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Background: Molecular changes associated with the initiation of the epithelial to mesenchymal transition (EMT) program involve alterations of large proteome-based networks. The role of protein products mapping to non-coding genomic regions is still unexplored. Objective: The goal of this study was the identification of an alternative protein signature in breast cancer cellular models with a distinct expression of EMT markers. Methods: We profiled MCF-7 and MDA-MB-231 cells using liquid-chromatography mass/spectrometry (LCMS/ MS) and interrogated the OpenProt database to identify novel predicted isoforms and novel predicted proteins from alternative open reading frames (AltProts). Results: Our analysis revealed an AltProt and isoform protein signature capable of classifying the two breast cancer cell lines. Among the most highly expressed alternative proteins, we observed proteins potentially associated with inflammation, metabolism and EMT. Conclusion: Here, we present an AltProts signature associated with EMT. Further studies will be needed to define their role in cancer progression.

Nucleos(t)ide analogues (NA) belong to a family of compounds widely used in anticancer/antiviral treatments. They generally exhibit a cell toxicity limited by cellular uptake levels and the resulting nucleos(t)ides metabolism modifications, interfering with the cell machinery for nucleic acids synthesis. We previously synthesized purine nucleos(t)ide analogues N7-coordinated to a platinum centre with unaltered sugar moieties of the type: [Pt(dien)(N7-dGuo)]2+ (1; dien = diethylenetriamine; dGuo = 2'-deoxy-guanosine), [Pt(dien)(N7-dGMP)] (2; dGMP = 5'-(2'-deoxy)-guanosine monophosphate), and [Pt(dien)(N7-dGTP)]2- (3; dGTP = 5'-(2'-deoxy)-guanosine triphosphate), where the indicated electric charge is calculated at physiological pH (7.4). In this work, we specifically investigated the uptake of these complexes (1-3) at the plasma membrane level. Specific experiments on HeLa cervical cancer cells indicated a relevant cellular uptake of the model platinated deoxynucleos(t)ide 1 and 3 while complex 2 appeared unable to cross the cell plasma membrane. Obtained data buttress an uptake mechanism involving Na+-dependent concentrative transporters localized at the plasma membrane level. Consistently, 1 and 3 showed higher cytotoxicity with respect to complex 2 also suggesting selective possible applications as antiviral/antitumor drugs among the used model compounds.

Hydroxytyrosol (HT), a peculiar olive and olive oil phenolic antioxidant, plays a significant role in the endothelial and cardiovascular protection associated with olive oil consumption. However, studies examining the effects of HT on the whole-genome expression of endothelial cells, which are prominent targets for vasculo-protective effects of olive oil polyphenols, have been lacking. This study aims to comprehensively evaluate the genomic effects exerted by HT, at the transcriptional level, in endothelial cells under resting or proinflammatory conditions. Human umbilical vein endothelial cells (HUVECs) were treated with 10 µmol/L HT for 1 h and then stimulated with 5 ng/mL interleukin (IL)-1β for 3 h. Total RNA was extracted, and gene expression profile assessed with microarray analysis. Functional enrichment analysis and pathway analysis were performed by Ingenuity Pathways Analysis. Microarray data were validated by qRT-PCR. Fixing a significance threshold at 1.5-fold change, HT affected the expression of 708 and 599 genes, respectively, in HUVECs under resting and IL-1β-stimulated conditions, among these, 190 were common to both conditions. Unfolded protein response (UPR) and endoplasmic reticulum stress resulted from the two top canonical pathways common between HT and HT-IL-1β affected genes. IL-17F/A signaling was found in the top canonical pathways of HT modified genes under resting unstimulated conditions, whereas cardiac hypertrophy signaling was identified among the pathways affected by HT-IL-1β. The transcriptomic analysis allowed pinpointing immunological, inflammatory, proliferative, and metabolic-related pathways as the most affected by HT in endothelial cells. It also revealed previously unsuspected genes and related gene pathways affected by HT, thus broadening our knowledge of its biological properties. The unbiased identification of novel genes regulated by HT improves our understanding of mechanisms by which olive oil prevents or attenuates inflammatory diseases and identifies new genes to be enquired as potential contributors to the inter-individual variation in response to functional food consumption.

