Purpose: Exosomes (EXOs) mediate local and systemic cell-tocell communication and regulate cell behavior by transferring mRNA, miRNAs and proteins to recipient cells. Recently, we demonstrated that bone marrow (BM) cancer associated fibroblasts (CAFs) promote tumor progression and drug resistance (DR) in multiple myeloma (MM) (1-3). In this study, we analysed the effect of MM-derived EXOs on CAFs in MGUS to MM transition and, in turn, the effect of CAFs-derived EXOs on endothelial (ECs) and MM cells. Methods: EXOs isolation was performed from conditioned medium (CM) of CAFs purified from BM aspirates of 8 active MM patients and from CM of cultured RPMI8226 and U266 MM cells. Electron microscopy (TEM), dual immunofluorescence-confocal laser-scanning microscopy, western blot (WB), flow cytometry (FC) and qRT-PCR studies were performed to evaluate the CAFsand MM-derived EXOs phenotypes, their miRs content and their mutual effect. Results: TEM analysis of CAFs- and MM-derived EXOs showed a vesicles population with heterogeneous aspect, 50-100 nm sized. WB analysis defined the expression of commonly used EXOs surface markers as CD63, Hsp70 for CAFs and CD63, Hsp70, Alix for MM cells. Confocal microscopy showed the ability both CAFs and MM cells to uptake respectively MM- and CAFs-derived EXOs labelled with fluorescent dyes. Functional studies showed that MM-derived EXOs induce a specific miRNA profile as overexpression of miR-27b-3p, -125b-5p, -214-3p and activated phenotype, as expression of FAP+ and SMA+ antigens, in MGUS and MM CAFs. In turn, MM CAFs released EXOs containing miR-27b-3p, -125b-5p, -214-3p are swallowed by MM cells and ECs. Functional studies also showed that CAFsderived EXOs are able to stimulate angiogenic ability in ECs and to induce bortezomib-resistance in MM cells. Discussions and Conclusions: Overall results suggest an important exosomal crosstalk among tumor cells, CAFs and ECs which lead to BM microenvironmental modifications, favouring MM progression and DR. References 1. Frassanito MA et al. Leukemia 2014; 28: 904-16. 2. Frassanito MA et al. Leukemia 2016; 30: 640-8. 3. Di Marzo L et al. Oncotarget 2016; 7: 60698-711.

The reinforcement of the defense mechanism of fish, through the administration of immunostimulants, is considered as a promising alternative to vaccines. Natural immunostimulants such as polyphenols, flavanoids, pigments and essential oils can modulate the innate immune response. In lower vertebrates, melano-macrophage centres, i.e. clusters of pigment-containing cells forming the extracutaneous pigment system, are wide-spread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. In the present study, we evaluated the effect of a polyphenol-enriched diet on the health status of European sea bass (Dicentrarchus labrax L.). Farmed sea bass were administered a feed containing a phytocomplex, rich in catechins and epigallocatechins, which was obtained from the seeds of Canosina Nero di Troia Vitis vinifera and mixed with conventional feed at two different concentrations. The effects of such a diet were investigated in juvenile and commercial size samples, i.e. undergoing a short- and long-term period of diet, respectively, focusing on their extracutaneous pigmentary system and, in more detail, on the enzymatic activities leading to melanin biosynthesis. Our results show that prolonged dietary treatments with higher concentration of polyphenols might modulate tyrosinase activity and gene expression in commercial size fishes. An increase of melano-macrophage activity is correlated to a stimulation of cytoprotective functions against antigenic stimulants and pathogens, as an expression of a robust and protective adaptive immune response.

