Peculiar Ca2+ Homeostasis, ER Stress, Autophagy, and TG2 Modulation in Celiac Disease Patient-Derived Cells.

Celiac disease (CD) is an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals by genetically predisposed individuals. Constitutive differences between cells from CD patients and control subjects, including levels of protein phosphorylation, alterations of vesicular trafficking, and regulation of type 2 transglutaminase (TG2), have been reported. In the present work, we investigated how skin-derived fibroblasts from CD and control subjects responded to thapsigargin, an endoplasmic reticulum ER stress inducer, in an attempt to contribute to the comprehension of molecular features of the CD cellular phenotype. We analyzed Ca2+ levels by single-cell video-imaging and TG2 activity by a microplate assay. Western blots and PCR analyses were employed to monitor TG2 levels and markers of ER stress and autophagy. We found that the cytosolic and ER Ca2+ level of CD cells was lower than in control cells. Treatments with thapsigargin differently activated TG2 in control and CD cells, as well as caused slightly different responses regarding the activation of ER stress and the expression of autophagic markers. On the whole, our findings identified further molecular features of the celiac cellular phenotype and highlighted that CD cells appeared less capable of adapting to a stress condition and responding in a physiological way.

Type 2 Transglutaminase in Coeliac Disease: A Key Player in Pathogenesis, Diagnosis and Therapy.

Type 2 transglutaminase (TG2) is the main autoantigen in coeliac disease (CD), a widespread inflammatory enteropathy caused by the ingestion of gluten-containing cereals in genetically predisposed individuals. As a consequence, serum antibodies to TG2 represent a very useful marker in CD diagnosis. However, TG2 is also an important player in CD pathogenesis, for its ability to deamidate some Gln residues of gluten peptides, which become more immunogenic in CD intestinal mucosa. Given the importance of TG2 enzymatic activities in CD, several studies have sought to discover specific and potent inhibitors that could be employed in new therapeutical approaches for CD, as alternatives to a lifelong gluten-free diet. In this review, we summarise all the aspects regarding TG2 involvement in CD, including its enzymatic reactions in pathogenesis, the role of anti-TG2 antibodies in disease management, and the exploration of recent strategies to reduce deamidation or to use transamidation to detoxify gluten.

Pro-Apoptotic and Pro-Autophagic Properties of Cardenolides from Aerial Parts of Pergularia tomentosa

Pergularia tomentosa L., a milkweed tropical plant belonging to the family Asclepiadaceae, is a rich source of unusual cardiac glycosides, characterised by transfused A/B rings and a sugar moiety linked by a double link, generating a dioxanoid structure. In the present report, five cardenolides isolated from the aerial parts of the plant (calactin, calotropin, 12ß-hydroxycalactin, 12ß,6'-dihydroxycalotropin, and 16α-hydroxycalotropin) were investigated for their biological effects on a human hepatocarcinoma cell line. Cell viability was monitored by an MTT assay. The occurrence of apoptosis was evaluated by detecting caspase-3 activation and chromatin fragmentation. The ability of these compounds to induce autophagy was analysed by monitoring two markers of the autophagic process, LC3 and p62. Our results indicated that all cardenolides had cytotoxic effects, with IC50 ranging from 0.127 to 6.285 µM. All compounds were able to induce apoptosis and autophagy, calactin being the most active one. Some of them also caused a reduction in cell migration and a partial block of the cell cycle into the S-phase. The present study suggests that selected cardenolides from aerial parts of P. tomentosa, particularly calactin, possess potentially desirable properties for further investigation as anticancer agents.

The mechanism of cytotoxicity of 4-nonylphenol in a human hepatic cell line involves ER-stress, apoptosis, and mitochondrial dysfunction.

