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Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Various key economic sectors of the National and Sicilian economy, in addition to contributing to the economic growth also cause a considerable production of waste which today are a problem both for companies then for the environment. For this reason, there is considerable interest in this waste and in the possibility of their reuse to extract bioactive molecules with added value, all in accordance with the objectives of the Blue Economy. In this regard, it has already been demonstrated that from the production waste of various economic sectors it is possible to obtain bioactive molecules with anticancer, antimicrobial, cosmetic, antioxidant and nutraceutical properties (Mauro et al., 2022). If we focus on farm animals and the feed used, it is known that the latter can be a source of antioxidants which help the body to prevent the harmful effects of free radicals and metabolic products. In this context, polyphenolic compounds (e.g. flavonoids) showed important antioxidant activities in vitro, although it seems that these are poorly absorbed in the intestine and their tissue concentrations are too low to contribute to the antioxidant defense (Surai, 2013). In light of all this, within the SMILING Project a study concerned the possibility of reusing wine production waste (pomace and grape seeds, the cause of the production of considerable quantities of waste) rich in fatty acids, triglycerides and polyphenols (Di Stefano et al., 2021; 2022) in the feeding of broiler chickens by evaluating the possi- ble effects of these diets on the animal’s health status also in terms of antioxidant capacity. Three experimental sets were carried out and concerned the administration of three different types of diets: Grape marc (0%, 3% and 6%), Grape seeds (0%, 3% and 6%) and a mix of grape marc and grape seed (0 and 3%). At the end of the experimental times, biomarkers typical of the evaluation of the state of health of the organisms were used for the preliminary analyzes of the blood and meat samples. Significant effects were observed in all diets depending on the concentration administered. Probably the phenolic content of the flours used contributes to the improvement of the health of the farmed animals.

Marine species are a rich source of bioactive molecules and among them, it is known that marine algae produce different secondary metabolites for which different biological activities such as: immunomodulatory (Raposo et al., 2016), antioxidant (Fisch et al., 2003) and antimicrobial (Pinteus et al., 2015) were demostrated. The aim of this study was chemical charac- terize the extracts of three macroalgae species: Carpodesmia crinite (Duby, Orellana & Sansón, 2019), Carpodesmia brachy- carpa (J. Agardh, Orellana & Sansón 2019, WoRMS, 2023), Ericaria brachycarpa (J. Agardh, Molinari & Guiry, 2020), Asparagopsis taxiformis (Delile) Trevisan 1845) to evaluate also their biological activities. The characterization of the secondary metabolites was performed by HPLC-MS and showed higher meroterpenoids levels. Then, the extracts were tested on the Arbacia lixula sea urchin and against the bacterial strains Listeria monocytogenes and Staphylococcus aureus. In the first case results showed a modulation in total and differential cell count demonstrating their involvement in immunity responses. In the second case important antimicrobial activities were observed against both bacterial strains tested. The results obtained, although preliminary, are certainly encouraging to understand better the biological potentiality of these metabolites.

Marine species represent a rich source of biologically active products that can be used in various fields. Among them, marine algae produce numerous secondary metabolites responsible for different biological activities such as: immunomodulatory [1], antioxidant [2], and antimicrobial [3]. The aim of this study was chemically characterizing the extracts of three macroalgae species: Carpodesmia crinite (Duby) Orellana & Sansón, 2019, Carpodesmia brachycarpa (J. Agardh) Orellana & Sansón 2019, Asparagopsis taxiformis (Delile) Trevisan 1845 and evaluate their biological activities. The characterization of the secondary metabolites was performed by HPLC-MS and the results obtained showed higher meroterpenoids levels. Moreover, the extracts tested against the Arbacia lixula sea urchin modulate the total and differential cellular count demonstrating their involvement in immunity responses. Furthermore, important antimicrobial activities were observed by testing these extracts against the bacterial strains Listeria monocytogenes and Staphylococcus aureus. For the first time our study shows the effects of macroalgae extracts on the immunomodulatory activity in Arbacia lixula sea urchin and important antimicrobial activity. The results obtained, although preliminary, are certainly encouraging and our purpose is also improved this information performing biochemical and molecular assays of extracts obtained to understand better the potential that these metabolites have towards the sea urchin Arbacia lixula that graze on these macroalga.

