Role of Bioactive Components in Inflammation and Oxidative Stress in Vascular Endothelium

An increasing number of scientific evidence supports the preventive value of dietary patterns that favor the consumption of plant food. Especially fruit, vegetables, grains and legumes; the correlation between the reduction of risk of chronic disease such as adherence to the Mediterranean diet is the most significant example. The central role of the endothelium in maintaining vascular homeostasis and the correlation between endothelial dysfunction and the development of cardiovascular diseases makes this tissue a primary target for dietary strategies aimed at cardiovascular diseases prevention. Research in the field of nutrition is therefore directed to the identification of food bioactive components with beneficial effects on the endothelium. This study first focused on the evaluation of the potential vascular protective effects of a wheat peptide belonging to the family of non-specific lipid transfer proteins type 2 (nsLTP2). nsLTP2, at physiological concentrations, showed antioxidant and cytoprotective effects in HUVECs undergoing oxidative/inflammatory stimulation and demonstrated modulatory capacity on the expression of adhesion molecules and heme oxygenase-1, both involved in endothelial inflammation. Polyphenols are widely studied antioxidant compounds and research supports the preventive/protective role of a polyphenol-rich diet. Despite experimental evidence of their positive influence on human health, to date there is no clear indication of the compunds responsible for this protective role. In fact, upon ingestion polyphenols are extensively metabolized and the molecule that will act at cellular level will more likely be a metabolite. For this reason the second part of the study focused on the protective effect of polyphenol metabolites belonging to two families: cinnamic acids and anthocyanins. Overall the tested compounds demonstrated antioxidant and cytoprotective activities at endothelial level in oxidative/inflammatory conditions, being also able to affect adhesion molecules expression. These observations may support and characterize biological activities of bioactive peptides and polyphenols metabolites beneficial to vascular health

Protective Effect of Wheat Derived Non-specific lipid-transfer Protein 2 on Vascular Endothelium Inflammation

The important functions of the endothelium and the relationship between cardiovascular risk factors and endothelial dysfunction suggest the primary role of this tissue as a target for dietary strategies aimed at the prevention from related diseases. Cereals are key component of a healthy and balanced diet, and the presence of non-specific lipid-transfer protein 2 (nsLTP2) in wheat represents an added value to contribute to maintain the functionality of the vascular endothelium and consequently of the cardiovascular system. Indeed, nsLTP2 downregulates the expression of the main cell adhesion molecules induced by a pro-inflammatory cytokine and, meanwhile, upregulates heme oxigenase-1, exerting a cytoprotective/anti-inflammatory activity. Therefore, nsLTP2 might represent a food-derived tool to protect the vascular system against several pathological condition

Effects of Vitamin E-Stabilized Ultra High Molecular Weight Polyethylene on Oxidative Stress Response and Osteoimmunological Response in Human Osteoblast

High Crosslink process was introduced in the development of joint prosthetic devices, in order to decrease the wear rate of ultrahigh molecular weight polyethylene (UHMWPE), but it also triggers the formation of free radicals and oxidative stress, which affects the physiological bone remodeling, leading to osteolysis. Vitamin E stabilization of UHMWPE was proposed to provide oxidation resistance without affecting mechanical properties and fatigue strength. The aim of this study is to evaluate the antioxidant effect of vitamin E added to UHMWPE on oxidative stress induced osteolysis, focusing in particular on the oxidative stress response in correlation with the production of osteoimmunological markers, Sclerostin and DKK-1, and the RANKL/OPG ratio compared to conventional UHMWPE wear debris. Human osteoblastic cell line SaOS2 were incubated for 96 h with wear particles derived from crosslinked and not crosslinked Vitamin E-stabilized, UHMWPE without Vitamin E, and growth medium as control. Cellular response to oxidative stress, compared to not treat cells, was evaluated in terms of proteins O-GlcNAcylation, cellular levels of OGA, and OGT proteins by immunoblotting. O-GlcNAcylation and its positive regulator OGT levels are increased in the presence of Vitamin E blended UHMWPE, in particular with not crosslinked Vit E stabilized UHMWPE. Conversely, the negative regulator OGA increased in the presence of UHMWPE not blended with Vitamin E. Vitamin E-stabilized UHMWPE induced a decrease of RANKL/OPG ratio compared to UHMWPE without Vitamin E, and the same effect was observed for Sclerostin, while DKK-1 was not significantly affected. In conclusion, Vitamin E stabilization of UHMWPE increased osteoblast response to oxidative stress, inducing a cellular mechanism aimed at cell survival. Vitamin E antioxidant effect influences the secretion of osteoimmunological factors, shifting the bone turnover balance toward bone protection stimuli. This suggests that Vitamin E-Stabilization of UHMWPE could contribute to reduction of oxidation-induced osteolysis and the consequent loosening of the prosthetic devices, therefore improving the longevity of total joint replacements