4-Nonylphenol (4-NP) is an emerging environmental pollutant widely diffused in waters and sediments. It mainly derives from the degradation of alkyl phenol ethoxylates, compounds commonly employed as industrial surfactants. 4-NP strongly contaminates foods and waters for human use; thus, it displays a wide range of toxic effects not only for aquatic organisms but also for mammals and humans. After ingestion through the diet, it tends to accumulate in body fluids and tissues. One of the main organs where 4-NP and its metabolites are concentrated is the liver, where it causes, even at low doses, oxidative stress and apoptosis. In the present study, we analyzed the effects of 4-NP on a human hepatic cell line (HepG2) to deepen the knowledge of its cytotoxic mechanism. We found that 4-NP, in a range of concentration from 50 to 100 µM, significantly reduced cell viability; it caused a partial block of proliferation and induced apoptosis with activation of caspase-3 and overexpression of p53. Moreover, 4-NP induced-apoptosis seemed to involve both an ER-stress response, with the appearance of high level of GRP78, CHOP and the spliced XBP1, and a dysregulation of mitochondrial physiology, characterized by an overexpression of main markers of mitochondrial dynamics. Our data support the idea that a daily consumption of 4-NP-contaminated foods may lead to local damages at the level of gastrointestinal system, including liver, with negative consequences for the organ physiology.

Effects of environmental cocaine concentrations on COX and caspase-3 activity, GRP-78, ALT, CRP and blood glucose levels in the liver and kidney of the European eel (Anguilla anguilla).

Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L-1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species.

Dose- and Time-Dependent Effects of Oleate on Mitochondrial Fusion/Fission Proteins and Cell Viability in HepG2 Cells: Comparison with Palmitate Effects.

Mitochondrial impairments in dynamic behavior (fusion/fission balance) associated with mitochondrial dysfunction play a key role in cell lipotoxicity and lipid-induced metabolic diseases. The present work aimed to evaluate dose- and time-dependent effects of the monounsaturated fatty acid oleate on mitochondrial fusion/fission proteins in comparison with the saturated fatty acid palmitate in hepatic cells. To this end, HepG-2 cells were treated with 0, 10 µM, 50 µM, 100 µM, 250 µM or 500 µM of either oleate or palmitate for 8 or 24 h. Cell viability and lipid accumulation were evaluated to assess lipotoxicity. Mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1) processes were evaluated by Western blot analysis. After 8 h, the highest dose of oleate induced a decrease in DRP1 content without changes in MFN2 content in association with cell viability maintenance, whereas palmitate induced a decrease in cell viability associated with a decrease mainly in MFN2 content. After 24 h, oleate induced MFN2 increase, whereas palmitate induced DRP1 increase associated with a higher decrease in cell viability with high doses compared to oleate. This finding could be useful to understand the role of mitochondria in the protective effects of oleate as a bioactive compound.

Interplay between Type 2 Transglutaminase (TG2), Gliadin Peptide 31-43 and Anti-TG2 Antibodies in Celiac Disease.

Celiac disease (CD) is a common intestinal inflammatory disease involving both a genetic background and environmental triggers. The ingestion of gluten, a proteic component of several cereals, represents the main hexogen factor implied in CD onset that involves concomitant innate and adaptive immune responses to gluten. Immunogenicity of some gluten sequences are strongly enhanced as the consequence of the deamidation of specific glutamine residues by type 2 transglutaminase (TG2), a ubiquitous enzyme whose expression is up-regulated in the intestine of CD patients. A short gluten sequence resistant to intestinal proteases, the α-gliadin peptide 31-43, seems to modulate TG2 function in the gut; on the other hand, the enzyme can affect the biological activity of this peptide. In addition, an intense auto-immune response towards TG2 is a hallmark of CD. Auto-antibodies exert a range of biological effects on several cells, effects that in part overlap with those induced by peptide 31-43. In this review, we delineate a scenario in which TG2, anti-TG2 antibodies and peptide 31-43 closely relate to each other, thus synergistically participating in CD starting and progression.

Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects.

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

Anti-type 2 transglutaminase antibodies as modulators of type 2 transglutaminase functions: a possible pathological role in celiac disease.

