% of CPA-8 treated decayed fruits produced spores (disease sporulation). Otherwise, pure thiophene showed no effect against any pathogen on disease incidence and disease sporulation. The results indicated that VOCs produced by B. amyloliquefaciens CPA-8 could develop an additive antifungal effect against postharvest fruit pathogens on stone fruit.

Development of a liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous analysis of intact glucosinolates and isothiocyanates in Brassicaceae seeds and functional foods.

A new high pressure liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of glucosinolates, as glucoraphanin and glucoerucin, and the corresponding isothiocyanates, as sulforaphane and erucin, was developed and applied to quantify these compounds in Eruca sativa defatted seed meals and enriched functional foods. The method involved solvent extraction, separation was achieved in gradient mode using water with 0.5% formic acid and acetonitrile with 0.5% formic acid and using a reverse phase C18 column. The electrospray ion source operated in negative and positive mode for the detection of glucosinolates and isothiocyanates, respectively, and the multiple reaction monitoring (MRM) was selected as acquisition mode. The method was validated following the ICH guidelines. Replicate experiments demonstrated a good accuracy (bias%<10%) and precision (CV%<10%). Detection limits and quantification limits are in the range of 1-400ng/mL for each analytes. Calibration curves were validated on concentration ranges from 0.05 to 50µg/mL. The method proved to be suitable for glucosinolates and isothiocyanates determination both in biomasses and in complex matrices such as food products enriched with glucosinolates, or nutraceutical bakery products. In addition, the developed method was applied to the simultaneous determination of glucosinolates and isothiocyanates in bakery product enriched with glucosinolates, to evaluate their thermal stability after different industrial processes from cultivation phases to consumer processing.