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The S100 gene family is the largest subfamily of calcium binding proteins of EF-hand type, expressed in tissue and cell-specific manner, acting both as intracellular regulators and extracellular mediators. There is a growing interest in the S100 proteins and their relationships with different cancers because of their involvement in a variety of biological events closely related to tumorigenesis and cancer progression. However, the collective role and the possible coordination of this group of proteins, as well as the functional implications of their expression in breast cancer (BC) is still poorly known. We previously reported a large-scale proteomic investigation performed on BC patients for the screening of multiple forms of S100 proteins. Present study was aimed to assess the functional correlation between protein and gene expression patterns and the prognostic values of the S100 family members in BC. By using data mining, we showed that S100 members were collectively deregulated in BC, and their elevated expression levels were correlated with shorter survival and more aggressive phenotypes of BC (basal like, HER2 enriched, ER-negative and high grading). Moreover a multi-omics functional network analysis highlighted the regulatory effects of S100 members on several cellular pathways associated with cancer and cancer progression, expecially immune response and inflammation. Interestingly, for the first time, a pathway analysis was successfully applied on different omics data (transcriptomics and proteomics) revealing a good convergence between pathways affected by S100 in BC. Our data confirm S100 members as a promising panel of biomarkers for BC prognosis.

Matrix metalloproteases (MMPS) are a family of well-known enzymes which operate prevalently in the extracellular domain, where they fulfil the function of remodeling the extracellular matrix. Within the about 26 family members, encoded by 24 genes in humans, MMP-2 and MMP-9, have been regarded as the primary responsibility for the basement membrane and pericellular ECM rearrangement. In cases of infiltrating carcinomas, which arise from the epithelial tissues of a gland or of an internal organ, a marked alteration of the expression and the activity levels of both MMPs is known to occur. Present investigation represents the continuation and upgrading of our previous studies, now focusing on the occurrence and intensity levels of MMP-2 and -9, and their proteomic correlations, in a cohort of 80 breast cancer surgical tissues

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPSaer) and anaerobic conditions (AgNPs-EPSanaer). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPSaer as the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPSaer mode of action. In particular, AgNPs-EPSaer induced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag+ accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPSaer toxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation.

This preliminary report shows eight complexes of triorganotin(IV): Ph3SnOH and (CH3)3SnOH with four chelating imines on new synthesis. Of these ligands, two are salen-like (four coordination sites, two imidic, two phenoxidic) [1], one is a tetradentate pyrrole derivative [2] while the fourth, a vita- min B6 derivative, is pentadentate [3]. Ligands have been characterized by means of FT-IR, UV-Vis, Fluorescence, 1H- and 13C-NMR, LC-MS ESI triple quadrupole; complexes by means FT-IR, 1H- and 119Sn-NMR, LC-MS ESI, using the isotopic distribution pattern as a discriminant [4]. Geometry and nature of coordination complexes have been also evaluated using the 119Sn chemical shifts. Solid-state synthesis of the complexes (with a ball mill [5]) was also explored; such method reduces both solvent consumption and time – from 8-10 h under controlled atmosphere to about 1 h, with results identical to the wet synthesis. Antitumoral and antibacterial activities of the triorganotin (IV) complexes (BS01M, BS01P, BS02M, BS02P, BS03M, BS03P, BS04M, BS04P) were tested in vitro. In both analyses, the Shiff bases alone showed no biological activity. Antimicrobial activity was evaluated by Kirby-Bauer method against the Gram-negative Escherichia coli, and the two Gram-positive Kocuria rizophila and Staphylococcus aureus strains. All the ML2 complexes were active in inhibiting bacterial growth, with BS02P and BS03P showing the best antibacterial performance. Among the ML complexes, BS01M was not active, BS02M showed a weak antibacterial activity only against the Gram-positive bacteria, BS04M was mainly active against the Gram-negative E. coli and BS03M was active against all the tested strains. Antitumor activity was evaluated by MTT assay against cervical (HeLa), colon adenocarcinoma (HT- 29) and breast (MDA-MB231) cancer cell lines. Results showed that ML2 complexes are more active than ML ones, with HeLa cells more sensitive to treatments. These complexes (especially the ML2) showed promising results; their mechanism of action is under investigation. References 1. K. Tayade, S.K. Sahoo, S. Chopra, N. Singh, Inorganica Chimica Acta, 421, 538-543 (2014); https://doi.org/10.1016/j. ica.2014.05.014 2. S. Meghdadi, M. Amirnasr, K. Mereiter, Polyhedron, 30, 1651-1656 (2011); https://doi.org/10.1016/j.poly.2011.03.041 3. D. Sharma, S.K. Sahoo, S. Chaudhary, R. Kanta Bera, J.F. Callan, Analyst, 138, 3646-3650 (2013); https://doi.org/10.1039/ C3AN00199G 4. G. Lawson, R.H. Dahm, N. Ostah, E.D. Woodland, Applied Organometallic Chemistry, 10, 125-133 (1996); https://doi. org/10.1002/(SICI)1099-0739(199603)10:23.0.CO;2-1 5. G.A. Bowmaker, Chemical Communications, 49, 334-348 (2013); http://doi.org/10.1039/C2CC35694E 6. R. Di Stefano, M. Scopelliti, C. Pellerito, G. Casella, T. Fiore, G.C. Stocco, R. Vitturi, L. Ronconi, I.D. Sciacca, L. Pellerito, Journal of Inorganic Biochemistry, 98, 534-546 (2004); https://doi.org/10.1016/j.jinorgbio.2003.12.013 7. C. Pellerito, L. Nagy, L. Pellerito, A. Szorcsik, Journal of Organometallic Chemistry, 691, 1733-1747 (2006); https://doi. org/10.1016/j.jorganchem.2005.12.025

Background: Nowadays, an optimal and effective medical surgery remains the gold standard for perianal fistulas. Hereby we reported preliminary results in favor of using Argentum-quartz solution for both primary and recurrent perianal fistulas. Methods: Three patients with intersphincteric and extrasphincteric fistulas were enrolled. Argentum-quartz solution was administrated twice a week for a period of 4 weeks, followed by a pause of 8 days and then another 4 weeks of treatment, totally 16 administrations. After treatment, all patients were monitored for a 4-month follow-up. Results: Complete closures of 2 extrasphincteric fistulas and a partial closure with absence of inflammation and superative phenomena in the intrasphincteric fistula were both manifested. Conclusion: Selective treatment of perianal fistulas with an argentum-quartz solution is safe and effective, and may represent a reliable alternative.