Auto-antibodies to the ubiquitous enzyme type-2 transglutaminase (TG2) are a specific hallmark of celiac disease (CD), a widely diffused, multi-factorial disease, affecting genetically predisposed subjects. In CD an inflammatory response, at the intestinal level, is triggered by diet consumption of gluten-containing cereals. Intestinal mucosa displays various degrees of atrophy and hyperplasia, with consequent global intestinal dysfunction and other relevant extra-intestinal symptoms. Through deamidation of specific glutamines of gluten-derived gliadin peptides, TG2 strongly enhances gliadin immunogenicity. In addition, TG2 cross-linking activity may generate complexes between TG2 itself and gliadin peptides, and these complexes seem to cause the auto-immune response by means of an apten-carrier-like mechanism of antigen presentation. Anti-TG2 antibodies can be early detected in the intestinal mucosa of celiac patients and are also abundantly present into the serum, thus potentially reaching other organs and tissues by blood circulation. Recently, the possible pathogenetic role of auto-antibodies to TG2 in CD has been investigated. Here, we report an overview about the genesis of these antibodies, their specificity, their modulating ability toward TG2 enzymatic or non-enzymatic activities and their biological effects exerted by interacting with extracellular TG2 or with cell-surface TG2. We also discuss the auto-immune response occurring in CD against other TG members (i.e. type 3 and type 6) and analyze the occurrence of anti-TG2 antibodies in other auto-immune CD-related diseases. Data now available let us to suppose that, even if antibodies to TG2 do not represent the triggering molecules in CD, they could be important players in disease progression and manifestations.

The toxic alpha-gliadin peptide 31-43 enters cells without a surface membrane receptor.

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Quercetin encapsulated polycaprolactone-polyvinylpyrrolidone electrospun membranes as a delivery system for wound healing applications.

The present work focuses on the production of electrospun membranes based on Poly(ε-caprolactone) (PCL) and Polyvinylpyrrolidone (PVP) for the topical release of Quercetin (Q). Membranes were prepared at 0.5, 1.0, 3.0, 7.0 and 15 % wt of Quercetin and studied from a morphological, physical, and biological point of view. The scanning electron microscopy (SEM) evidences micrometric dimensions of the fibres with a good dispersion of the functional molecule. The retention degree of liquids was evaluated by testing four different liquid media while the radical scavenging activity of Quercetin-loaded membranes was evaluated through DPPH analysis. The release kinetics of Quercetin highlights the presence of an initial burst followed by slower release up to attaining an equilibrium state, after roughly 50 h, showing the possibility of a fine-tuning of drug release. Diffusion coefficients were then evaluated by using Fick's law. Finally, to verify the actual biocompatibility of the systems produced and the possible application in the repair of tissue injury, the biological activity of Quercetin released from drug-loaded membranes was analysed in an immortalized human keratinocyte cell line HaCaT by a wound healing assay. So, the reported preliminary data confirm the possibility of applying the electrospun Quercetin-loaded PCL-PVP membranes for wound healing applications.

Anti-tissue transglutaminase antibodies activate intracellular tissue transglutaminase by modulating cytosolic Ca2+ homeostasis.

Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca(2+) mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca(2+) release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca(2+), respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca(2+) level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca(2+) and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.

Quercetin-Loaded Polycaprolactone-Polyvinylpyrrolidone Electrospun Membranes for Health Application: Design, Characterization, Modeling and Cytotoxicity Studies.

Fibrous membranes of polycaprolactone (PCL)-polyvinylpyrrolidone (PVP) encapsulating 15% wt of quercetin are fabricated by a uniaxial electrospinning technique. Morphological analysis of the electrospun systems proved the fabrication of micrometric fibers (1.58 µm for PCL/PVP and 2.34 µm for quercetin-loaded membrane). The liquid retention degree of the electrospun membranes is evaluated by testing four different liquid media. The contact angle estimation is performed by testing three liquids: phosphate buffer solution, basic solution (pH = 13) and acidic solution (pH = 3), showing high hydrophobicity degree (contact angles > 90°) in all cases. The release of quercetin from the nanofibers in PBS (phosphate buffer solution) and pH = 3 medium, modeled through different models, shows the possibility of a fine tuning of drug release (up to 7 days) for the produced materials. The release profiles attained a plateau regime after roughly 50 h up to 82% and 71% for PBS and pH = 3 media, respectively. Then, since quercetin is known to undergo photooxidation upon UV radiation, release tests after different UV treatment times are carried out and compared with the untreated membrane, demonstrating that the release of the active drug changes from 82% for no-irradiated sample up to 57% after 10 h of UV exposure. The biology activity of released quercetin is evaluated on two human cell lines. The reported results demonstrate the ability of the quercetin-loaded membranes to reduce cell viability of human cell lines in two different conditions: direct contact between cells and quercetin-loaded membranes and cells treatment with culture medium previously conditioned with quercetin-loaded membranes. Therefore, the reported preliminary data confirm the possibility of applying the electrospun quercetin-loaded PCL-PVP membranes for health applications.

Nutritional Quality of Wholegrain Cereal-Based Products Sold on the Italian Market: Data from the FLIP Study.

The consumption of wholegrains (WG) is encouraged worldwide, but the lack of a common legal definition of such products leads to an unclear classification and identification on the grocery store shelf. In Italy, several products are generally sold as WG, but it cannot be determined if they are made entirely with all WG cereal(s) or if they are partially produced with WG ingredients (PWG). The aims of this study were to (a) survey the number of cereal-based food items formulated with WG, PWG, or refined (RG) present on the Italian market; and (b) analyse the nutritional quality, intended as nutrition facts, of WG products in comparison to PWG and RG. Nutritional information and declarations were retrieved from packs of 3040 products belonging to five different categories: breakfast cereals, biscuits, sweet snacks, bread, and bread substitutes. A descriptive analysis of the products and comparison of energy, macronutrients, fibre and salt among RG, PWG and WG products within each category was performed. In all categories, a major portion of the products did not contain WG ingredients. Results showed that the nutritional quality of RG, PWG, and WG products varied in relation to the product category and that WG inclusion cannot be always considered a marker of the overall nutritional quality of foods. Instead, it is necessary to evaluate the global product characteristics, and it is important to pay attention to differences between WG and PWG products that can be perceived by consumers as equivalent.

miRNA 34a, 100, and 137 modulate differentiation of mouse embryonic stem cells.

MicroRNAs (miRNAs) play an important role in proper function and differentiation of mouse embryonic stem cells (ESCs). We performed a systematic comparison of miRNA expression in undifferentiated vs. differentiating ESCs. We report that 138 miRNAs are increased on the induction of differentiation. We compared the entire list of candidate mRNA targets of up-regulated miRNAs with that of mRNA down-regulated in ESCs on induction of differentiation. Among the candidate targets emerging from this analysis, we found three genes, Smarca5, Jarid1b, and Sirt1, previously demonstrated to be involved in sustaining the undifferentiated phenotype in ESCs. On this basis, we first demonstrated that Smarca5 is a direct target of miR-100, Jarid1b of miR-137, and we also confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. The suppression of these three miRNAs by anti-miRs caused the block of ESC differentiation induced by LIF withdrawal. On the other hand, the overexpression of the three miRNAs resulted in an altered expression of differentiation markers. These results demonstrate that miR-34a, miR-100, and miR-137 are required for proper differentiation of mouse ESCs, and that they function in part by targeting Sirt1, Smarca5, and Jarid1b mRNAs.

1,1,1-trichloro-2,2-bis (p-chlorophenyl)-ethane (DDT) and 1,1-Dichloro-2,2-bis (p, p'-chlorophenyl) ethylene (DDE) as endocrine disruptors in human and wildlife: A possible implication of mitochondria.

1,1,1-trichloro-2,2-bis (p-chlorophenyl)-ethane (DDT) and its main metabolite 1,1-Dichloro-2,2-bis (p, p'-chlorophenyl) ethylene (DDE) act as endocrine disruptors in humans and wildlife. Immunomodulatory functions have also been attributed to both xenobiotics. DDT was banned in the 1970s due to its toxicity, but it is still produced and used for indoor residual spraying with disease vector control purposes. Due to their persistence and lipophilic properties, DDT and DDE can bioaccumulate through the food chain, being stored in organisms' adipose depots. Their endocrine disruptor function is mediated by agonist or antagonist interaction with nuclear receptors. Present review aimed to provide an overview of how DDT and DDE exposure impacts reproductive and immune systems with estrogen-disrupting action in humans and wildlife. Studies showing DDT and DDE impact on mitochondrial function and apoptosis pathway will also be reviewed, suggesting the hypothesis of direct action on mitochondrial steroid receptors.