A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and translation of the transcripts. Notably, most RBPs contain intrinsically disordered regions (IDRs) that are thought to be involved in the formation of membrane-less structures, probably due to liquid–liquid phase separation (LLPS). Such structures are evidenced as a variety of granules that contain proteins and different classes of RNAs. The other side of the peculiar properties of IDRs is, however, that, under altered cellular conditions, they are also prone to form aggregates, as observed in neurodegeneration. Interestingly, RBPs, as part of both normal and aggregated complexes, are also able to enter extracellular vesicles (EVs), and in doing so, they can also reach cells other than those that produced them.

Brain cancers are complex and heterogeneous; most of them derive from glial cells[1], and are called gliomas, further subdivided into astrocytomas, oligodendrogliomas, ependymomas and glioastrocytomas[2]. The malignant cells undergo modifications of their metabolism and behaviour, and acquire the ability to migrate along the blood vessels in small groups (model of the guerrilla war)[3], thus invading the surrounding brain parenchyma. Most important, they have the capacity to affect the surrounding microenvironment, by altering both the extracellular matrix and the properties of the normal cells present in the brain, including glial-, endothelial-, and immune-cells, further promoting cancer cell growth and migration. Most of these effects are probably due to molecules (nucleic acids, proteins, lipids etc.) released from cancer cells into their environment via extracellular vesicles (EVs)[1]. Although a lot of efforts have been done, over the years, in order to acquire a better understanding of gliomas at the molecular level, and to set combined therapeutic approaches, brain cancers are mostly incurable. In order to gain a better knowledge of the cellular and molecular events that accompany their transformation, we started from primary cultures of rat cortical astrocytes, and selected three clones that showed increasingly high cell division rates. Then we analyzed both normal cells and their clones at the cytogenetic, cytogenomic and epigenetic level, and found that the most modified astrocytes (A-FC6 clone) have epigenetic and chromosomal alterations typical of cancer, such as an isochromosome (i8q) and that the other two clones (A-GS1 and A-VV5) have intermediate properties. We also analyzed the expression of the linker histone H1.0, normally expressed in differentiated cells[4]. Surprisingly, we found that the somatic histone H1.0 steadily increases from normal astrocytes to the A-FC6 clone. These results suggested that the normal cell cultures together with their three clones may constitute a potential model for studying glioma development. We are now analyzing the expression of other proteins normally expressed in higher amount in cancer cells (e.g. PDI and FABP7), and we are studying how much different is the ability of transformed cells to release EVs respect to normal astrocytes. One of the aim of this study is also to identify proteins and RNAs specifically sorted to EVs from both normal and transformed astrocytes.

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

Since the realization that the cellular homologs of a gene found in the retrovirus that contributes to erythroblastosis in birds (v-erbA), i.e. the proto-oncogene c-erbA encodes the nuclear receptors for thyroid hormones (THs), most of the interest for THs focalized on their ability to control gene transcription. It was found, indeed, that, by regulating gene expression in many tissues, these hormones could mediate critical events both in development and in adult organisms. Among their effects, much attention was given to their ability to increase energy expenditure, and they were early proposed as anti-obesity drugs. However, their clinical use has been strongly challenged by the concomitant onset of toxic effects, especially on the heart. Notably, it has been clearly demonstrated that, besides their direct action on transcription (genomic effects), THs also have non-genomic effects, mediated by cell membrane and/or mitochondrial binding sites, and sometimes triggered by their endogenous catabolites. Among these latter molecules, 3,5-diiodo-L-thyronine (3,5-T2) has been attracting increasing interest because some of its metabolic effects are similar to those induced by T3, but it seems to be safer. The main target of 3,5-T2 appears to be the mitochondria, and it has been hypothesized that, by acting mainly on mitochondrial function and oxidative stress, 3,5-T2 might prevent and revert tissue damages and hepatic steatosis induced by a hyper-lipid diet, while concomitantly reducing the circulating levels of low density lipoproteins (LDL) and triglycerides. Besides a summary concerning general metabolism of THs, as well as their genomic and non-genomic effects, herein we will discuss resistance to THs and the possible mechanisms of action of 3,5-T2, also in relation to its possible clinical use as a drug.

In this study we focused on four taxa of the genus Calendula (C. maritima, C. suffruticosa subsp. fulgida, C. arvensis, and the hybrid between the first two ones), collected in Mediterranean area (Sicily). Six extracts for each species were obtained using solvents with increasing polarity (hexane, ethanol 80%, acetone 70%, and water) and through extraction by supercritical fluids (SFE). It has been observed that the solvent with the highest extraction efficiency was ethanol 80% for all species. However, SFE extracts showed high antioxidant activity comparable to the ethanol 80% extract (polyphenol, DPPH, and reducing power method). These findings were confirmed by in vitro analysis (MTT assay) where it was observed that the tested concentration (24 &micro, g/mL), obtained from ethanol 80% and SFE extracts, showed a protective effect comparable to that induced by a synthetic antioxidant. Extraction with SFE ensured a great selectivity by avoiding the use of toxic organic solvents and thus consisted of a promising technique for sustainable production of Calendula extracts.