We investigated the effects of natural antioxidants and modified atmosphere packaging (MAP) on the quality and shelf-life of Coryphaena hippurus (common dolphinfish) fillets. Fillets of the control group (CO) were simply placed on trays and not sealed. Fillets of the second group (MAP) were preserved in modified atmosphere (45% CO2, 50% N2, 5% O2), and fillets of the third group (MAP-AOX) were preserved by combining natural antioxidants from halophytes with modified atmosphere. All samples were stored in a refrigerator at 1 ± 0.5 C for 18 days. Analyses were conducted at T0 and after 3, 6, 12, 15 and 18 days of storage. According to sensory tests and colour analyses, the fillets in the MAP and MAP-AOX groups maintained the best appearance. In the CO group, we observed a significant decrease of omega-3 polyunsaturated fatty acids (FAs) (P < 0.05), which indicates the oxidation of these FAs. FAs in the MAP and MAP-AOX groups were protected against oxidation. Further, lower peroxide value (PV) and malondialdehyde (MDA) content were observed in the MAP and MAP-AOX groups compared to the CO group. The sensory properties of the fillets were correlated with changes in FA content, PV and MDA content.

Here we report differences between: 1) a heterogeneous population of primary rat brain astrocytes (Primary), in culture since several years ago, and 2) a cloned cell line (Clone), obtained from the Primary cells. Both populations maintain astrocyte morphology but, according to cytogenetic and epigenomic characterization, differ for the chromosomal asset from rat normal cells (42 chromosomes): Primary cells show mostly a bimodal karyotype with 41 or 43 chromosomes, and Clone has a unique-modal karyotype of 43 chromosomes. Interestingly, we also found that both cell lines show genome-wide DNA hypomethylation, with Clone showing even more pronounced demethylation respect to Primary cells. These features, together with a faster doubling time, confer to Clone an altered proliferation control phenotype. Conversely, the Primary cell population is more similar to normal cells. Used together the two cell populations are a promising model to investigate in vitro modifications of genome, epigenome and others ‘omics’, mimicking tumor clonal evolution-derived heterogeneity, particularly useful in studies on CNS cancers, which derive mostly from glial cells.

The Hsp70 family is one of the best conserved and abundant member of the heat shock proteins (HSP). This family includes several members and in particular one constitutively expressed member (Hsc70) and another one inducibly expressed under several stress conditions (Hsp70). To date, the intracellular functions of Hsp70 are well defined, and increasing evidences establish its roles in the extracellular environment, such as cytoprotection and immunomodulation. Increasing evidences suggest that several cell types are able to release Hsp70 in the extracellular environment, both under physiological and stress conditions. At the same time many release mechanisms have been identified. This chapter briefly reviews recent advances in our understanding on extracellular Hsp70 role in both physiological and pathological conditions. A better comprehension will be useful to take advantage of its potential as a therapeutic target.

Anthropic noise is considered a real pollutant, in particular the submarine noise. The impact on biodiversity is not yet sufficiently understood. Further research is needed to evaluate any negative effects. The noises associated with anthropogenic activities are increasing: shipments, seismic surveys, sonar, recreational rowing and future mineral extraction activities from ocean depths (DSM). These noises are having an impact on the welfare of many marine species. The understanding of the effects on biodiversity could be important for the creation of guidelines, laws or rules for the most environmentally sustainable exploitation of natural resources. Our study aims to investigate the motility, biochemical and molecular responses of Arbacia lixula exposed to an acoustic stimulus produced by anthropogenic activities and perhaps perceptible by invertebrates. The animals were divided into a control tank and experimental tank. The specimens were exposed to sonic stress for 3 hours after which biological sampling was performed. The sonic stress used was a linear sweep from 100 to 200 kHz emitted every 1 second. The Sound Pressure Level ranges between 173 and 181 dBrms (re 1μPa), with a peak at 150 kHz. To measure the motility, on the top of the tanks two cameras were placed to make photo each 8 seconds during acoustic stimulus. The bottom of the tanks were divided virtually by squares and the vertical walls were divided by two quote. At each photo we assessed the position of the animals and count the number of squares/levels crossed comparing to the precedent photo. In this way we obtained the motility of the specimens in the three directions (cm/s). After three hours of stimulus projection, the sea urchins were captured and the coelomatic fluid was extracted. The protein concentration and the enzymatic activities of esterase, phosphatase, catalase and peroxidase were measured on the celomocytes and on the supernatant of the celomatic fluid. The gene expression of HSP70 and HSP90 with RT-PCR was evaluated on celomocytes. Exposure to this type of noise produced a significant changing in motility and an increase in the expression of HSPs gene, more so for HSP70. No statistical difference was observed in the extent of enzymatic activities and protein concentration. The results obtained indicate that this type of acoustic stimulus has effects on the behavior and on the gene expression of HSPs of individuals of Arbacia lixula.

