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In the past decade, there has been an epochal change in the way that diseases are investigated and diagnosed [...]

miRNAs are small noncoding RNAs that control gene expression at the posttranscriptional level. It has been recognised that miRNA dysregulation reflects the state and function of cells and tissues, contributing to their dysfunction. The identification of hundreds of extracellular miRNAs in biological fluids has underscored their potential in the field of biomarker research. In addition, the therapeutic potential of miRNAs is receiving increasing attention in numerous conditions. On the other hand, many operative problems including stability, delivery systems, and bioavailability, still need to be solved. In this dynamic field, biopharmaceutical companies are increasingly engaged, and ongoing clinical trials point to anti-miR and miR-mimic molecules as an innovative class of molecules for upcoming therapeutic applications. This article aims to provide a comprehensive overview of current knowledge on several pending issues and new opportunities offered by miRNAs in the treatment of diseases and as early diagnostic tools in next-generation medicine.

What is the central question of this study? Are differential patterns of circulating miRNAs associated with sleep duration in normal-weight European children and adolescents? What is the main finding and its importance? Differences in the expression level of circulating miR-26b-3p and miR-485-5p are positively associated with total sleep duration in healthy normal-weight children and adolescents.It is commonly recognized that sleep is essential for children's health, and that insufficient sleep duration is associated with negative health outcomes. In humans, sleep duration and quality are influenced by genetic, environmental and social factors. Epigenetic mechanisms, likewise, regulate circadian rhythms and sleep patterns. In the present study, we aimed to identify circulating microRNAs associated with sleep duration in a subsample of normal-weight European children/adolescents (n = 111) participating in the I.Family Study. Subjects were divided into two groups based upon self-reported sleep duration, according to the recommended amount of sleep for paediatric populations. Sleep needs for children 13 years were at least 9 h per day, and for children 13 were at least 8 h per day. There were group differences (short sleepers versus normal sleepers) in circulating levels of miR-26b-3p (mean (95% CI) = 2.0 (1.3-2.7) versus 2.3 (1.9-2.7), P = 0.05) and miR-485-5p (mean (95% CI) = 0.6 (0.3-0.9) versus 0.9 (0.7 - 1.0), P 0.001), adjusting for country of origin, age, sex, pubertal status, screen time and highest educational level of parents. Our findings show for the first time that sleep duration reflects the profile of specific circulating microRNAs in school-aged children and adolescents. It is conceivable that epigenetic modifications, mainly related to circadian rhythm control, may be modulated or interfere with sleep duration.

In recent years, the link between regulatory microRNAs (miRNAs) and diseases has been the object of intensive research. miRNAs have emerged as key mediators of metabolic processes, playing crucial roles in maintaining/altering physiological processes, including energy balance and metabolic homeostasis. Altered miRNAs expression has been reported in association with obesity, both in animal and human studies. Dysregulation of miRNAs may affect the status and functions of different tissues and organs, including the adipose tissue, pancreas, liver, and muscle, possibly contributing to metabolic abnormalities associated with obesity and obesity-related diseases. More recently, the discovery of circulating miRNAs easily detectable in plasma and other body fluids has emphasized their potential as both endocrine signaling molecules and disease indicators. In this review, the status of current research on the role of miRNAs in obesity and related metabolic abnormalities is summarized and discussed.

Background: Nearly 10 years ago, the World Health Organization reported the increasing prevalence of overweight and obesity worldwide as a challenge for public health due to the associated adverse consequences. Epidemiological studies established a firm relationship between an elevated body mass index and chronic conditions such as diabetes, dyslipidemia, hypertension, heart disease, non-alcoholic fatty liver disease, and some types of cancer. Omic studies demonstrated that microRNA (miRNA) profile changes in tissues correlate with a number of diseases, including obesity. Recent studies showed a remarkable stability of miRNAs also in blood, emphasizing their potential as theranostic agents for a variety of disorders and conditions. A number of miRNAs enriched in homeostasis of obesity and metabolic disorders have been characterized in previous researches.Aim: This work was finalized to investigate the differential circulating miRNAs signature in early childhood obesity. Our cross-sectional study analyzed the signature of circulating miRNAs in plasma samples of normal weight (n = 159) and overweight/obese (n = 149) children and adolescents participating to the I.Family study, an EC-funded study finalized to investigate the etiology of overweight, obesity and related disorders and the determinants of food choice, lifestyle, and related health outcomes in children and adolescents of eight European countries (www.ifamilystudy.eu).Results: Differences in miRNA signature with respect to anthropometric and biochemical variables were analyzed. A high degree of variability in levels of circulating miRNAs was identified among children from different countries, in line with recent reports supporting the hypothesis that these molecules are likewise affected by environmental and lifestyle factors. A panel of miRNAs differentially expressed in overweight/low-grade obesity children was characterized (miR-551a and miR-501-5p resulted upregulated; miR-10b-5p, miR-191-3p, miR-215-5p, and miR-874-3p resulted downregulated). ROC curves were also constructed for experimentally confirmed miRNAs. Single miRNAs generally exhibited low AUC values with the highest values for miR-874-3p and miR-501-5p which in combination provided an interesting value (AUC = 0.782). Pearson's analysis confirmed that miR-10b-5p, miR-215-5p, miR-501-5p, miR-551a, and miR-874-3p significantly correlated with BMI z-score. Molecular interactions of obesity-associated miRNAs were also predicted by bioinformatics tools.Conclusions: Our work showed that several circulating miRNAs are differentially represented in overweight/low-grade obesity children and adolescents. Although causal pathways cannot be firmly inferred, it is conceivable that circulating miRNAs may be new biomarkers of early childhood obesity.Trial registration: ISRCTN, ISRCTN62310987. Registered 23/02/2018 - Retrospectively registered.