Microbiological, nutritional and bioactive properties of edible powders obtained from Acheta domesticus (house cricket) and Tenebrio molitor (mealworm) were investigated. Except for the enterobacteria, viable bacteria were at a higher concentration in mealworm flour. The diversity evaluation carried out using MiSeq Illumina that mainly identified Citrobacter and Enterobacteriaceae in mealworm powder and members of the Porphyromonadaceae family in house cricket powder. Enterococci were identified and characterized for their safety characteristics in terms of the absence of antibiotic resistance and virulence. Both powders represent a good source of proteins and lipids. The fatty acid profile of mealworm powder was characterized by the predominance of the monounsaturated fatty acids and house cricket powder by saturated fatty acids. The enzymatic hydrolysis produced the best results in terms of percentage of degree of hydrolysis with the enzyme Alcalase, and these data were confirmed by SDS-PAGE electrophoresis. Furthermore, the results showed that the protein hydrolysate of these powders produces a significant antioxidant power.

Physical activity (PA) has been central in the life of our species for most of its history, and thus shaped our physiology during evolution. However, only recently the health consequences of a sedentary lifestyle, and of highly energetic diets, are becoming clear. It has been also acknowledged that lifestyle and diet can induce epigenetic modifications which modify chromatin structure and gene expression, thus causing even heritable metabolic outcomes. Many studies have shown that PA can reverse at least some of the unwanted effects of sedentary lifestyle, and can also contribute in delaying brain aging and degenerative pathologies such as Alzheimer’s Disease, diabetes, and multiple sclerosis. Most importantly, PA improves cognitive processes and memory, has analgesic and antidepressant effects, and even induces a sense of wellbeing, giving strength to the ancient principle of “mens sana in corpore sano” (i.e., a sound mind in a sound body). In this review we will discuss the potential mechanisms underlying the effects of PA on brain health, focusing on hormones, neurotrophins, and neurotransmitters, the release of which is modulated by PA, as well as on the intra- and extra-cellular pathways that regulate the expression of some of the genes involved.

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co-culture systems.

Opuntia ficus-indica fruit and cladodes are a source of mucilage, a carbohydrate complex, as well as of phytochemicals and other nutrients, useful in the food industry. Despite the environmental-based composition variability has been studied, there is no information on its seasonal variability, which is particularly important for its industrial use. In this study, some technological, nutritional, and bioactive properties of O. ficus-indica cladodes mucilage were analyzed during the seasonal growth period. Total proteins did not change during the whole period and the lipid content significantly decreased from winter to the onset of summer. The fatty acid profile showed high levels of palmitic (16:0), oleic (18:1n9), and linoleic (18:2n6) acids and that the polyunsaturated fatty acid class (PUFA) is the most abundant fatty acid class, which showed a significant increase during the growth period. In this class, the presence of 20:5n-3 (EPA) and 22:6n-3 (DHA), two of the most important human nutrition essential fatty acids, is worth noting as well as the positive effect of the omega-3 series founder, α-linolenic acid 18:3 n-3. Total polyphenol values indicated that extract quality changes with the sampling period and is not closely related to the total extract yield. All the descriptors of the antioxidant power seem to indicate that the best properties are exhibited during the summer. The principal component analysis can significantly help identify the technical, nutritional, and bioactive factors that make it possible to distinguish the quality of O. ficus-indica mucilage, in order to harvest it in the most appropriate season.

The Mediterranean Diet (MD), UNESCO Intangible Cultural Heritage of Humanity, has become a scientific topic of high interest due to its health benefits. The aim of this review is to pick up selected studies that report nutrigenomic or nutrigenetic data and recapitulate some of the biochemical/genomic/genetic aspects involved in the positive health effects of the MD. These include (i) the antioxidative potential of its constituents with protective effects against several diseases; (ii) the epigenetic and epigenomic effects exerted by food components, such as Indacaxanthin, Sulforaphane, and 3-Hydroxytyrosol among others, and their involvement in the modulation of miRNA expression; (iii) the existence of predisposing or protective human genotypes due to allelic diversities and the impact of the MD on disease risk. A part of the review is dedicated to the nutrigenomic effects of the main cooking methods used in the MD and also to a comparative analysis of the nutrigenomic properties of the MD and other diet regimens and non-MD-related aliments. Taking all the data into account, the traditional MD emerges as a diet with a high antioxidant and nutrigenomic modulation power, which is an example of the “Environment-Livings-Environment” relationship and an excellent patchwork of interconnected biological actions working toward human health.