Stem cells are used in regenerative medicine, but their therapeutic efficacy is compromised by their huge death during the first days post-transplantation. Indeed, the microenvironment within damaged tissues is hostile for stem cell survival mainly due to oxidative stress. H2O2 may play a relevant role in inducing death of the injected cells. The aim of our study was to determine the mechanism of mesoangioblast (A6) cell death after an H2O2 treatment. FACS analysis with annV/PI showed that H2O2 induced a dose and time-dependent decrement in A6 viability. We have also found an increase in caspases 8, 9 and 3 activity after the treatment. To assess their involvement in cell death, the pan caspase inhibitor Z-VAD was used. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by H2O2 was caspase-independent. Then, we tested whether H2O2 is responsible for the autophagy activation.To study autophagy we evaluated the expression of specific markers. H2O2 decreased beclin1, Atg5, Atg7 and the ratio LC3II/I, in a dose dependent way. At the same time it increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of pAKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data A6 treatment with H2O2 seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, a specific form of caspase-independent, non-apoptotic or necrotic cell death. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annV/PI staining. FACS analysis showed an increase in percentage of both late apoptotic/necrotic and necrotic cells, which were further increased by pretreatment with Z-VAD. To investigate the relationship between physiological autophagy and necroptosis, cells were treated with H2O2 in the presence of the autophagic inhibitor 3MA. AnnV/PI staining showed that the inhibition of autophagy by 3MA significantly enhanced necroptosis in A6 treated cells. Conversely, 3MA had no effect on apoptosis. In conclusion, our data indicate that the cytotoxicity of H2O2 in A6 mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.

The aim was to correlate specific biological aspects of cumulus cells isolated from individual cumulus-oocyte complexes (COCs) with the clinical outcome of the related embryos.

Skeletogenesis is a key morphogenetic event in the life of marine invertebrates. Marine calcifiers secrete their calcareous skeletons taking up ions from seawater. Marine biominerals include aragonite and calcite, the latter of which in some taxa (e.g. echinoderms, coralline algae) can have a substantial magnesium (Mg) component. Echinoderms have an extensive endoskeleton composed of high magnesian calcite and occluded matrix proteins1. As biomineralization in sea urchin larvae is sensitive to the Magnesium:Calcium ratio of sea water, we investigated the effects of magnesium deprivation on development and skeletogenesis in the Mediterranean sea urchin Arbacia lixula. Microscopic inspection revealed that embryos reared in Mg-free seawater exhibited developmental delay from 6 hours post-fertilization, complete lack of skeleton formation at 24 hours, and severe skeleton malformations in larvae (48-72 hours). We subsequently focused on the localization of the skeletogenic cells (primary mesenchyme cells) and the spatial expression of associated genes. Immunocytochemistry revealed abnormal ectopic location of the primary mesenchyme cells (PMCs) and of the developing skeleton of treated embryos. Expression of msp130, an important skeleton matrix protein gene expressed only in PMCs, detected by in situ hybridization, was normal at 24 hours, but this gene was not down-regulated at 48 hours, as in controls2. Strikingly, development of the pigment cells, immune cells that, like the skeleton, are mesodermal derivatives, was also impaired. These results suggest the essential role of Mg in skeleton formation in sea urchin embryos with an indication that this element is also generally important for development of mesoderm. 1. Smith AM et al. Mar Ecol Prog Ser 2016, 561:1–16 2. Martino C et al. Aq tox 2018, 194:57-66.

The aim of this article is to show methods for dissection of the neck. In the summer of 2017 a group of students of the University of Palermo that have already passed the exam of Human Anatomy took a 4 weeks dissection course at the University of Malta. The students were provided with a dissection kit, video recording equipment and cameras for taking pictures. They dissected the skin, the subcutaneous tissue, the muscular bundles, the muscles, the vascular and nervous bundles, the nerves, the larynx, the trachea and the esophagus. This paper presents the results of the dissection course and a small and simple guide to young students and medical doctors who want to learn the bases of neck dissection.

The earth’ resources are running out, the population will increase and further sources will be needed. These, were found in the deep ocean. To date the mining activities (Deep Sea Mining, DSM) they have not started. One impact of DSM could be underwater noise. Considering the noise frequencies of anthropic activities in the ocean, and the European directive on underwater noise control, we stressed the animals with four acoustic 1/3 band noises around: 63 Hz, 125 Hz, 500 Hz, 1kHz. We study the effects on Sparus aurata juveniles, and on Mytilus galloprovincialis. We will analyse behavioural., molecular and biochemical responses Los recursos de la tierra se están agotando, la población aumentará y se necesitarán más fuentes. Esaos fueron encontradas en el océano profundo. Hasta ahora, las actividades mineras (Deep Sea Mining, DSM) no han empezado. Un impacto del DSM podría ser el ruido subacuático. Teniendo en cuenta las frecuencias de ruido de las actividades antrópicas en el océano, y la directiva europea sobre el control del ruido submarino, molestamos los animales con cuatro ruidos acústicos de 1/3 de banda alrededor: 63 Hz, 125 Hz, 500 Hz, 1kHz. Estudiamos los efectos en juveniles de Sparus aurata y en Mytilus galloprovincialis. Analizaremos respuestas comportamiental,moleculares y bioquímicas.

Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.

Malignant glioma cells invade the surrounding brain parenchyma, by migrating along the blood vessels, thus promoting cancer growth. The biological bases of these activities are grounded in profound alterations of the metabolism and the structural organization of the cells, which consequently acquire the ability to modify the surrounding microenvironment, by altering the extracellular matrix and affecting the properties of the other cells present in the brain, such as normal glial-, endothelial- and immune-cells. Most of the effects on the surrounding environment are probably exerted through the release of a variety of extracellular vesicles (EVs), which contain many different classes of molecules, from genetic material to defined species of lipids and enzymes. EV-associated molecules can be either released into the extracellular matrix (ECM) and/or transferred to neighboring cells: as a consequence, both deep modifications of the recipient cell phenotype and digestion of ECM components are obtained, thus causing cancer propagation, as well as a general brain dysfunction. In this review, we first analyze the main intracellular and extracellular transformations required for glioma cell invasion into the brain parenchyma; then we discuss how these events may be attributed, at least in part, to EVs that, like the pawns of a dramatic chess game with cancer, open the way to the tumor cells themselves.

Jay Amin Hydroxamic Acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA’s cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 M. JAHA’s lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction and autophagy mechanisms. In order to glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in anti-oxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs.

