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Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.

The geochemistry of thermal spring waters in the Acquasanta Terme area, located on the Adriatic side of central Italy, has been investigated in order to characterize the geothermal feeding system. The springs discharge more than 100 L/s at temperature ranging between 20 and 30 °C. They occur in a tectonic window of Mesozoic limestones in the central sector of the Acquasanta anticline within the Laga foredeep Basin. Chemical and isotopic compositions of thermal and cold fluids were investigated, most of them monitored for one year, in order to understand the thermal fluid circulation paths. The chemistry of the major elements defines the existence of Na–Cl and Ca–Cl–SO42- hot discharging waters and permits the characterization of the thermal end-member hosted in carbonate-dominated reservoir (Burano Anhydrites Fm–Calcare Massiccio Fm). This deep fluid is well represented by the sample T1 and shows high temperature and electrical conductivity (EC), very stable over time, and not affected by mixing phenomena. This is also confirmed by tritium results (0 T.U.). Close to the surface at different depth, such water undergoes mixing or dilution processes with HCO3− rich freshwaters, driven by the complex structural setting of the area and by diffuse karst caves. This is identified as the main reason for observed compositional variations of sampled springs, and three areas affected by different mixing phenomena have been defined at the surface. The concentrations of SO42− and H2S suggest redox processes affecting sulfur after interaction with evaporitic formations, identified with the Burano Anhydrites, at the base of the Umbria–Marche sedimentary sequence (∼3500-m-deep). Contribution from this reservoir is also supported by characteristic Sr isotope signature. δ18O and δD values indicate a meteoric origin of the thermal waters and allow estimation of an average infiltration altitude ranging between 1500 and 1700 m a.s.l. This datum, supported by structural data, suggests the Laga Mountains as the recharge area of the system. Reservoir temperature inferred by SO42−–HCO3−–F, Ca/Mg and SO42−/F geothermometers is about 80 °C, consistent with the geothermal gradient of the foredeep basin, and deserving further investigations for potential economic implications about this low-enthalpy geothermal area.

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration. (C) 2016 Elsevier B.V. All rights reserved.

Although H2O2 is traditionally known to have cytotoxic effects, recent studies argue about its regulatory role on lipid metabolism. However, the mechanism underlying the induction of lipid biosynthesis by oxidative stress still remains unknown. To shed light on this aspect we investigated the H2O2-induced lipogenesis in rat liver BRL-3A cells. We found that a short-term exposition of cells to 35μM H2O2 didn't cause any significant sign of cell damage measured by following diene formation and lactate dehydrogenase release from cells. However, in this stressful condition, a significant increase of [1-(14)C]acetate incorporation into fatty acids and cholesterol, associated to an increase in the activity and expression of key enzymes of fatty acid and cholesterol synthesis, were measured. mRNA and protein contents of the transcription factors SREBP-1 and SREBP-2, involved in the activation of lipid synthesis, increased as well. The analysis of molecular mechanism of SREBP-1 activation revealed, in treated compared to control cells, a higher SREBP-1a mRNA translation involving an internal ribosome entry side (IRES), present in the leader region of its mRNA. Longer exposition to the pro-oxidant induced a progressive loss of cell viability together with an increase of cell triacylglycerol content.

Cholestasis is one of the major causes of liver diseases. A chronic accumulation of toxic bile acids in the liver, which occurs in this condition, can induce fibrosis and cirrhosis. Inflammation is a fundamental component of acute and chronic cholestatic liver injury.Platelet-activating factor (PAF) is a proinflammatory lipid which may be generated by two independent pathways called the de novo and remodeling pathway being the last responsible for the synthesis of PAF during inflammation. In recent years a key role in PAF remodeling has been attributed to lysophosphatidylcholine acyltransferase (LPCAT) enzymes. Although the knowledge on their characteristic is growing, the exact mechanism of LPCAT in pathological conditions remains still unknown.Here, we reported that the level of lyso-PAF and PAF significantly increased in the liver of cirrhotic vs. control rats together with a significant decrease in both mRNA abundance and protein level of both LPCAT1 and LPCAT2. Acyltransferase activities of both LPCAT1 and LPCAT2 were parallel decreased in the liver of cirrhotic animals. Interestingly, treatment with silybin strongly decreased the level of both pro-inflammatory lipids and restored the activity and expression of both LPCAT1 and LPCAT2 of cirrhotic liver. Silybin effect was specific for LPCAT1 and LPCAT2 since it did not affect LPCAT3 mRNA abundance of cirrhotic liver.