A wide range of contaminants, industrial by-products, plastics, and pharmaceutics belonging to various categories, have been found in sea water. Although these compounds are detected at concentrations that might be considered as sub-lethal, under certain conditions they could act synergistically producing unexpected effects in term of toxicity or perturbation of biochemical markers leading to standard pathway. In this study, the Sparus aurata fibroblast cell line SAF-1, was exposed to increasing concentrations of carbamazepine (CBZ), polybrominated diphenyl ether 47 (BDE-47) and cadmium chloride (CdCl2) until 72 h, to evaluate the cytotoxicity and the expression of genes related to antioxidant defense, cell cycle and energetic balance. In general, both vitality and gene expression were affected by the exposure to the different toxicants, in terms of antioxidant defense and cell cycle control, showing the most significant effects in cells exposed to the mixture of the three compounds, respect to the single compounds separately. The synergic effect of the compounds on the analyzed biomarkers, underlie the potential negative impact of the contaminants on health of marine organisms.

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.

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.

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.

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.

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.

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.

Brain primary tumors are among the most diverse and complex human cancers, and they are normally classified on the basis of the cell-type and/or the grade of malignancy (the most malignant being glioblastoma multiforme (GBM), grade IV). Glioma cells are able to migrate throughout the brain and to stimulate angiogenesis, by inducing brain capillary endothelial cell proliferation. This in turn causes loss of tight junctions and fragility of the blood–brain barrier, which becomes leaky. As a consequence, the most serious clinical complication of glioblastoma is the vasogenic brain edema. Both glioma cell migration and edema have been correlated with modification of the expression/localization of different isoforms of aquaporins (AQPs), a family of water channels, some of which are also involved in the transport of other small molecules, such as glycerol and urea. In this review, we discuss relationships among expression/localization of AQPs and brain tumors/edema, also focusing on the possible role of these molecules as both diagnostic biomarkers of cancer progression, and therapeutic targets. Finally, we will discuss the possibility that AQPs, together with other cancer promoting factors, can be exchanged among brain cells via extracellular vesicles (EVs).

Distribution of aquaporin-4 (AQP4) was studied by western analysis and immunofluorescence in rat astrocytes exposed to either hypothermic (30 °C) or hyperosmolar (0.45 M sucrose) stress, and in the cerebrospinal fluid (CSF) of patients who suffered traumatic brain injury (TBI). CSF was obtained from 5 healthy subjects and from 20 patients suffering from severe TBI. CSF samples were taken at admission and on days 3 and 5-7. Here we report that, in response to both hypothermia and hyperosmolar stress, AQP4 was markedly reduced in cultured astrocytes. We also found that AQP4 significantly increased in patients with severe brain injury in respect to healthy subjects (P < 0.002). AQP4 in CSF remained unchanged in patients with elevated intracranial pressure (ICP), whereas there was a clear tendency to further increase in those patients whose ICP could be controlled within the normal range. We conclude that AQP4 levels in CSF are elevated after TBI and it might serve as a useful biochemical marker to assess brain water metabolism in clinical settings.

H1 linker histones are a class of DNA-binding proteins involved in the formation of supra-nucleosomal chromatin higher order structures. Eleven non-allelic subtypes of H1 are known in mammals, seven of which are expressed in somatic cells, while four are germ cell-specific. Besides having a general structural role, H1 histones also have additional epigenetic functions related to DNA replication and repair, genome stability, and gene-specific expression regulation. Synthesis of the H1 subtypes is differentially regulated both in development and adult cells, thus suggesting that each protein has a more or less specific function. The somatic variant H1.0 is a linker histone that was recognized since long ago to be involved in cell differentiation. Moreover, it has been recently found to affect generation of epigenetic and functional intra-tumor heterogeneity. Interestingly, H1.0 or post-translational forms of it have been also found in extracellular vesicles (EVs) released from cancer cells in culture, thus suggesting that these cells may escape differentiation at least in part by discarding H1.0 through the EV route. In this review we will discuss the role of H1.0 in development, differentiation, and stem cell maintenance, also in relation with tumorigenesis, and EV production.