Environmental factors could have a key role in the remarkable and continuous decline of sperm quality observed in the last fifty years. This study addressed the gap of knowledge on the effect of air pollutants on sperm DNA fragmentation, comparing the seminal parameters from men living in locations with different levels of air pollution. The detrimental effects of environmental pollution in the Taranto area are alarming: the high level of pollutants released from the steel plants in the atmosphere can cause health and fertility issues. Our study analyzed sperm samples from three groups of patients: i) workers of Taranto steel plants; ii) Taranto residents; iii) Palermo residents, assumed as controls. Results demonstrate that patients from the “steel plants workers” group, constantly exposed to environmental pollutants for professional reasons, show a mean percentage of sperm DNA fragmentation above 30%. In contrast, patients from the “Taranto residents” group and controls show mean percentages ranging between 16.8% and 25%, respectively. We suggest that sperm DNA evaluation can either be an indicator of individual reproductive health, and a suitable tool to connect the surrounding environment with its effects. On the other end, preliminary data of the Eco Food Fertility project3 indicated an impairment of several semen quality parameters, including increased sperm DNA damage, in clinically healthy male volunteers living in areas with high environmental impact. Since the methods to study pollutants effects have still to be validated, we suggest that studying sperm DNA fragmentation could serve as a valuable biomarker of the presence and effects of pollution, and we candidate the human sperm cell as an early bioindicator of male health and reproduction.

Cold-shock domain containing protein C2 (CSD-C2; also known as PIPPin) is an RNA-binding protein conserved in the evolution that interacts with the 3’-untranslated region (3’-UTR) of rat H1.0 and H3.3 histone messengers. Biolayer interferometry (BLI) is a technique that measures changes in an interference pattern generated from visible light, reflected from an optical layer, and a biolayer which contains molecules of interest. In this study, we used the BLI methodology in order to analyze and describe the binding properties of CSD-C2 and the mRNA encoding the rat brain histone protein H3.3. Recombinant CSD-C2 was incubated with in vitro transcribed, and biotinylated H3.3 RNA fragments bound to streptavidin-conjugated Octet optical biosensors. In order to define the RNA region involved in binding, we used RNA probes corresponding to different portions of H3.3 RNA 3’-UTR. In this study, we showed that CSD-C2 binds to the last 199 nucleotides of the H3.3 RNA 3’-UTR, and that the apparent affinity constant of the interaction is in the nanomolar range. In addition, this study confirmed that BLI can be a very efficient and reliable method for studying RNA-protein interactions.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The specimens were selected from patients with invasive ductal carcinoma of the breast, the most frequent and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers potentially useful for clinical applications. The proteomic screening; by 2D-IPG and mass spectrometry; allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients; and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is reasonably supported by concurrent events: the inhibition of apoptosis and stimulation of cellular proliferation, and the increased expression of glycolytic enzymes with multiple functions. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. We suggest that this approach on large-scale 2D-IPG proteomics of breast cancer is currently a valid tool that offers the opportunity to evaluate on the same assay the presence and recurrence of individual proteins, their isoforms and short forms, to be proposed as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.

Gadolinium (Gd) concentration is constantly increasing in the aquatic environment, becoming an emergent environmental pollutant. We investigated the effects of Gd on Paracentrotus lividus sea urchin embryos, focusing on skeletogenesis and autophagy. We observed a delay of biomineral deposition at 24 hours post fertilization (hpf), and a strong impairment of skeleton growth at 48 hpf, frequently displayed by an asymmetrical pattern. Skeleton growth was found partially resumed in recovery experiments. The mesodermal cells designated to biomineralization were found correctly migrated at 24 hpf, but not at 48 hpf. Western blot analysis showed an increase of the LC3-II autophagic marker at 24 and 48 hpf. Confocal microscopy studies confirmed the increased number of autophagolysosomes and autophagosomes. Results show the hazard of Gd in the marine environment, indicating that Gd is able to affect different aspects of sea urchin development: morphogenesis, biomineralization, and stress response through autophagy.

SummarySea urchin represents an ideal model for studies on fertilization and early development, but the achievement of egg competence and mitochondrial behaviour during oogenesis remain to be enlightened. Oocytes of echinoid, such as sea urchin, unlike other echinoderms and other systems, complete meiotic maturation before fertilization. Mitochondria, the powerhouse of eukaryotic cells, contain a multi-copy of the maternally inherited genome, and are involved directly at several levels in the reproductive processes, as their functional status influences the quality of oocytes and contributes to fertilization and embryogenesis. In the present paper, we report our latest data on mitochondrial distribution, content and activity duringParacentrotus lividusoogenesis. The analyses were carried out using confocal microscopy,in vivoincubating oocytes at different maturation stages with specific probes for mitochondria and mtDNA, and by immunodetection of Hsp56, a well known mitochondrial marker. Results show a parallel rise of mitochondrial mass and activity, and, especially in the larger oocytes, close to germinal vesicle (GV) breakdown, a considerable increase in organelle activity around the GV, undoubtedly for an energetic aim. In the mature eggs, mitochondrial activity decreases, in agreement with their basal metabolism. Further and significant information was achieved by studying the mitochondrial chaperonin Hsp56 and mtDNA. Results show a high increase of both Hsp56 and mtDNA. Taken together these results demonstrate that during oogenesis a parallel rise of different mitochondrial parameters, such as mass, activity, Hsp56 and mtDNA occurs, highlighting important tools in the establishment of developmental competence.

The sea urchin embryo is a suitable model that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium accumulates in a dose- and time-dependent manner into embryonic cells, activating different stress and defence mechanisms, including the synthesis of HSPs and the onset of apoptosis and/or autophagy. In this paper we investigated the functional relationship between autophagy and apoptosis, evaluating apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy. We found that the inhibition of autophagy produced the concurrent reduction of apoptosis, suggesting that the two phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain the relationship was evaluated. Using a substrate for ATP production, we found that apoptosis, assessed by TUNEL and cleaved caspase-3 immunocytochemistry, was substantially restored in cadmium-treated embryos where autophagy was inhibited by 3-Methyladenine. On the basis of these results, we propose that, autophagy could play a crucial role in stress response of this organism because autophagy could energetically contribute to apoptotic execution through its catabolic role.