Food intake is a vital process that supplies necessary energy and essential nutrients to the body. Information regarding luminal composition in the gastrointestinal tract (GIT) collected through mechanical and nutrient sensing mechanisms are generally conveyed, in both mammals and fish, to the hypothalamic neurocircuits. In this context, ghrelin, the only known hormone with an orexigenic action, and the intestinal peptide transporters 1 and 2, involved in absorption of dietary di- and tripeptides, exert important and also integrated roles for the nutrient uptake. Together, both are potentially involved in signaling pathways that control food intake originating from different segments of the GIT. However, little is known about the role of different paralogs and their response to fasting. Therefore, after 3 weeks of acclimatization, 12 Atlantic salmon (Salmo salar) post-smolt were fasted for 4 days to explore the gastrointestinal response in comparison with fed control (n = 12). The analysis covered morphometric (weight, length, condition factor, and wet content/weight fish %), molecular (gene expression variations), and correlation analyses. Such short-term fasting is a common and recommended practice used prior to any handling in commercial culture of the species. There were no statistical differences in length and weight but a significant lower condition factor in the fasted group. Transcriptional analysis along the gastrointestinal segments revealed a tendency of downregulation for both paralogous genes slc15a1a and slc15a1b and with significant lowered levels in the pyloric ceca for slc15a1a and in the pyloric ceca and midgut for slc15a1b. No differences were found for slc15a2a and slc15a2b (except a higher expression of the fasted group in the anterior midgut), supporting different roles for slc15 paralogs. This represents the first report on the effects of fasting on slc15a2 expressed in GIT in teleosts. Transcriptional analysis of ghrelin splicing variants (ghrl-1 and ghrl-2) showed no difference between treatments. However, correlation analysis showed that the mRNA expression for all genes (restricted to segment with the highest levels) were affected by the residual luminal content. Overall, the results show minimal effects of 4 days of induced fasting in Atlantic salmon, suggesting that more time is needed to initiate a large GIT response.

The increasing access to antihypertensive medications has improved longevity and quality of life in hypertensive patients. Nevertheless, hypertension still remains a major risk factor for stroke and myocardial infarction, suggesting the need to implement management of pre- and hypertensive patients. In addition to antihypertensive medications, lifestyle changes, including healthier dietary patterns, such as the Dietary Approaches to Stop Hypertension (DASH) and the Mediterranean diet, have been shown to favorably affect blood pressure and are now recommended as integrative tools in hypertension management. An analysis of the effects of nutritional components of the Mediterranean diet(s) on blood pressure has therefore become mandatory. After a literature review of the impact of Mediterranean diet(s) on cardiovascular risk factors, we here analyze the effects of olive oil and its major components on blood pressure in healthy and cardiovascular disease individuals and examine underlying mechanisms of action. Both experimental and human studies agree in showing anti-hypertensive effects of olive oil. We conclude that due to its high oleic acid and antioxidant polyphenol content, the consumption of olive oil may be advised as the optimal fat choice in the management protocols for hypertension in both healthy and cardiovascular disease patients.

Simple Summary Current research on nanocomposite materials with tailored physical–chemical properties is increasingly advancing in biomedical applications for bone regeneration. In this study, occurrence of differential responsiveness to dextran-grafted iron oxide (DM) nanoparticles and to their hybrid nano-hydroxyapatite (DM/n-HA) counterpart was investigated in human-derived, osteoblast-like cells. Sensitivity of cells in the presence of DMs or DM/n-HAs was evaluated in terms of cytoskeletal dynamics. Remarkably, it was shown that effects triggered by the DM are no more retained when DM is embedded onto DM/n-HA nanocomposites. In parallel, analyses on the expression of genes involved in (a) intracellular signaling pathways triggered by ligands or cell interactions with elements of the extracellular matrix, (b) modulation of processes such as cell cycle arrest, apoptosis, senescence, DNA repair, metabolism changes, and (c) iron homeostasis and absorption through cell membranes, indicated that the DM/n-HA-treated cells retain tracts of physiological responsiveness unlike DM-treated cells. Overall, a shielding effect by the n-HA was assumed (masking the DM’s cytotoxicity), and a modular biomimicry of the DM/n-HA nanocomposites. On these bases, the biocompatibility of n-HA associated to DM’s magnetic responsiveness offer a combination of structural/functional features of these nano-tools for bone tissue engineering, for finely acting within physiological ranges. Abstract The development of nanocomposites with tailored physical–chemical properties, such as nanoparticles containing magnetic iron oxides for manipulating cellular events at distance, implies exciting prospects in biomedical applications for bone tissue regeneration. In this context, this study aims to emphasize the occurrence of differential responsiveness in osteoblast-like cells to different nanocomposites with diverse features: dextran-grafted iron oxide (DM) nanoparticles and their hybrid nano-hydroxyapatite (DM/n-HA) counterpart. Here, responsiveness of cells in the presence of DMs or DM/n-HAs was evaluated in terms of cytoskeletal features. We observed that effects triggered by the DM are no more retained when DM is embedded onto the DM/n-HA nanocomposites. Also, analysis of mRNA level variations of the focal adhesion kinase (FAK), P53 and SLC11A2/DMT1 human genes showed that the DM/n-HA-treated cells retain tracts of physiological responsiveness compared to the DM-treated cells. Overall, a shielding effect by the n-HA component can be assumed, masking the DM’s cytotoxic potential, also hinting a modular biomimicry of the nanocomposites respect to the physiological responses of osteoblast-like cells. In this view, the biocompatibility of n-HA together with the magnetic responsiveness of DMs represent an optimized combination of structural with functional features of the DM/n-HA nano-tools for bone tissue engineering, for finely acting within physiological ranges.