Molecular dynamics of lipid bilayer based supramolecular systems

L’analisi strutturale accurata per la previsione delle interazioni intermolecolari tramite metodi computazionali , si presenta oggi come un dato imprescindibile sia in diversi protocolli di laboratorio, che in molti ambiti scientifici come la chimica , la farmaceutica, la biologia e la scienza dei materiali. Il Docking è un potente strumento ampiamente utilizzato nella progettazione razionale di nuovi e sempre più potenti farmaci. Accanto ad essa, la dinamica molecolare permette una più accurata analisi della cinetica dei sistemi biologici, non ottenibile dalle sola risoluzione delle strutture cristallografiche. Coadiuvati da più moderni software sviluppati per il trattamento dei sistemi biologici virtuali, ci siamo addentrati negli ambiti del Gene Terapy e del Drug Design. Con l'obiettivo di sintetizzare nuovo lipidi funzionalizzati che potessero complessare più efficacemente il DNA nella struttura multilamellare del vettore liposomiale, e quindi rilasciare di conseguenza l’acido nucleico all'interno della cellula, il nostro laboratorio di sintesi ha sviluppato un nuovo protocollo per la preparazione di nuovi composti lipidici chimerici: lipidi funzionalizzati con eteri corona e lipidi sensibili al pH. Per tentare di spiegare e migliorare i risultati ottenuti dalla controparte sperimentale, abbiamo approntato un totale di diciotto simulazioni di dinamica molecolare, effettuate su modelli di membrane miste di POPC e DOPC con i lipidi sintetici. Man mano che la concentrazione di etere corona aumenta, si ha un aumentato disordine del doppio strato lipidico. I dati raccolti per le simulazioni sul lipide sensibile al pH, in presenza di CaCl2, mostrano una distribuzione omogenea dei lipidi a concentrazioni basse di lipide sintetico. Nel momento in cui la concentrazione di lipide sintetico aumenta, tende ad accrescersi anche il disordine del sistema. Il sale agisce tramite un effetto di coordinazione intermolecolare, impedendo alla membrana di raggiungere una adeguata fluidità , per tutto il corso della simulazione. I recettori accoppiati a proteine G ( GPCR ) costituiscono una vasta e diversificata famiglia di proteine la cui funzione primaria è quella di trasdurre stimoli extracellulari in segnali intracellulari . Il recettore 5HT2c è un target di molti farmaci usati per trattare una varietà di condizioni fisiologiche e psichiatriche. Questo studio si è concentrato su diversi aspetti relativi all’analisi della suddetta proteina di membrana: una prima fase di docking molecolare mirato sul ligando naturale , vari agonisti e antagonisti, e considerando inoltre la presenza di SNP che potrebbero influenzare la risposta dei pazienti al farmaco; una seconda fase di dinamica molecolare effettuata sul complesso ligando/recettore immerso nell'ambiente idrofobico del doppio strato lipidico, al fine di valutare le variazioni conformazionali indotte dalla presenza di quei composti precedentemente sottoposti a docking; parallelamente abbiamo approntato una dinamica molecolare in cui il ligando naturale serotonina viene lasciato libero nel solvente, per studiare la captazione nello spazio extracellulare ad opera del recettore stesso; in ultimo, una simulazione di dinamica molecolare tramite un modello semplificato Coarse Grained, effettuata su più recettori 5-HT2c immersi nell'ambiente lipidico , al fine di rilevare la formazione di oligomeri (la struttura quaternaria biologicamente attiva evidenziata per quei recettori). Insieme, tutti questi dati ci danno una panoramica più completa dei diversi aspetti che devono essere presi attentamente in considerazione per la progettazione razionale di nuovi farmaci mirati alla 5-HT2c . Lo scopo di questo lavoro è di presentare le prospettive di innovazione offerte da un'analisi in silico, sia in termini di "migliore comprensione ", sia in termini di "suggerimento" : la prima come complementazione di dati sperimentali , la seconda come un nuovo modo di progettare l'esperimento stesso

Biomarkers of Oxidative Stress in Acute and Chronic Diseases

Molecular biomarkers consent to apply individual decisions in the complex management of both acute or chronic diseases, and their identification constitutes a fundamental phase for achieving the important object to develop personalized therapies [...]