Most aspects of nervous system development and function rely on the continuous crosstalk between neurons and the variegated universe of non-neuronal cells surrounding them. The most extraordinary property of this cellular community is its ability to undergo adaptive modifications in response to environmental cues originating from inside or outside the body. Such ability, known as neuronal plasticity, allows long-lasting modifications of the strength, composition and efficacy of the connections between neurons, which constitutes the biochemical base for learning and memory. Nerve cells communicate with each other through both wiring (synaptic) and volume transmission of signals. It is by now clear that glial cells, and in particular astrocytes, also play critical roles in both modes by releasing different kinds of molecules (e.g., D-serine secreted by astrocytes). On the other hand, neurons produce factors that can regulate the activity of glial cells, including their ability to release regulatory molecules. In the last fifteen years it has been demonstrated that both neurons and glial cells release extracellular vesicles (EVs) of different kinds, both in physiologic and pathological conditions. Here we discuss the possible involvement of EVs in the events underlying learning and memory, in both physiologic and pathological conditions.

Our previous studies documented that human fibroblast-limbal stem cells (f-LSCs) possess immunosuppressive capabilities, playing a role in regulating T-cell activity. This study highlights the molecular activities by which human f-LSCs can attenuate the inflammatory responses of self-reactive peripheral blood mononuclear cells (PBMCs) collected from patients with autoimmune endocrine diseases (AEDs). Anti-CD3 activated PBMCs from twenty healthy donors and fifty-two patients with AEDs were cocultured on f-LSC monolayer. 2D-DIGE proteomic experiments, mass spectrometry sequencing and functional in vitro assays were assessed in cocultured PBMCs. We identified the downmodulation of several human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) isoforms in healthy and AED activated PBMCs upon f-LSC interaction. The reduction of hnRNPA2/B1 protein expression largely affected the cycling ki67+, CD25+, PD-1+ reactive cells and the double marked CD8+/hnRNPA2B1+ T cell subset. Anti-PD1 blocking experiments evoked hnRNPA2/B1 overexpression, attributing putative activation function to the protein. hnRNPA2/B2 transient silencing inverted immunopolarization of the self-reactive PBMCs from AEDs toward a M2/Th2-type background. Pharmacological inhibition and co-immunoprecipitation experiments demonstrated the involvement of NF-ĸB in hnRNPA2/B activity and turnover. Our data indicate cardinal involvement of hnRNP A2/B1 protein in peripheral mechanisms of tolerance restoration and attenuation of inflammation, identifying a novel immunoplayer potentially targetable in all AEDs.

The readthrough of nonsense mutations by small molecules like Ataluren is considered a novel therapeutic approach to overcome the gene defect in several genetic diseases as cystic fibrosis (CF). This pharmacological approach suppresses translation termination at premature termination codons (PTCs readthrough) thus restoring the expression of a functional protein. However, readthrough might be limited by the nonsense-mediated mRNA decay (NMD), a cell process that reduces the amount/level of PTCs containing mRNAs. Here we investigate the combined action of Ataluren and caffeine to enhance the readthrough of PTCs. IB3.1 CF cells with a nonsense mutation were treated with caffeine to attenuate the Nonsense-Mediated mRNA Decay (NMD) activity and thus enhance the stability of the nonsense (ns)-CFTR-mRNA to be targeted by Ataluren. Our results show that NMD attenuation by caffeine enhances mRNA stability and more importantly when combined with Ataluren increase the recovery of the full-length CFTR protein.

Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation.

The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream.

INTRODUCTION Secretion of extracellular vesicles (EVs) and EV-mediated exchange of molecules among brain cells, under physiological conditions, has been described years ago. At the same time, ability of EVs to transfer proteins and nucleic acids to the surrounding cells, thus modifying their phenotypes, has been clearly demonstrated for tumor cells of different origins, including brain cancers. More recently, involvement of EVs in pathological processes affecting the Central Nervous System (CNS), and their capacity of transferring damaging cargoes have been also suggested. To explore these functions, we exposed primary cultures of astrocytes to EVs isolated from cerebral spinal fluid (CSF) of patients affected by multiple sclerosis (MS), in order to study the potential ability of these EVs to induce in receiving cells morphological and functional changes, such as modifications of proliferation rate, apoptosis or cell death. METHODS Our study was carried out on 10 individuals who, after clinical diagnosis, were separated into two different groups: controls and MS patients. The diagnosis has always been confirmed by a neurologist and the patients gave informed consent to participate in a molecular study on MS. EV population is actually composed of two kind of vesicles: (a) exosomes (Es), which originate from the endosomal compartment and are smaller, and larger membrane vesicles (MVs), which directly bud from the plasma membrane. To separate Es from MVs, before ultracentrifugation, CSF was filtered through a 0.2-μm filter and centrifuged at 10,000 × g for 30 min. The pellet containing MVs was saved, while the supernatant was further centrifuged at 105,000 × g for 90 min to obtain Es. Pelleted vesicles were suspended with phosphate-buffered saline, pH 7.5 (PBS) and used to treat astrocytes for 24 hours. RESULTS MVs from MS patients induced a higher degree of cell death than those prepared from the CSF of control group (99,35% dead cells vs 89,33), but the difference was not significant (p value=0,142). A dramatic effect was instead noticed when treating astrocytes with Es from patients, which were able to induce a highly significant (p value=0,0001) degree of cell death respect to Es from controls (94,38% vs 71,21). CONCLUSIONS Our study suggests that EVs present in the CSF contain molecules which can be toxic to astrocytes and this is probably the reason why brain cells discard such molecules via different kinds of EVs. However, EVs, and in particular Es, present in CSF of MS patients are even more toxic for cells than EVs present in the CSF of healthy controls. Further studies are now necessary to identify the active molecules present in EVs.

An array of diimidazolium salts has been synthesized and used to investigate their anti-microbial and anti-proliferative activities. In particular, salts based on the 3,30-di-n-alkyl-1,10-(1,n-phenylenedimethylene)- diimidazolium cation and differing in the alkyl chain length on the imidazolium ion, the isomeric substitution on the aromatic spacer and in the anion nature were used. The anti-proliferative activity was evaluated against cervical (HeLa), colon adenocarcinoma (HT-29) and breast (SKBR3) cancer cell lines. In the latter case, also a morphological assessment after treatment with salts was performed. All salts were tested for their hemolytic activity against human erythrocytes. On the other hand, the anti-microbial activity was investigated using both Gram-negative (Escherichia coli) and Gram-positive (Kokuria rhizophila, Staphilococcus aureus and Bacillus subtilis) strains. On the whole, the data collected demonstrate that biological activity is the result of the combined action of both cation and anion structures. In general, the cation hydrophobicity plays the most significant role with structural features of the anion becoming more relevant in the presence of a shorter alkyl chain on the cationic head.

Introduction Mouse mesoangioblasts are vessel associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. We have already demonstrated that these stem cells, as all the other stem cells, are able to release in the extracellular milieu membrane vesicles (EV) containing biological active molecules, such as FGF2, MMP2/91 and Hsp702. Today takes hold the idea that the vesicles can replace stem cells opening a new scenario in regenerative medicine3. To this aim, we investigated the possible paracrine interaction of mesoangioblast EV on different cell types and their effects. Results and conclusions We have showed that mesoangioblast EV interact with human endothelial cells by in vitro inducing their differentiation versus capillary-like structures, and increasing their migration capability. In addition, we have analyzed the immunomodulatory effect of EV on human lymphocytes. We have demonstrated that EV are able to inhibit both lymphocyte activation and proliferation. Finally, we started to investigate the mechanisms of interaction between EV and target cells. In particular, we have observed the involvement of EV saccharidic residues in cell targeting. The enzymatic removal of EV N-lynked glycans by PNGase F, which cleaves between asparagine and GlcNAc in all types of glycan chains, induces a substantial reduction in EV-target cell interaction. Conversely, EndoH that is responsible for the cleavage between two residues of GlcNAc increases this interaction. In conclusion, saccharidic residues exert a role in EV-cell interplay. References 1 Candela ME et al. J Cell Physiol 2010, 224: 144-51. 2 Barreca MM et al. J Cell Physiol. 2017, 232: 1845-61. 3 Tetta C et al. Endocrine 2013, 44: 11-19.

Study question: The effect of HPV infection on semen characteristics was investigated using a new method that allow to evaluate virus localization in the different semen components. Summary answer: Our data indicate a correlation between HPV infection of exfoliated epithelial cells (from spermatic ducts and accessory glands) and reduction of sperm cell motility. What is known already: HPVs are agents of the most common sexually transmitted diseases. Both in men and in women, HPVs cause a variety of clinical symptoms ranging from warts to cancer. It has been shown (IARC, 2009) that infection with 12 oncogenic HPVs is the major cause of development of cervical cancer in women and it is also related to penile cancers in men. HPV infects epithelial cells and can bind other cell types. Its presence in semen is widely known, but the effects on fertility and reproductive function are still controversial. Study design, size, duration: From January 2015 to December 2015, twenty men were enrolled. From each subject seminal parameters were evaluated according to WHO guidelines. HPV was detected and genotyped both in total semen and in swim up-fraction. HPV detection was performed also in separated semen fractions: sperm cells from total semen and from swim-up and epithelial cells. Semen parameters of positive samples were compared with parameters of negative samples. Moreover, HPV genome wholeness in sperm cells was evaluated. Participants/materials, setting, methods: Partners of women with high-grade squamous intraepithelial lesions. DNA was purified after differential lysis. HPV genotyping was performed by a conventional reverse hybridization and HPV detection in the different semen components was performed by nested PCR with the use of universal primers (MY09 / 11, GP5+ / 6+). HPV genome wholeness was evaluated by Real Time-PCR using six different target genes. Main results and the role of chance: Samples were distributed into HPV positive and negative based on the conventional genotyping (total DNA extraction and reverse hybridization method) or on the new method (differential lysis, DNA extraction from separate fractions and nested PCR method) for every different fraction. In any case, we did not find any statistically significant difference on semen volume, sperm concentration, morphology. On the contrary, only when we compared HPV-positive epithelial cell samples to negative samples in the same fraction, we found a difference in sperm motility. The average percentage of sperm linear progressive motility amounted to 10% ± 11.5 in the positive samples and 54.4% ± 23.3 in the negative samples (two-tailed P value=0.0003). Moreover the HPV genome wholeness was tested by RT-PCR. Results performed on DNA extracted from sperm cells of a sample showed that the copy number of E1 and E2 genes was lower than LCR (Long Control Region) and E6 copy numbers (copy number ratio: 0.4 ± 0.05). These data indicate that in this sample more than half viral genomes are defective, suggesting that a recombination event could be occurred into the sperm heads. Limitations, reasons for caution: Low number of samples because of the sporadic frequency of infection with a single high-risk HPV genotype. Wider implications of the findings: The data support the proposed role of HPV in decreased male and couple fertility. The presence of defective viral genomes rises the idea that these defective forms could be integrated in the sperm genome. In turn, this risk prompts new possible consequences of the viral infection in semen.

Extremely halophilic archaea (EHA) might play an important role in salted fish production. So far, limited information has been available on the effect of EHA and salt concentration on the safety and quality characteristics of salted anchovies. Eight Halobacterium salinarum strains were isolated from different sea salt samples and subjected to phenotypic and genotypic characterization. The strains were then inoculated into fresh salt before addition to anchovies. A total of 18 experimental productions were performed. The inoculated trials showed the lowest counts of undesired microorganisms. In particular, salted anchovies produced with Hbt. salinarum H11 showed the lowest histamine concentration as well as the highest sensory scores. Differences in terms of volatile organic compounds (VOCs) were estimated among trials. Furthermore, multivariate analysis showed that experimental production performed with a reduced amount of salt (175 g of sea salt per kg of anchovies) did not affect the final quality of salted anchovies. The strain Hbt. salinarum H11 produced salted anchovies with well-appreciated organoleptic features. Thus, the addition of EHA and the use of a lower amount of sea salt might represent a valuable alternative to the traditional method for production of salted anchovies.

Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI) in U group (patients treated with Highly Purified human Menopausal Gonadotrophin) than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH) combined with recombinant human Luteinizing Hormone (r-hLH)). Both groups R (treated with r-hFSH alone) and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients.

Astaxanthin is a major marine carotenoid with powerful antioxidant and neuroprotective properties. The in vitro protective effect of astaxanthin in adult retinal cells of the type-2 diabetic model Psammomys obesus in hyperglycemic conditions was investigated. Primary retinal cells were cultured in normal (5 mM) or high concentrations of glucose (25 and 40 mM) for 5 days and treated with 1-20 μM astaxanthin for the last 48 h of culturing. Mitochondrial dehydrogenase activity and cell viability were assessed using MTT test and trypan blue exclusion dye. Retinal cells were characterized by immunohistochemistry. The results showed that mitochondrial function increased significantly (P

Gli obiettivi delle ricerche condotte sono stati duplici: 1) verificare l’eventuale comparsa o aumento di fenomeni apoptotici nelle cellule follicolari, negli oociti e nelle strutture stromali, di tessuto ovarico sottoposto a criopreservazione. 2) verificare la presenza di apoptosi in oociti non fecondati dopo trattamento ICSI (Intra Citoplasmic Sperm Injection), per comprendere se pregressi eventi apoptotici siano già in atto in oociti umani trattati con tecniche avanzate di fecondazione assistita, e possano pertanto essere la causa della mancata fecondazione di circa il 30-35 % degli oociti trattati.

Jay Amin Hydroxamic Acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA’s cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 M. JAHA’s lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction and autophagy mechanisms. In order to glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in anti-oxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs.

Both prokaryotic and eukaryotic cells release into the extracellular matrix membrane-bound structures of different sizes, origin and composition, collectively called extracellular vesicles (EVs) [1]. Tumor cells, in particular, use EVs to transfer both nucleic acids and proteins to the surrounding normal cells, thus inducing in them transformed behaviours or killing them. G26/24 oligodendroglioma cells, for example, transfer by EVs pro-apoptotic proteins, such as TRAIL and Fas-Ligand [2], extracellular matrix remodelling proteases (such as ADAMTS) [3], and even the H1.0 histone protein [4]. Another tumour cell line, with a different tissue origin (A375 melanoma cells) releases into the medium EVs containing a sumoylated form of H1.0 histone and even H1.0 mRNA [5]. By a T1 RNaseprotection assay, we evidenced, in these EVs, three main H1.0 RNA-protein complexes, the most abundant of which had an apparent molecular mass of about 65 kDa. By affinity chromatography on biotinylated H1.0 RNA, we isolated a group of proteins, then analysed by mass spectrometry. One of these proteins is the myelin expression factor-2 (MYEF2). The presence of this protein in EVs was also confirmed by Western blot analysis [5]. MYEF2 was already known as a transcription factor with putative RNA-binding domains. Our demonstration of its actual ability to bind RNA, together with the well accepted ability of EVs to transfer microRNAs (miRNAs) and long non coding RNAs (lncRNAs), rises the possibility that different classes of RNA can function as carriers of factors that, once in receiving cells, epigenetically change their transcriptional potential. [1] Schiera G, et al. 2015. Biomed Res Int. 2015:152926. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [4] Schiera G et al. 2013, Int J Oncol 43:1771-6 [5] Schiera G. et al. 2016. Int J Oncol. 49:1807-1814.

Most cells release into the extracellular space membrane-bound structures of different sizes, origin and composition, collectively called extracellular vesicles (EVs) [1]. Tumor cells are much more active than normal cells in producing EVs. Because of this property, they are able to transfer both nucleic acids and proteins to the surrounding normal cells, thus inducing in these latter at least some transformed behavior. We previously showed that EVs produced by G26/24 oligodendroglioma cells can horizontally transfer to their neighbours radioactive proteins [2]. In addition, EVs released by these cells contain pro-apoptotic proteins, such as TRAIL and Fas-Ligand, able to induce apoptosis in rat cortical neurons and astrocytes in culture [2]. Moreover these population of EVs also contain extracellular matrix remodelling proteases (such as ADAMTS) [3], as well as the H1.0 histone protein, and even H1.0 mRNA [4]. Another tumor cell line, with a different tissue origin (A375 melanoma cells), was then demonstrated to release into the medium EVs containing a sumoylated form of H1.0 histone [5]. By a T1 RNase-protection assay, using as target the H1.0 RNA, three main complexes were evidenced in these EVs, the most abundant of which had an apparent molecular mass of about 65 kDa. By affinity chromatography on a biotinylated H1.0 RNA, we isolated a group of proteins, among which we identified by mass spectrometry a protein of about 60 kDa: myelin expression factor-2 (MYEF2). Western blot analyses confirmed the presence of MYEF2 in EVs released from A375 melanoma cells [5]. MYEF2 was actually already known as a protein with putative RNA-binding domains and a demonstrated ability to function as a transcription factor. Our demonstration of its actual ability to bind RNA rises the interesting possibility that RNA can function as a carrier of factors that, once in the receiving cells, can change their transcriptional activity. Starting from this hypothesis, we are now analyzing cells of cerebral origin for the presence of MYEF2 and/or other transcription factors which are transferred among cells via EVs. [1] Schiera G, et al. 2015.Biomed Res Int. 2015:152926. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [4] Schiera G et al. 2013, Int J Oncol 43:1771-6 [5] Schiera G. et al. 2016. Int J Oncol. 49:1807-1814.

The release of molecules from damaged tissues stimulates both resident and circulating stem cells to initiate a tissue repair programme. 1 However, during transplantation procedures the therapeutic efficacy of stem cells is compromised by reduced homing capability towards the target site.2,3 Furthermore, cell survival is very low and many studies focused on improving cell viability upon implantation. In this study, we performed in vitrosevere oxidative stress to select some more resistant mouse mesoangioblasts, vessel-associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. We found that the selected subpopulation retains selfrenewal and myogenic differentiation capabilities under physiological growth conditions, showing, also, an enhancement in cell survival and migration capabilities under stress conditions respect to the unselected cells. In fact, following oxidative stress treatment the isolated cell subpopulation showed more resistance, survival and recovery properties. To evaluate whether or not the isolated cell clone showed selective advantages over the parental mesoangioblasts, we carried out in vivo experiments using immunocompromised dystrophic mice. We injected intramuscularly the Tibialis Anterior with both the selected cells and the parental cells. Actually resistant mesoangioblasts displayed markedly enhanced survival and integration capabilities into the host damaged skeletal muscle, displaying more than 70% increase in integration compared to the non-selected mesoangioblast cell population. In conclusion, the positive effects of sorting on mesoangioblast cells suggest that a selection step using oxidative stress preconditioning may provide a novel methodology to select for resistant cells that can be used in regenerative tissue applications to prevent high mortality rates upon transplantation.

Extracellular vesicles (EV) represent an important mediator of cell-to-cell communication and are involved in both autocrine and paracrine signaling, with a critical role in a number of physiological and pathological conditions.1 The bioactive molecules contained within EV simultaneously activate several different pathways resulting in the synergistic stimulation of target cells. The discovery and characterization of EV have added a novel understanding to regenerative medicine, namely the finding that stem cells are an abundant source of EV.1-2 A6 mouse mesoangioblasts, vessel-associated multipotent progenitor stem cells that are capable of differentiating into different mesodermal cell types, are able to release in the extracellular environment membrane vesicles, which contain structural proteins, FGF-2 and the two gelatinases MMP2 and MMP9.3 Moreover, we have already demonstrated that EV released by these cells contain Hsp70 as a transmembrane protein, which is involved in an autocrine signaling responsible for increased cell migration.4 In this study we have investigated the possible paracrine effects of A6 derived EV with other cell types, and the effects of these interactions. Firstly, we have focused our attention on their interaplay capacity with human endothelial cells, which are induced to form capillary-like structures in vitro and to increase their motility. Furthermore, we have analyzed EV immunomodulatory effect on Jurkat lymphocytes, demonstrating that they are able to inhibit both their activation and proliferation. Finally, we have investigated the role of sugar residues on the membrane of A6 derived EV in their interaction with other cell types, by enzymatic removing of N-linked glycans on their membrane. In particular, PNGase F that is responsible for the cleavage between asparagine and GlcNAc in all types of glycan chains induces a substantial reduction in EV-target cell interaction. On the contrary, the use of EndoH, which is responsible for the cleavage between two residues of GlcNAc, increase target cell-EV interaction.

The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune-relevant genes (igm, tcr-β, csfr1 and bd) were measured in the head-kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune-associated genes in the head-kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture.

The main aim of this research was to investigate the biopreservative effects of lemon essential oil (EO) micro-emulsions on salted sardines. The experimental design included two experimental trials, SR1 and SR2 carried out with 25 ml of lemon EO micro-emulsion at 0.3 and 1.0% (v/v), respectively, and a control trial performed without EO addition. Chemical analyses on salted sardines inoculated with the EOs clearly showed a substantial persistence of several volatile organic compounds (VOCs) belonging to groups of monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene hydrocarbons derived from EOs during the entire period of ripening. In particular, the molecules mostly represented were limonene, p-cymene and (beta-pinene. Immediately after the addition of EOs, the concentrations of all microbial groups decreased. The presence of Enterobacteriaceae, staphylococci and rod lactic acid bacteria (LAB) observed in the trials SR1 and SR2 was significantly lower than that registered for the control. trial during the entire period of monitoring. Furthermore, the addition of EOs determined a lower accumulation of histamine in sardines compared to those of the control trial. The highest scores of sensory evaluation were registered for flavour and overall acceptability of the experimental trials in presence of EOs. On the basis of the increasing interest toward novel food preservatives, we conclude that the use of EOs to produce salted fishes represents a valid strategy to improve safety and sensory characteristics of salted sardines. This work has also economic implications, since the flavour improvement due to the addition of lemon EOs might increase the consumption of sardines by regular and new consumers. (C) 2016 Elsevier Ltd. All rights reserved.

SummaryTo select from a single patient the best oocytes able to reach the blastocyst stage, we searched for valuable markers for oocytes competence. We evaluated the DNA fragmentation index (DFI) and the level of some survival molecules, such as AKT, pAKT and pERK1/2, in individual cumulus cell–oocyte complexes (COC). The study included normo-responder women. The average age of the patients was 34.3. DFI in cumulus cells was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assayin situ. AKT, pAKT and pERK1/2 were measured by immunological assay and densitometric analysis of fluorescent signals using NIS-Elements BR 3.10 image software. Statistical analysis was performed using STATA SE/14.1. The study focused on 53 patients involved after informed consent. Out of 255 MII oocytes, 197 were fertilized and the derived embryos had the following evolution: 117 completed the development to blastocyst and were transferred to uterus; 57 were vitrified at the blastocyst stage; and 23 were arrested duringin vitroculture at different stages of cleavage. We found a significant statistical difference between the DFI of cumulus cells of the arrested embryos and the transferred blastocysts (P= 0.004), confirming that DFI could be considered as a valuable marker of oocyte competence. In addition, the pAKT/DFI ratio was higher in cumulus cells of oocytes able to produce blastocysts, indicating that DFI is significantly lower when pAKT is higher (P= 0.043). This study demonstrates for the first time that the relationship between apoptosis and survival molecules can be used as a marker to select the best oocytes.