Polyamines (PAs) are linear polycations that are involved in many biological functions. Putrescine, spermidine and spermine are highly represented in the nucleus of eukaryotic cells and have been the subject of decades of extensive research. Nevertheless, their capability to modulate the structure and functions of DNA has not been fully elucidated. We found that polyamines self-assemble with phosphate ions in the cell nucleus and generate three forms of compounds referred to as Nuclear Aggregates of Polyamines (NAPs), which interact with genomic DNA. In an in vitro setting that mimics the nuclear environment, the assembly of PAs occurs within well-defined ratios, independent of the presence of the DNA template. Strict structural and functional analogies exist between the in vitro NAPs ( iv NAPs) and their cellular homologues. Atomic force microscopy showed that iv NAPs, as theoretically predicted, have a cyclic structure, and in the presence of DNA, they form a tube-like arrangement around the double helix. Features of the interaction between iv NAPs and genomic DNA provide evidence for the decisive role of “natural” NAPs in regulating important aspects of DNA physiology, such as conformation, protection and packaging, thus suggesting a new vision of the functions that PAs accomplish in the cell nucleus.

A growing interest in developing new strategies for preventing coeliac disease has motivated efforts to identify cereals with null or reduced toxicity. In the current study, we investigate the biological effects of ID331 Triticum monococcum gliadin-derived peptides in human Caco-2 intestinal epithelial cells. Triticum aestivum gliadin derived peptides were employed as a positive control. The effects on epithelial permeability, zonulin release, viability, and cytoskeleton reorganization were investigated. Our findings confirmed that ID331 gliadin did not enhance permeability and did not induce zonulin release, cytotoxicity or cytoskeleton reorganization of Caco-2 cell monolayers. We also demonstrated that ID331 omega-gliadin and its derived peptide omega(105-123) exerted a protective action, mitigating the injury of Triticum aestivum gliadin on cell viability and cytoskeleton reorganization. These results may represent a new opportunity for the future development of innovative strategies to reduce gluten toxicity in the diet of patients with gluten intolerance. (C) 2016 Elsevier Ltd. All rights reserved.

Natural polyamines (putrescine, spermidine, and spermine) self-assemble in a simulated physiological environment (50 mm sodium phosphate buffer, pH 7.2), generating in vitro nuclear aggregates of polyamines (ivNAPs). These supramolecular compounds are similar in structure and molecular mass to naturally occurring cellular nuclear aggregates of polyamines, and they share the ability of NAPs to interact with and protect the genomic DNA against nuclease degradation. Three main ivNAP compounds were separated by gel permeation chromatography. Their elution was carried out with 50 mm sodium phosphate buffer supplemented with 150 mm NaCl. Freezing and thawing of selected chromatographic fractions obtained by GPC runs in which the mobile phase was sodium phosphate buffer not supplemented with NaCl yielded three different microcrystallites, specifically corresponding to the ivNAPs, all of which were able to bind DNA. In this study, we demonstrated that in vitro self-assembly of polyamines and phosphates is a spontaneous, reproducible and inexpensive event, and provided the indications for the production of the ivNAPs as a new tool for manipulating the genomic DNA machinery.

Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 mu g/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells.

Selectively targeting death receptors to trigger apoptosis in cancer cells appears ideal in cancer therapy. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is of great interest since it has been shown to predominantly kill cancer cells without toxic effects on normal counterparts, thus representing a promising anticancer agent. However, resistance towards TRAIL/Apo2L treatment has also been described. To overcome this obstacle, co-administration of TRAIL/Apo2L plus several compounds, including histone deacetylase inhibitors (HDACi), has been attempted as a strategy to restore cancer cell sensitivity to TRAIL-induced apoptosis. In recent years, the clinical application of HDACi has been largely explored for their ability to modulate gene transcription, block cell division cycle, inhibit cell proliferation, induce cellular differentiation and apoptosis.The ability of valproic acid (VPA), a well-known HDACi, to sensitise the K562 cell line, derived from a human leukemia, to TRAIL/Apo2L-mediated apoptosis was evaluated. VPA was selected since it is currently used in clinical practice and its pharmacokinetic, pharmacodynamic and bioavailability are known.When applied with TRAIL/Apo2L, VPA increased cell death and caspase-3 activity by 4-fold compared to the treatment with TRAIL/Apo2L alone. VPA sensitized K562 cells to TRAIL/Apo2L-mediated apoptosis by increasing the expression of DR4 and DR5 by 3- and 14-fold respectively. In addition, VPA per se, in the absence of TRAIL/Apo2L, reduced the expression of antiapoptotic factors, such as c-FLPs, associated with DISC, and Bcl-2/Bcl-X(L), associated with mitochondria, acting on both extrinsic and intrinsic apoptotic pathways.Our results demonstrated the ability of VPA to sensitize TRAIL/Apo2L-resistant cells to apoptosis, thus providing an attractive approach for the treatment of leukemias and other proliferative malignancies.