Streptomycetes are gram-positive bacteria that produce about 2/3 of pharmaceutically active secondary metabolites, such as antibiotics, and anti-tumor, immunosuppressive, antifungal and antiparasitic agents. In this study, we investigated the possible effects of Streptomyces coelicolor extracts and putative vesicular fraction on primary cultures of rat astrocytes, in both physiological and stressed conditions, induced by treatment with hydrogen peroxide. Briefly, crude extracts and putative vesicular fractions were prepared from two S.coelicolor strains (M145 wild-type strain and bold F166 strain), and used to treat primary astrocytes, which were then also treated with hydrogen peroxide. Data obtained show that both the general protein pattern of S.coelicolor and putative vesicular fraction vary at different times of growth. M145 strain vesicles and the crude extracts from both strains have a protective effect on stressed astrocytes. The protective effects are higher with the vesicular fraction from bacteria at 18 and 72h of growth. The 166 strain crude extract has a greater protective effect than M145 strain, especially in the initial stages of growth: 18h and 24h. Although they need further studies, these observations are very promising and suggest that, in the future, molecules produced by these bacteria may offer a protection against the high ROS production observed in many pathological conditions, such as in neurodegenerative diseases.

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body. In this communication we report setting of three-dimensional structures able to mimic the extracellular matrix of the nervous system: we prepared Poly-L-Lactic Acid (PLLA) porous scaffolds via thermally induced phase separation (TIPS), and investigated the parameters that influence porosity, average pore size and degree of interconnection, i.e. polymer concentration, temperature and time of process. Astrocytes and brain capillary endothelial cells (BCECs) were cultured on these three-dimensional structures and tested for their ability to grow and survive on PLLA scaffolds. We analyzed in parallel the cell growth in 2D and 3D culture systems and observed the differences in cell morphology by fluorescence analysis: three-dimensional scaffolds have the ability to guide cell growth, provide support, encourage cell adhesion and proliferation. Astrocytes and BCECs adapted well to these porous matrices, not only remaining on the surface, but also penetrating inside the scaffolds. This 3D cell culture system could be further enriched to host two or three different brain cell types, in order to set an in vitro model of blood brain barrier, that may be useful for drug delivery studies, and for the formulation of new therapeutic strategies, to be used for the treatment of neurological diseases.

Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies.

Tyrosine nitration of proteins is emerging as a post-translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor-induced neuronal differentiation, we now find that nitration occurs on the microtubule-associated protein tau. In differentiated PC12 cells, we have identified as tau a nitrated protein that co-immunoprecipitates with alpha-tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of tau occurs in a physiological process and suggests that it could play a role in neuronal differentiation.

Extracellular vesicles (EVs) are shed in the extracellular environment by both prokaryotes and eukaryotes. Although produced from both normal and cancer cells, malignant cells release a much higher amount of EVs, which also contain tumor-specific proteins and RNAs. We previously found that G26/24 oligodendroglioma cells shed EVs that contain the pro-apoptotic factors FasL and TRAIL1-2. Interestingly, G26/24 release, via EVs, extracellular matrix remodelling proteases3, and H1° histone protein4, and mRNA. To shed further light on the role of EVs in discarding proteins and mRNAs otherwise able to counteract proliferative signals, we studied a melanoma cell line (A375). We found that also these cancer cells produce H1° and release it into the extracellular space by EVs. Interestingly, H1° sorted to vesicles has a molecular mass higher than expected, and is probably sumoylated. By T1 RNase-protection assay with the H1° RNA, three main complexes were evidenced in EVs, the most abundant of which has a molecular mass of about 65 kDa. By using a biotinylated H1° RNA to fish interacting factors, we isolated from EVs a few proteins which have been then identified by mass spectrometry: the most abundant is 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. 1. D’Agostino et al. 2006, Int J Oncol 29:1075-85. 2. Lo Cicero A et al. 2011, Int J Oncol 39:1353-57. 3. Lo Cicero A et al. 2012, Matrix Biol 31: 229-33. 4. Schiera G et al. 2013, Int J Oncol 43: 1771-76.