Polyphenols, as food antioxidants, are of great interest due to their health benefits as they decrease the risks of cancer and coronary cardiopathy (1). Moreover they influence the quality and organoleptic characteristics of foods (2). Lastly, some neurotransmitters are phenolic compounds. Hence the need to work out a sensitive, portable and inexpensive detection methods to monitor these compounds (3). We developed a disposable paper-based bioassay for the detection of phenolic compounds; the assay was successfully applied for the determination of polyphenols in a real matrix such as wine. The colorimetric quantification of the analyte is based on an enzymatic assay. The tyrosinase enzyme has been immobilized on a filter paper by simple over-spotting with 3-methyl-2-benzothiazolinone hydrazone (MBTH), that allows the detection of phenols by forming stable colored adducts with their enzymatic oxidation products. The color intensity of the adduct (developed after 5 min of reaction) was found to increase proportionally with the increase of the phenolic substrate concentrations. Analyte detection can be achieved by eye and quantification can be simply obtained by using a camera phone and an image analysis software. The response, characteristics of the sensor were determined using l-3,4-dihydroxyphenyl-alanine (l-DOPA), an archetype substrate of tyrosinase, as the analyte. This disposable paper-based biosensor relies on a rapid and simple method, without need of sophisticated instrumentation and trained personnel and could be extremely useful in remote locations or developing countries which does not have ready access to laboratory facilities and where simple, sensitive and low cost bioassays are essentials. 1) D. Del Rio, L.G. Costa, M.E. Lean, A. Croizer. Nutr. Metab. Cardiovasc. Dis. 20 (2010) 1-6. 2) I. Ignat, I. Volf, V.I. Popa. Food Chem. 126 (2011) 1821-1835. 3) M. Arciuli, G. Palazzo, A. Gallone, A. Mallardi. Sensors & Actuators: B. Chemical (2013)In press.

In this study the development of a low cost, portable and disposable paper-based bioassay for phenolic compounds is described. The colorimetric detection of the analyte is based on an enzymatic assay. The tyrosinase enzyme has been immobilized on a filter paper by simple over-spotting with 3-methyl-2-benzothiazolinone hydrazone (MBTH), that allows the detection of phenols by forming stable colored adducts with their enzymatic oxidation products. The color intensity of the adduct (developed after 5 min of reaction) was found to increase proportionally with the increase of the phenolic substrate concentrations. Analyte detection can be achieved by eye and quantification can be simply obtained by using a camera phone and an image analysis software. The response characteristics of the sensor were determined using L-3,4-dihydroxyphenyl-alanine (L-DOPA), an archetype substrate of tyrosinase, as the analyte. The sensor gave a LOD of 5 M and a linear response up to 0.5 mM. The polyphenol content in wine was determined by the biosensor and results were compared with that obtained for the phenolic-pool determined by tyrosinase enzymatic assay in solution and for the antioxidant-pool probed by the Folin-Ciocalteau assay. This paper based platform holds potential for the detection of different types of phenolic compounds. The proposed assay has the advantage of rapidity and simplicity over other detection methods, without need of sophisticated instrumentation and trained personnel.