Chronic inflammation of the adipose tissue (AT) is a major contributor to obesity-associated cardiometabolic complications. The olive oil polyphenol hydroxytyrosol (HT) contributes to Mediterranean diet cardiometabolic benefits through mechanisms still partially unknown. We investigated HT (1 and 10 &mu, mol/L) effects on gene expression (mRNA and microRNA) related to inflammation induced by 10 ng/mL tumor necrosis factor (TNF)-&alpha, in human Simpson&ndash, Golabi&ndash, Behmel Syndrome (SGBS) adipocytes. At real-time PCR, HT significantly inhibited TNF-&alpha, induced mRNA levels, of monocyte chemoattractant protein-1, C-X-C Motif Ligand-10, interleukin (IL)-1&beta, IL-6, vascular endothelial growth factor, plasminogen activator inhibitor-1, cyclooxygenase-2, macrophage colony-stimulating factor, matrix metalloproteinase-2, Cu/Zn superoxide dismutase-1, and glutathione peroxidase, as well as surface expression of intercellular adhesion molecule-1, and reverted the TNF-&alpha, mediated inhibition of endothelial nitric oxide synthase, peroxisome proliferator-activated receptor coactivator-1&alpha, and glucose transporter-4. We found similar effects in adipocytes stimulated by macrophage-conditioned media. Accordingly, HT significantly counteracted miR-155-5p, miR-34a-5p, and let-7c-5p expression in both cells and exosomes, and prevented NF-&kappa, B activation and production of reactive oxygen species. HT can therefore modulate adipocyte gene expression profile through mechanisms involving a reduction of oxidative stress and NF-&kappa, B inhibition. By such mechanisms, HT may blunt macrophage recruitment and improve AT inflammation, preventing the deregulation of pathways involved in obesity-related diseases.

The small intestine is the central component of the gastrointestinal (GI) tract (gut) where nutrients are absorbed into the body. Its functional structure is mainly based on its extremely extended surface area, further increased by a specific carpet of villi, responsible for the translocation of nutrients from the GI lumen into the bloodstream. Also, in the small intestine, the absorption processes of the orally administered drugs are basically related to the pharmacokinetics [1]. The deficit of cell culture methods to maintain in vivo–like functions forces researchers to optimize and apply methods in which cells are seeded and cultured under controlled and dynamic fluid flow [2]. Moreover, the lack of predictive human organ models has increased the necessity of approaches for proper mimicking of organ function in vitro, studying physiological parameters that regard mechanical, chemical and physical stimuli crucial for differentiation, morphology and function of the epithelia [3]. In this work we present a Gut-On-Chip (GOC) device, equipped with ITO (Indium tin Oxide) electrodes patterned by wet etching techniques, as a multifunctional microsystem for monitoring epithelial parameters. The potential to support cells adhesion, growth and polarization of a functional monolayer is also investigated in the Caco-2 epithelial-like cell line by in-device seeding and culture. In a perspective, this first prototype has established the basis for several technology integrations to study complex cellular phenomena targeted in key physiological topics (e.g. the tight interplay of different physical effects during mechanotransduction processes) and in pharmacological open issues such as drug absorption and metabolism.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System, is a pluripotent humoral agent whose biological actions include short-term modulations and long-term adaptations. In fish, short-term cardio-tropic effects of AngII are documented, but information on the role of AngII in long-term cardiac remodelling is not fully understood. Here, we describe a direct approach to disclose long-term morpho-functional effects of AngII on the zebrafish heart. Adult fish exposed to waterborne teleost analogue AngII for 8 weeks showed enhanced heart weight and cardio-somatic index, coupled to myocardial structural changes (i.e. augmented compacta thickness and fibrosis), and increased heart rate. These findings were paralleled by an up-regulation of type-1 and type-2 AngII receptors expression, and by changes in the expression of GATA binding protein 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and superoxide dismutase 1 soluble mRNAs, as well as of cytochrome b-245 beta polypeptide protein, indicative of cardiac remodelling. Our results suggest that waterborne AngII can sustain and robustly affect the cardiac morpho-functional remodelling of adult zebrafish.