Extracellular vesicles (EVs) are produced by most prokaryotic and eukaryotic cells; tumour cells, however, release much higher amounts of EVs, which contain cancer-specific proteins and RNAs. Molecules carried by EVs are captured by surrounding cells, which then undergo profound phenotypic modifications. G26/24 oligodendroglioma cells release, for example, EVs containing FasL and TRAIL, which induce apoptosis in rat cortical neurons and astrocytes in culture. By metabolic labelling of cells, EV-mediated horizontal transfer of radioactive proteins was clearly demonstrated. Among the proteins present in EVs produced by oligodendroglioma cells, extracellular matrix remodelling proteases, and the linker histone H1°, a differentiation-specific histone, were identified. We also found that A375 melanoma cells release EVs which, like the once produced by oligodendroglioma cells, contain H1°. Interestingly, H1° histone sorted to vesicles has a molecular mass higher than expected, and is probably sumoylated. More recently, by a T1 RNase-protection assay, done by mixing an in-vitro transcribed, H1°- encoding RNA, and EVs, three main RNA-protein complexes were evidenced, the most abundant of which had an apparent molecular mass of about 65 kDa. We then synthesized a biotinylated H1° RNA, with the aim to fish, by affinity chromatography, the evidenced proteins. The RNA-bound fraction was finally analysed by mass spectrometry. The most abundant protein identified was the myelin expression factor-2 (MYEF2), which has indeed a molecular mass of about 60 kDa. The presence of MYEF2 in EVs released from A375 melanoma cells was finally confirmed by Western blot analysis.

INTRODUCTION: Post-transcriptional regulation of gene expression relies on RNA-binding proteins (RBPs), which regulate intracellular transport, stability, and translation of mRNAs [1]. We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 histones [2-5]. All these proteins are probably part of a ribonucleoprotein particle [6]. Here we report more details on the expression and intracellular localization of some of these RBPs, during rat brain development and in isolated rat astrocytes. METHODS: Affinity chromatography was performed as already described [6]. Preparation of total lysates and cellular sub-fractions was done as reported in [3]. Possible co-localization of Hsc70 with CSD-C2 in cultured astrocytes was analysed by immunofluorescence microscopy. RESULTS: The presence of Hsc70 chaperone in the already identified ribonucleoprotein complex [6] was confirmed by affinity chromatography. We also found that the complex itself is present not only in the nuclear extracts, but also in the cytoplasmic fraction. Moreover, A1 and K hnRNPs, previously found in the complexes, were found to be differentially expressed and localized during rat brain maturation; an increase in A1 expression was also demonstrated in cultured astrocytes grown on a fibronectin-containing substrate. We finally report that sumoylated PIPPin, already found in neurons, is also present in the nuclei of cultured astrocytes. CONCLUSIONS: We confirmed the existence of a group of proteins able to interact with H1°/H3.3 mRNAs. These proteins are, however, differentially expressed during brain maturation and also show different subcellular localization. [1] Di Liegro et al. 2014, Int J Mol Med 33:747-62. ; [2] Scaturro et al. 1998, J Biol Chem 273:22788-91.; [3] Nastasi et al. 1999, J Biol Chem 274:24087-93. [4] Sala et al. 2007, Int J Mol Med 19:501-9. [5] Saladino et al. 2012, Int J Mol Med 29:141-5 [6] Di Liegro et al. 2013, Neuroscience 229:71-6.

We previously demonstrated that G26/24 oligodendroglioma cells release EVs that contain proteins, such as FasL and TRAIL, which induce apoptosis in rat cortical neurons [1] and astrocytes [2]. We also reported that cancer cells use EVs for transferring, into the environment [3], proteins such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. In particular, by releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used as a model A375 melanoma cells. METHODS EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait. RESULTS We found that also melanoma cells synthesize H1° and secrete it via EVs. Interestingly, H1° histone sorted to vesicles is probably sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot. CONCLUSIONS We demonstrated that EVs released from melanoma cells contain the H1° linker histone, which is probably sumoylated before sorting to the vesicles. In addition, EVs contain H1° RNA-binding proteins, one of which seems to be MYEF2. [1] D’Agostino et al. 2006, Int J Oncol 29:1075-85. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Di Liegro et al. 2015, Biomed Res Int, in press [4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [5] Schiera G et al. 2013, Int J Oncol 43:1771-6 [6] Scaturro et al. 1998, J Biol Chem 273:22788-91