Dose-Dependent Response to the Environmental Pollutant Dichlorodipheniletylhene (DDE) in HepG2 Cells: Focus on Cell Viability and Mitochondrial Fusion/Fission Proteins.

Dichlorodiphenyldichloroethylene (DDE), the primary persistent metabolite of dichlorodiphenyltrichloroethane (DDT), has toxic effects on cells, but its dose-dependent impact on mitochondrial proteins involved in mitochondrial fusion and fission processes associated with cell viability impairment has not yet been analysed. Mitochondrial fusion and fission processes are critical to maintaining the mitochondrial network and allowing the cell to respond to external stressors such as environmental pollutants. Fusion processes are associated with optimizing mitochondrial function, whereas fission processes are associated with removing damaged mitochondria. We assessed the effects of different DDE doses, ranging between 0.5 and 100 µM, on cell viability and mitochondrial fusion/fission proteins in an in vitro hepatic cell model (human hepatocarcinomatous cells, HepG2); the DDE induced a decrease in cell viability in a dose-dependent manner, and its effect was enhanced in conditions of coincubation with dietary fatty acids. Fusion protein markers exhibited an inverted U-shape dose-response curve, showing the highest content in the 2.5-25 µM DDE dose range. The fission protein marker was found to increase significantly, leading to an increased fission/fusion ratio with high DDE doses. The low DDE doses elicited cell adaption by stimulating mitochondrial dynamics machinery, whereas high DDE doses induced cell viability loss associated with mitochondrial dynamics to shift toward fission. Present results are helpful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants.

A flexible method to study neuronal differentiation of mouse embryonic stem cells.

Embryonic Stem Cells (ESCs) represent an invaluable tool for the study of early mammalian development, for regenerative medicine and for drug discovery. To fulfill these promises, efficient and easy protocols to differentiate ESCs have to be developed. Most of these protocols results in low efficiency of neural induction and/or requires extended in vitro culture. Here we describe in detail an easy and efficient method to differentiate ESCs into neurons, that can be used to identify molecules required for proper neuronal differentiation. Moreover, we present a modification of this method that allows to clearly evaluate the ability of some molecules to favor neuron formation in vitro. These methods can represent an efficient platform for studying the molecular mechanisms underlying early events of neural induction and differentiation in ESCs, as well as for testing molecule efficacy in the pharmaceutical testing.

Analysis of Food Labels to Evaluate the Nutritional Quality of Bread Products and Substitutes Sold in Italy: Results from the Food Labelling of Italian Products (FLIP) Study.

Bread is one of the most common staple foods, despite the increasing consumption of the so-called "bread substitutes". The aim of the present work is to survey the nutritional quality intended as a nutrition declaration of 339 pre-packed bread products and 1020 bread substitutes sold in the major retailers present on the Italian market. Comparisons of energy, macronutrient, and salt content within product types, and between regular and gluten-free (GF) products and products with or without nutrition claim (NC) and health claim (HC) declarations, were performed. A high inter-product variability was detected. The median energy contents were 274 (interquartile range 255-289) and 412 (380-437) kcal/100 for bread products and substitutes, respectively. Irrespective of the category, GF products had lower amounts of energy than their gluten-containing counterpart (p < 0.001), whereas products carrying NC had lower energy, sugar and salt amounts than the products without these declarations on the pack (p < 0.001 for all). A strong positive correlation was observed between energy and carbohydrate in bread (rho = 0.73, p < 0.001), but not in substitutes (rho = 0.033, p = 0.29). The present work highlighted a high variability in the apparent nutritional quality of bread products and substitutes sold on the Italian market, and suggested that bread alternatives should not be considered tout court as substitutes from a nutritional point of view.