The properties of fatty acid chain elongation synthesis have been investigated in liver mitochondria of the European eel (Anguilla anguilla). The incorporation of [1-(14)C]acetyl-CoA into fatty acids shows a specific activity of 0.43+/-0.05 nmol/min x mg protein (n=6), which is more than twice higher than that previously reported in rat liver mitochondria. Label incorporation into fatty acids was, in mitochondria disrupted by freezing and thawing, much higher than in intact organelles thus suggesting a probable localization of this pathway inside mitochondria. Only a negligible acetyl-CoA incorporation into fatty acids occurs in the absence of ATP, Mg2+ or reduced pyridine nucleotides; NADH alone seems to be as effective as NADH + NADPH as a hydrogen donor for the reducing steps. CoASH, without effect up to 10 microM, showed a strong inhibition at higher concentrations. From the ratio of total radioactivity and radioactivity in carboxyl carbon it can be inferred that in eel-liver mitochondria only chain elongation of preexisting fatty acids occurs. A significant fatty acid chain elongation activity is also present when, instead of acetyl-CoA, [2-(14)C]malonyl-CoA is used as a carbon unit donor. Moreover, the synthesized fatty acids were actively incorporated into phopholipids, mainly phosphatidylcholine, phosphatidylethanolamine and sphyngomyelin.

The tricarboxylate carrier was purified to homogeneity from liver mitochondria of European eel at the silver and the yellow stage and functionally reconstituted into liposomes. Unexpectedly, the molecular activity of the tricarboxylate carrier obtained from silver eel was about twofold higher than that of the same protein from yellow eel, although eels at the silver stage stop feeding. Parallel changes were found in the activities of the lipogenic enzymes in silver eels. This suggests a functional coordination between all these proteins sequentially involved in hepatic lipogenesis. Cardiolipin added to proteoliposomes strongly stimulated the activity of the purified tricarboxylate carrier from yellow eels, whereas it slightly reduced the activity of the same protein from silver eels. The higher activity of the tricarboxylate carrier from silver eels could therefore be ascribed, at least in part, to a different composition of the lipid domain surrounding the carrier protein, possibly in response to the hormonal alterations accompanying metamorphosis from yellow to silver stage.

‘Annurca’ apple fruit, which is commonly cultivated in Southern Italy and undergoes, after harvest, a typical reddening treatment to turn the apples’ skin red, is noticeable for its high firmness. An ‘Annurca’ variety, called ‘Bella del Sud’ is examined for its pectic composition after reddening and just after cold storage. Pectins, extracted with water and potassium oxalate, change their composition after different storage regimes with respect to unstored fruit. ‘Annurca’ pectin, as analyzed by gel-permeation chromatography, is composed of three types of polymers (high MW, >350 kDa, medium MW 350–110 kDa, low MW 40–9 kDa). Gel-permeation chromatograms revealed that the medium MW polymers decreased after normal atmosphere storage. Eluates were subjected to enzymatic and chemical hydrolysis, then to HPLC analysis. The high and medium MW polysaccharides were rich in galacturonic acid. Low MW were rich in neutral sugars. The storage-induced differences in sugar composition revealed that the most important changes are loss of galacturonic acid in medium MW fragments, increase of glucose, rhamnose and arabinose and depletion of mannose–galactose units in low MW fragments.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with additional proteins among which catenins that bridge it to the actin cytoskeletal. Loss of E-cadherin expression is considered a crucial step in the epithelial–mesenchymal transition process (EMT) and it is implicated with the capacity of cancer cells to invade and metastasize.