Post-transcriptional regulation of gene expression depends on RNA-binding proteins (RBPs), which are able to regulate translation, stability and subcellular localization of mRNAs [1]. RNA-protein complexes start to be built up since transcription; some proteins remain then bound to the transcript, while others behave as only transient components. In the developing nervous system of mammals, the postnatal production of the histone variants H1° and H3.3 is mainly regulated at the post-transcriptional level. Synthesis and incorporation into chromatin of the two histone proteins has been suggested to be involved in the epigenetic regulation of gene expression, both in normal brain development and brain cancer. We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 mRNA [2-5]. Recently, we demonstrated that these proteins are probably part of a ribonucleoprotein particle which contains both unspecific and specific RBPs [6]. Here we confirm, by affinity chromatography, the presence of Hsc70 chaperone in the complex and, in addition, we report that the complex is present not only in the nuclear extracts, as previously shown, but also in a membrane-containing cytoplasmic pellet, obtained by centrifugation at 105.000 x g (P105). In parallel, we demonstrated that sumoylated PIPPin, already found in neurons, is present also in the nuclei of cultured astrocytes. Finally, we found that two of the hnRNPs previously found in the complexes show a differential expression and localization during rat brain maturation. [1] Di Liegro CM et al. 2014, Int J Mol Med 33:747-62. [2] Scaturro M et al. 1998, J Biol Chem 273:22788-91. [3] Nastasi T et al. 1999, J Biol Chem 274:24087-93. [4] Sala A et al. 2007, Int J Mol Med 19:501-9. [5] Saladino P et al. 2012, Int J Mol Med 29:141-5 [6] Di Liegro CM et al. 2013, Neuroscience 229:71-6.

Extracellular vesicles (EVs) are released into the extracellular space from both tumor and normal brain cells. By releasing EVs which contain FGF2 and VEGF1-2, astrocytes and neurons, co-cultured with brain capillary endothelial cells, are for example able to induce them to form a blood-brain barrier-like monolayer. On the other hand, membrane microvesicles (MVs) shed from G26/24 oligodendro­glioma cells, when added to primary cultures of rat cortical neurons, induce neuronal damage; the damaging effects include a strong reduction of neurite outgrowth, and apoptosis in about 75% of the cells3. The same amount of shed MVs induce apoptosis in about 40% of astrocytes4. These effects are probably due to Fas Ligand and TRAIL, two proteins, present in G26/24 vesicles, with well-known cell death inducing ability5-6. EV-mediated horizontal transfer of labeled proteins from oligodendroglioma cells to astrocytes in culture was also noticed4. We found that, in cultured astrocytes, as previously found in developing rat brain, the amount of the H1° linker histone increases during differentiation, while the level of its mRNA decreases, suggesting that its expression is mainly regulated at the post-transcriptional level7. On the other hand, G26/24 maintain high levels of both H1° protein and mRNA. We recently found that these tumor cells release both H1° protein and mRNA, through EVs, into the culture medium8. We suggest that G26/24 oligodendroglioma cells, and possibly other tumor cells, can escape differentiation cues, and continue to proliferate by eliminating proteins, such as the H1° linker histone (and its mRNA) into the extracellular space. Schiera G et al. J Cell Mol Med 2007, 138-94. Proia P et al. Int J Mol Med 2008, 21: 63-7. D’Agostino S et al. Int J Oncol 2006, 29:1075-85. Lo Cicero A et al. Int J Oncol 2011, 39: 1353-57. Albanese J et al. Blood 1998, 91: 3862-74. Abrahams VM et al. Cancer Res 2003, 63: 5573-81. Castiglia D et al. Neurochem Res 1994, 19:1531-37. Schiera G et al. Int J Oncol. 2013, 43:1771-76.

Release of extracellular vesicles (EVs) is a process conserved from prokaryotes to eucaryotes. Although EVs are produced from both normal and cancer cells, malignant cells release a much higher amount of EVs, which contain tumour-specific proteins and RNAs. We previously found that G26/24 oligodendroglioma cells shed EVs that contain the pro-apoptotic factors FasL and TRAIL and are able to inhibit neurite outgrowth, and induce apoptosis in about 75% of rat cortical neurons [1] and 40% of astrocytes [2] in culture. By labelling proteins synthesized in one cell type, we also demonstrated EV-mediated horizontal transfer of proteins among brain cells. Interestingly, G2624 release, via EVs, extracellular matrix remodelling proteases [3], and H1° histone protein [4]. We suggested that by releasing H1°, a differentiation-specific histone, cancer cells may escape differentiation cues [4]. To shed further light on the role of EVs in discarding proteins and mRNAs otherwise able to counteract proliferative signals, we studied a melanoma cell line (A375). We found that also these cancer cells produce H1° and release it into EVs. Interestingly, H1° sorted to vesicles shows a molecular mass higher than expected, and is probably sumoylated. By T1 RNase-protection assay with the H1° RNA, three main complexes were evidenced in EVs, the most abundant of which has a molecular mass of about 65 kDa. By using a biotinylated H1°RNA to fish interacting factors, we isolated from EVs a few proteins which have been then identified by mass spectrometry: the most abundant is a protein of about 60 kDa, recognized as myelin expression factor-2 (MYEF2). Western blot analyses confirmed the presence of MYEF2 in EVs released from A375 melanoma cells. [1] D’Agostino et al- 2006. Int J Oncol 29:1075-85. [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