Salts Influence Cathechins and Flavonoids Encapsulation in Liposomes: A Molecular Dynamics Investigation

Cathechins and flavonoids are responsible of numerous health benefits. Two of the most representatives' compounds for their antioxidant and therapeutic effects are Epigallocatechin 3-Gallate (EGCG), from green tea extracts, and morelloflavone (MF), from Garcinia dulcis. Here we explore, by atomistic Molecular Dynamics simulations, how EGCG and MF interact with lipid bilayers and we show the salts' influence on their encapsulation degree in neutral liposomes. As a result, we found out that EGCGs naturally bind to the hydrophilic regions of phospholipids, positioning themselves mostly at the interface between water and lipid phases. The presence of a salt clearly influences the EGCG molecules' absorption and the total effect depends strongly on the salt nature and concentration. Beside, for MF, we observed a high stability of the intermolecular MFs aggregates in water that strongly penalizes the flavonoid's interaction with the lipid polar heads. However, salts can influence MF's liposomal penetration, even if they are not able to promote completely its absorption inside the bilayer. For both compounds, the increase of penetration is more marked in presence of magnesium chloride, whilst calcium chloride showed the opposite effect. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Motion-induced blindness measured objectively.

During central fixation, a moving pattern of nontargets induces repeated temporary blindness to even salient peripheral targets: motion-induced blindness (MIB). Hitherto, behavioral measures of MIB have relied on subjective judgments. Here, we offer an objective alternative that builds on earlier findings regarding the effects of MIB on the detectability of physical target offsets. We propose a small modification of regular MIB displays: Following a variable duration (lead time), one of the targets is physically removed. Subjects are to respond immediately afterward. We hypothesize that illusory target offsets, caused by MIB, are mistaken for physical target offsets and that errors should thus increase with lead time. Indeed, for both nonsalient and salient targets, we found that detection accuracy for physical target offsets dramatically decreased with lead time. We conclude that target offset detection accuracy is a valid objective measure of MIB. With our method, effects of guessing are minimal, and the fitting of psychometric functions is straightforward. In principle, a staircase extension--for more efficient data collection--is also possible

Cholesterol-mediated oligomerization pathways of serotonin G-coupled receptor 5-HT2C

Serotonin (5-HT) receptors have been shown to homodimerize and heterodimerize with other G protein-coupled receptors (GPCRs), although the details of this process have not yet been elucidated. Here we use coarse-grained molecular dynamics on monomeric 5-HT2C receptors to predict the transmembrane (TM) helices involved in such associations. All these simulations were carried out both in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers and in mixed composition POPC-Cholesterol ones, to show whether the presence of cholesterol could directly influence and drive the dimeric association. The goal is to get insights on the self-assembly pathway leading to GPCRs 5-HT2C oligomerization, which is supposed to be the basis of its constitutional activity. From the analysis of the molecular dynamics trajectories, we observed the formation of 5-HT2C oligomers through self-assembly and we identified the main domains involved in the receptor dimerization. In particular, dimers and oligomers from the two different environments show TM4-TM5 and TM1-TM7-H8 as the preferential dimerization interfaces. Nevertheless, substantial differences arise for oligomers in POPC and in POPC-Chol membranes: in POPC-Chol the variability of dimers interfaces is strictly limited to the TM1-TM7-H8 and TM4-TM5 interfaces and the dimorphism depends on cholesterol that directly participates in its formation. These results are in agreement with both experimental evidences and other computational studies conducted on other GPCRs oligomerization

Salts Influence Cathechins and Flavonoids Encapsulation in Liposomes: A Molecular Dynamics Investigation

Cathechins and flavonoids are responsible of numerous health benefits. Two of the most representatives' compounds for their antioxidant and therapeutic effects are Epigallocatechin 3-Gallate (EGCG), from green tea extracts, and morelloflavone (MF), from Garcinia dulcis. Here we explore, by atomistic Molecular Dynamics simulations, how EGCG and MF interact with lipid bilayers and we show the salts' influence on their encapsulation degree in neutral liposomes. As a result, we found out that EGCGs naturally bind to the hydrophilic regions of phospholipids, positioning themselves mostly at the interface between water and lipid phases. The presence of a salt clearly influences the EGCG molecules' absorption and the total effect depends strongly on the salt nature and concentration. Beside, for MF, we observed a high stability of the intermolecular MFs aggregates in water that strongly penalizes the flavonoid's interaction with the lipid polar heads. However, salts can influence MF's liposomal penetration, even if they are not able to promote completely its absorption inside the bilayer. For both compounds, the increase of penetration is more marked in presence of magnesium chloride, whilst calcium chloride showed the opposite effect. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim