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Pharmacological therapy for nonalcoholic fatty liver disease (NAFLD) is not approved at the present time. For this purpose, the effect of combined eicosapentaenoic acid (EPA; 50 mg/kg/day) modulating hepatic lipid metabolism and hydroxytyrosol (HT; 5 mg/kg/day) exerting antioxidant actions was evaluated on hepatic steatosis and oxidative stress induced by a high-fat diet (HFD; 60% fat, 20% protein, and 20% carbohydrates) compared to a control diet (CD; 10% fat, 20% protein, and 70% carbohydrates) in mice fed for 12 weeks. HFD-induced liver steatosis (i) was reduced by 32% by EPA, without changes in oxidative stress-related parameters and mild recovery of Nrf2 functioning affording antioxidation and (ii) was decreased by 42% by HT, concomitantly with total regain of the glutathione status diminished by HFD, 42% to 59% recovery of lipid peroxidation and protein oxidation enhanced by HFD, and regain of Nrf2 functioning, whereas (iii) combined EPA + HT supplementation elicited 74% reduction in liver steatosis, with total recovery of the antioxidant potential in a similar manner than HT. It is concluded that combined HT + EPA drastically decreases NAFLD development, an effect that shows additivity in HT and EPA effects that mainly relies on HT, strengthening the impact of oxidative stress as a central mechanism underlying liver steatosis in obesity.

High-fat-diet (HFD) feeding is associated with liver oxidative stress (OS), n-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) depletion, hepatic steatosis and mitochondrial dysfunction.

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.

Cationic phosphonium salts are interesting because they inhibit the proliferation of carcinoma cells more than untransformed epithelial cells in vitro. This differential anti-proliferative effect has been used to identify phosphonium salts and other lipophilic cations that later demonstrated effects in animals. Using 6 carcinoma-derived and 2 untransformed epithelial cell lines, tetraphenylphosphonium chloride (TPP) and other cationic aryl phosphonium salts (CAPS) demonstrated a growth inhibition pattern similar to that of cation rhodamine 123, suggesting that CAPS may inhibit mitochondrial function. We tested this hypothesis for the effect of phosphonium salt TPP on FaDu human hypopharyngeal carcinoma cells. TPP inhibited the proliferation of FaDu carcinoma cells at submicromolar concentrations. Uptake of 3H-TPP by FaDu cells was partially inhibited in medium containing high K+ and fully inhibited by valinomycin in this medium, indicating that TPP accumulates preferentially in mitochondria, and to a lesser extent in the cytoplasm. FaDu cells exposed to TPP exhibited damage to mitochondrial inner membranes, reduced ATP/ADP ratios, decreased oxygen uptake rates and decreased mitochondrial membrane potentials. The treated cells secreted lactate more rapidly than untreated controls and exhibited hypersensitivity to 2-deoxyglucose, an inhibitor of glycolysis. TPP's antimitochondrial effects apparently enhance cytoplasmic glycolysis. In conclusion, TPP inhibits FaDu carcinoma cell growth by inhibiting mitochondrial respiration and ATP synthesis. Cationic phosphonium salts that inhibit carcinoma cell growth through antimitochondrial effects might be used to treat solid tumors without the risk of secondary tumors associated with agents affecting nuclear DNA.

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Pomegranate peels and seeds are industrial residues considered interesting sources of punicalagin (PU) and punicic acid (PA), respectively. To optimise their extraction process and protect them against environmental factors, pomegranate residues were extracted with supercritical CO 2 (SC-CO 2 ) using a Box-Behnken design and then optimal extracts encapsulated by spray-drying applying a 2 2 central composite design. Peel extracts showed a PU content of 0.4-9.5% with optimal extraction conditions of 400 bar, 43°C and 20% ethanol. On the other hand, SC-CO 2 seed extracts showed a PA content of 65.1-78.4% with 450 bar, 48°C and 10% ethanol as optimal extraction parameters. Otherwise, the encapsulation efficiency of SC-CO 2 extracts was significantly affected by core/wall material ratio and its quadratic effect. This parameter ranged from 35.1% to 72.4% for peel extracts and from 68.2% to 92.7% for seed extracts. Results showed that the proposed technologies are a feasible approach to the integral utilisation of residues from the pomegranate industry.