Oxidative stress is involved in the genesis and progress of many disorders of the gastrointestinal (GI) tract. In particular, the colon epithelium is one of the GI tract segments more exposed to pro-oxidant conditions. We aimed to study the intracellular antioxidant activity of hydroxytyrosol (HT), one of the most potent natural antioxidant phenolic compounds typically present in olive oil, directly on the colon epithelium, under basal physiological and pro-oxidant conditions. Our approach was based on the application of in situ confocal microscopy on rat colon explants loaded with the fluorescent probe 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluoresceindiacetate, which is sensitive to intracellular oxidative stress. In the intact mucosa, HT exerted a dose-dependent decrease of the basal intracellular reactive oxygen species (ROS) production of superficial colonocytes. Also, it induced a direct dose-dependent antioxidant action on the colon mucosa exposed to a pro-oxidant condition such as the H2O2 challenge. The effect of 100 µM HT was comparable to that of 10 µM Trolox, which is widely used as a standard in in vitro assays for the determination of antioxidant activity. The intracellular antioxidant activity of HT on the intact mucosa was also tested against tert-butyl peroxide, another pro-oxidant. The results show that HT can directly contribute to the redox balance of colonic epithelium by reducing ROS in both basal and pro-oxidant conditions, and support the potential of HT as a functional food ingredient with applications in protecting the intestinal mucosa against oxidative stress.

Background Recent data support the hypothesis that genetic damage occurring early in life during childhood can play an important role in the development of chronic diseases in adulthood, including cancer. Objectives The objective of this paper, part of the MAPEC_LIFE project, is to describe the frequency of micronuclei and meta-nuclear alterations in exfoliated buccal cells of 6–8year-old Italian children recruited in five Italian towns (i.e., Brescia, Torino, Pisa, Perugia and Lecce) with different air pollution levels. Methods About 200 children per town were recruited from primary schools. Biological samples were collected twice from the same children, in two different seasons (winter 2014-15 and late spring 2015). Cytogenetic damage was evaluated by the buccal micronucleus cytome assay. Results Overall,n = 1046 children represent the final cohort of the MAPEC_LIFE study. On the whole, the results showed a higher mean MN frequency in winter (0.42 ± 0.54‰) than late-spring (0.22 ± 0.34‰). MN frequency observed among the five Italian towns showed a trend that follows broadly the levels of air pollution in Italy: the highest MN frequency was observed in Brescia during both seasons, the lowest in Lecce (winter) and Perugia (late-spring). Conclusions To the best of our knowledge, the number of recruited children included in the analysis (n = 1046) is the highest compared to previous studies evaluating the frequency of MN in exfoliated buccal cells so far. MN frequency was associated with winter season and living in towns at various levels of air pollution, suggesting an important role of this exposure in determining early cytogenetic effects.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

The zebrafish (Danio rerio) is a small vertebrate ideally suited to the modeling of human diseases. Large numbers of genetic alterations have now been modeled and could be used to study organ development by means of a genetic approach. To date, limited attention has been paid to the possible use of the zebrafish toolbox in studying human mitochondrial disorders affecting the nervous system. Here, we review the pertinent scientific literature discussing the use of zebrafish in modeling gene mutations involved in mitochondria-related neurological human diseases. A critical analysis of the literature suggests that the zebrafish not only lends itself to exploration of the pathological consequences of mitochondrial energy output on the nervous system but could also serve as an attractive platform for future drugs in an as yet untreatable category of human disorders.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.