Chemical,Pharmacological, and in vitro Metabolic Stability Studies on Enantiomerically Pure RC‐33 Compounds: Promising Neuroprotective Agents Acting as σ1 Receptor Agonists

Our recent research efforts identified racemic RC‐33 as a potent and metabolically stable σ₁ receptor agonist. Herein we describe the isolation of pure RC‐33 enantiomers by chiral chromatography, assignment of their absolute configuration, and in vitro biological studies in order to address the role of chirality in the biological activity of these compounds and their metabolic processing. The binding of enantiopure RC‐33 to the σ₁ receptor was also investigated in silico by molecular dynamics simulations. Both RC‐33 enantiomers showed similar affinities for the σ₁ receptor and appeared to be almost equally effective as σ₁ receptor agonists. However, the R‐configured enantiomer showed higher in vitro hepatic metabolic stability in the presence of NADPH than the S enantiomer. Overall, the results presented herein led us to select (R)‐RC‐33 as the optimal candidate for further in vivo studies in an animal model of amyotrophic lateral sclerosis.     

New Anti-Nodal Monoclonal Antibodies Targeting the Nodal Pre-Helix Loop Involved in Cripto-1 Binding

Nodal is a potent embryonic morphogen belonging to the TGF-β superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44–67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (K_D 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.     

Effect of Post-harvest UV-B Irradiation on Polyphenol Profile and Antioxidant Activity in Flesh and Peel of Tomato Fruits

In the present study, the possibility of enhancing phenolic and flavonoid concentration in tomato (Solanum lycopersicum L.) fruits by post-harvest irradiation with UV-B light was assessed. Fruits of the commercial cv Money Maker (MM) and the mutant genotype high pigment-1 (hp-1), constitutively rich in these compounds, were harvested at mature green and turning stages and left to ripen within climatic chambers where they were daily treated with UV-B radiation (1 h, 6.08 kJ/m² day). In control chambers, UV-B radiation was screened by benzophenone-treated polyethylene film. The treatment was generally effective in increasing phenolic, flavonoid and flavonol concentration in both peel and flesh of MM and hp-1 fruits, although in this latter the positive response to UV-B treatment was mainly evident in fruits harvested at mature green stage. Following UV-B treatment, antioxidant activity increased in the peel of both genotypes independently from the harvesting stage and in the flesh of hp-1 fruits harvested at mature green stage. Hydroxycinnamic acids of both genotypes reacted to UV-B treatment differently depending on harvesting stage and tissue localisation, generally showing an increase in the peel of fruits harvested at mature green stage. With few exceptions, UV-B irradiation also induced a higher accumulation of individual flavonoids both in the peel and in the flesh of MM and hp-1 fruits independently from harvesting stage. Based on these results, UV-B irradiation can be considered a promising technique to increase the nutraceutical potential of tomato fruits by non-molecular tools.     

One-pot melt synthesis of resorcinol based polyarylates for UV-stable coatings

A new one-pot method for the melt synthesis of polyarylates based on resorcinol and phthalic acids has been developed. The method consists in the reaction of diphenyl carbonate, phthalic acids and resorcinol using titanium butoxide as catalyst. Model reactions have shown that the diaryl ester is generated in situ by reaction of the aromatic diacid and of the aromatic diol with diphenyl carbonate. It is thus possible to obtain polymers with tailored end-groups and molecular weight, by changing the molar ratio of the monomers and the reaction conditions. More specifically, we applied this method to the synthesis of low molecular weight polymers with carboxylic end-groups, suitable for powder coating applications. The literature actually reports that resorcinol based polyarylates present an outstanding long-term UV stability and therefore COOH terminated oligomers are highly promising materials for the preparation of super-durable powder coatings.     

Polyphenol and nutrient release from skin of almonds during simulated human digestion

The bioaccessibility of nutrients and phytochemicals from almond skin has not been previously evaluated. We quantified the release of lipid, protein and polyphenols during simulated human digestion from natural (NS) and blanched (BS) skins, the latter being a by-product of the almond industry. Higher percentages of polyphenols were released from NS compared to BS during in vitro digestion. Most of the limited release of lipid and protein occurred during gastric digestion, with no significant differences between NS and BS. The total dietary fibre content was 45% for NS and 46% for BS, glucose and galacturonic acid being the major sugars present. No changes in dietary fibre composition and distribution of autofluorescent phenolics were observed in the cell walls of almond skin after simulated digestion. In the GI tract, the cell walls may therefore function as a useful source of fermentable fibre with beneficial implications for gut health.     

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L.

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC₅₀ values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.     

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.     

Obesity and sleep disturbance: the chicken or the egg?

Epidemiological studies suggested an association between obesity and sleep disturbances. Obstructive sleep apnea is the most prevalent type of obesity-related sleep disorder that lead to an increased risk for numerous chronic health conditions. In addition the increased visceral adipose tissue might be responsible for the secretion of inflammatory cytokines that could contribute to alter the sleep-wake rhythm. Unhealthy food characterized by high consumption of fat and carbohydrate seems to negatively influence the quality of sleep while diet rich of fiber is associated to more restorative and deeper sleep. Although obesity could cause through several pathogenetic mechanisms an alteration of sleep, it has been reported that subjects suffering from sleep disorders are more prone to develop obesity. Experimental laboratory studies have demonstrated that decreasing either the amount or quality of sleep increase the risk of developing obesity. Experimental sleep restriction also causes physiological, hormonal and food behavioral changes that promote a positive energy balance and a compensatory disproportionate increase in food intake, decrease in physical activity, and weight gain. Thus, the aim of this review is to provide observational evidence on the association of obesity with sleep disturbances and viceversa with emphasis on possible pathophysiological mechanisms (hormonal and metabolic) that link these two pathological conditions.