BACKGROUND: Selectively targeting death receptors to trigger apoptosis in cancer cells appears ideal in cancer therapy. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is of great interest since it has been shown to predominantly kill cancer cells without toxic effects on normal counterparts, thus representing a promising anticancer agent. However, resistance towards TRAIL/Apo2L treatment has also been described. To overcome this obstacle, co-administration of TRAIL/Apo2L plus several compounds, including histone deacetylase inhibitors (HDACi), has been attempted as a strategy to restore cancer cell sensitivity to TRAIL-induced apoptosis. In recent years, the clinical application of HDACi has been largely explored for their ability to modulate gene transcription, block cell division cycle, inhibit cell proliferation, induce cellular differentiation and apoptosis. MATERIALS AND METHODS: The ability of valproic acid (VPA), a well-known HDACi, to sensitise the K562 cell line, derived from a human leukemia, to TRAIL/Apo2L-mediated apoptosis was evaluated. VPA was selected since it is currently used in clinical practice and its pharmacokinetic, pharmacodynamic and bioavailability are known. RESULTS: When applied with TRAIL/Apo2L, VPA increased cell death and caspase-3 activity by 4-fold compared to the treatment with TRAIL/Apo2L alone. VPA sensitized K562 cells to TRAIL/Apo2L-mediated apoptosis by increasing the expression of DR4 and DR5 by 3- and 14-fold respectively. In addition, VPA per se, in the absence of TRAIL/Apo2L, reduced the expression of antiapoptotic factors, such as c-FLPs, associated with DISC, and Bcl-2/Bcl-X(L), associated with mitochondria, acting on both extrinsic and intrinsic apoptotic pathways. CONCLUSION: Our results demonstrated the ability of VPA to sensitize TRAIL/Apo2L-resistant cells to apoptosis, thus providing an attractive approach for the treatment of leukemias and other proliferative malignancies.

We previously demonstrated that quercetin, a naturally occurring flavonoid with strong antioxidant properties, was able to enhance programmed cell death in HPB-acute lymphoblastic leukemia (ALL) cell line, derived from a human tymoma, when associated with the agonistic anti-CD95 monoclonal antibody. Here, we report that HPB-ALL cells are normally resistant to CD95-mediated apoptosis, and quercetin is able to sensitize this cell line through a mechanism independent of its antioxidant properties. In fact, other compounds structurally and functionally similar to quercetin, when associated with anti-CD95 antibody did not induce any CD95-mediated apoptosis, still maintaining their antioxidant capacity. We found that quercetin effects are mediated by the activation of PKCalpha. Treatment of HPB-ALL cells with quercetin slightly decreased PKCalpha activity, but when the flavonoid was associated with anti-CD95, the kinase activity increased by 12-fold with respect to the treatment with quercetin. In addition, overexpression of PKCalpha induced programmed cell death in the absence of any additional stimulus, while a kinase-defective mutant of PKCalpha was ineffective. Our data confirm the involvement of specific PKC isoforms in CD95 signaling and suggest, for the first time, that quercetin targets this pathway increasing apoptogenic response in a cell line resistant to CD95-mediated apoptosis.

The protective effect of red wine polyphenols against hydrogen peroxide (H2O2)-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H2O2, were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system. © Elsevier Science Inc. 2000

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1–2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Background MicroRNAs (miRNAs) are small non-coding RNAs involved in the modulation of gene expression and in the control of numerous cell functions. Alterations of miRNA patterns frequently occur in cancer and metabolic disorders, including obesity. Recent studies showed remarkable stability of miRNAs in both plasma and serum making them suitable as potential circulating biomarkers for a variety of diseases and conditions. The aim of this study was to assess the profile of circulating miRNAs expressed in plasma samples of overweight or obese (OW/Ob) and normal weight (NW) prepubertal children from a European cohort (www.ifamilystudy.eu). The project, aimed to assess the determinants of eating behavior in children and adolescents of eight European countries, is built on the IDEFICS cohort (www.ideficsstudy.eu), established in 2006. Among the participants of the I.Family Italian Cohort, ten OW/Ob (age 10.7 ± 1.5 years, BMI 31.6 ± 4.3 kg/m2) and ten NW (age 10.5 ± 2.7 years, BMI 16.4 ± 1.7 kg/m2) children were selected for the study. Gene arrays were employed to differentially screen the expression of 372 miRNAs in pooled plasma samples. Deregulated miRNAs (p