G26/24 oligodendroglioma cells produce EVs that contain pro-apoptotic proteins, such as FasL and TRAIL, able to induce neuronal- [1] and astrocytic- [2] death. Cancer cells release EVs [3] through which transferring proteins, such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. By releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used A375 melanoma cells. EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait [7]. Melanoma cells were found to synthesize H1° and secrete it via EVs. Moreover, EVs also contain H1° mRNA. Interestingly, H1° histone sorted to vesicles seems to be sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot. [1] D’Agostino et al. 2006, Int J Oncol 29:1075-85. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Di Liegro et al. 2015, Biomed Res Int, 2015, Article ID 152926. [4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [5] Schiera G et al. 2013, Int J Oncol 43:1771-6 [6] Scaturro et al. 1998, J Biol Chem 273:22788-91 [7] Scaturro et al. 2003, Int J Mol Med 11:509-511

Post-transcriptional regulation of messenger RNA (mRNA) metabolism and subcellular localization is of the utmost importance both during development and in cell differentiation. Besides carrying genetic information, mRNAs contain cis-acting signals (zip codes), usually present in their 5'- and 3'-untranslated regions (UTRs). By binding to these signals, trans-acting factors, such as RNA-binding proteins (RBPs), and/or non-coding RNAs (ncRNAs), control mRNA localization, translation and stability. RBPs can also form complexes with non-coding RNAs of different sizes. The release of extracellular vesicles (EVs) is a conserved process that allows both normal and cancer cells to horizontally transfer molecules, and hence properties, to neighboring cells. By interacting with proteins that are specifically sorted to EVs, mRNAs as well as ncRNAs can be transferred from cell to cell. In this review, we discuss the mechanisms underlying the sorting to EVs of different classes of molecules, as well as the role of extracellular RNAs and the associated proteins in altering gene expression in the recipient cells. Importantly, if, on the one hand, RBPs play a critical role in transferring RNAs through EVs, RNA itself could, on the other hand, function as a carrier to transfer proteins (i.e., chromatin modifiers, and transcription factors) that, once transferred, can alter the cell's epigenome.

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes [1]. The role of this process in tumorigenesis and tumor progression is controversial: in the early stages, it can block tumor growth and conversely it can promote its progression in the later stages [2]. The tumor suppressor BECN1 gene, encodes the protein Beclin 1, a marker of autophagy down-regulated in several types of cancer, such as colorectal cancer [3]. There are a lot of both genetic and environmental risk factors for colorectal cancer, including diet: for this reason, in accordance with epidemiological studies, consumption of foods rich in phytochemicals is widely promoted. The betalain indicaxantin (Ind) is a phytochemical from the Opuntia Ficus-Indica fruit having several biological activities, such as antioxidant, anti-inflammatory. It showed antiproliferative and proapoptotic effects in colorectal adenocarcinoma (Caco2) cells where was able to regulate gene expression through modulation of methylation state of DNA at CpG islands [4]. For the first time, using Methylation-Sensitive Restriction Endonuclease PCR (MSRE-PCR), we report that Ind (50 e 100 µM) decreases the methylation of BECN1 promoter in Caco2 cells, to the same extent as 5-azacytidine (Zcyd, positive control). Interestingly, colorimetric detection of DNA Methyltransferases activity, indicates that Ind reduced the activity of these enzymes, like Zcyd did. These preliminary data, indicating that Ind is able to decrease the methylation of BECN1 gene, allow us to propose an epigenetic hypothesis of autophagy regulation in Caco2 cells.

Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5' or 3' to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3'-untranslated (3'-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (. Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1. mg/l to 1. mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (. p

Synthesis of H1° histone, in the developing rat brain, is also regulated at post-transcriptional level. Regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs); therefore, we searched for H1° mRNA-interacting proteins. With this aim, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of cold shock domain-containing protein 2 (CSD-C2, also known as PIPPin), a brain-enriched RBP previously described in our laboratory. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70. Overall, we have contributed to the molecular characterization of a ribonucleoprotein particle possibly controlling H1° histone expression in the brain.