FAO and WHO encourage studies that implement in vitro experimental protocols to pre-screen novel probiotics and/or attest the efficacy of claimed probiotic strains [1]. To be effective, orally-delivered probiotics should reach the human intestine in relatively high numbers and in a viable state; therefore, they have to tolerate the stresses associated to the gastro-intestinal (GI) environment. All along the different GI sections, bacteria are challenged by several sources of stress, including the action of digestive enzymes, low pH and emulsifying bile salts. Bacterial cells are naturally equipped with various defense mechanisms to enhance survival in hostile environments [2]; these include chaperones to assist the re-folding of denatured proteins and proteases which degrade irreversibly damaged proteins. The food matrix used to deliver microbes may considerably contribute to their probiotic action, e.g. by enhancing survival to stress, stimulating selective growth and favouring gut colonization. Lactobacillus plantarum (Lp) is a widespread lactic acid bacterium which is recognized as a probiotic and is traditionally employed as a food starter. The stress tolerance of Lp WCFS1 was examined in a previously developed in vitro system that simulates the human GI tract [3]. Different carrier matrices were used to assess their protective and buffering properties. Higher survival was observed for bacteria included in complex and/or nutrient-rich matrices, and when potential prebiotics were added, thus highlighting the relevance of matrix composition in shielding and growth-promoting effects. The molecular response of Lp WCFS1 to the simulated GI system was analysed by studying the transcriptional pattern of bacterial genes involved both in stress response (i.e. chaperones, shsp, proteases) and in exerting beneficial effects on the host (i.e. bacteriocins, adhesion factors). Transcription of bacterial stress-related genes remarkably matched the extent of stress during the GI transit, as revealed by the observed survival rate. Indeed, the GI steps of higher mortality corresponded to major induction of typical stress genes, thus implying their involvement in the mechanisms of cellular adaptation to GI stress. GI environment consistently up-regulated probiosis-associated genes. Data obtained with the different vehicle matrices may be valuable for the development of protective and host/bacteria-friendly carriers in the design of functional food. Transcriptional analysis paves the way to the use of some bacterial genes as molecular markers for the screening of strains with potential probiotic applications. Our results also suggest cues to improve reliability and performance of in vitro GI simulators. References 1. FAO/WHO (2002) ftp://ftp.fao.org/es/esn/food/wgreport2.pdf. 2. van de Guchte M. et al. (2002) Antonie Van Leeuwenhoek 82, 187 - 216. 3. Bove P. et al. (2012) Appl. Microb. Biotechnol. DOI 10.1007/s00253-012-4031-2.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

Plant extracts are a rich source of bioactive compounds and have been traditionally used for both cosmetic and therapeutic purposes. In plants and fungi, the presence of a variety of phenolic compounds leads to the undesirable browning of fruits and vegetables (1). In humans, disorders in the amount and distribution of melanin pigments can be associated to disease states. Plant-derived chemicals have been shown to exert various biological activities and pharmacological effects (2). Because tyrosinase catalyzes the key steps of melanogenesis, most of the strategies for developing skin-lightening and/or anti-food-browning agents are based on the inhibition of tyrosinase activity (i.e. oxidation of L-DOPA) (3). In this study, the essential oils obtained by hydrodistillation from nine different plants were investigated for their potential inhibitory activity on mushroom tyrosinase (MT) and on B16 melanoma cell line tyrosinase (MCLT). The essential oils composition was assessed by GC-mass-spectrometry techniques, anyway we decided to investigate their possible anti-tyrosinase activity considering the oil in its entirety because the biological activity of plant extracts often derives from the combined effect of their different components (4,5). Our results indicate a different inhibitory efficacy on MT and MCLT activities. While extracts from Cinnamonum zeylanicum, Syzigium aromaticum and Citrus aurantium amara appear to be the best inhibitors of both MT and MCLT, with an oil dose-dependent effect, oils from Origanum vulgaris and Rosmarinus officinalis have major inhibitory effect on MT. Our findings indicate that cinnamon, clove and bitter orange essential oils have high potentials in applications as skin-whitening agents of natural source. (1) Martinez M.V. et al. Trends Food Sci Technol 1995, 6: 195-200. (2) Craig W.J. Am J Clin Nutr 1999, 70: 491S-9S. (3) Briganti S. et al. Pigment Cell Res 2003,16: 101-110. (4) Burt S. Int J Food Microbiol 2004, 94: 223-253.

The quality of fresh-cut fruit and vegetable products includes a combination of attributes, such as appearance, texture, and flavor, as well as nutritional and safety aspects that determine their value to the consumer. Nutritionally, fruit and vegetables represent a good source of vitamins, minerals, and dietary fiber, and fresh-cut produce satisfies consumer demand for freshly prepared, convenient, healthy food. However, fresh-cut produce deteriorates faster than corresponding intact produce, as a result of damage caused by minimal processing, which accelerates many physiological changes that lead to a reduction in produce quality and shelf-life. The symptoms of produce deterioration include discoloration, increased oxidative browning at cut surfaces, flaccidity as a result of loss of water, and decreased nutritional value. Damaged plant tissues also represent a better substrate for growth of microorganisms, including spoilage microorganisms and foodborne pathogens. The risk of pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in fresh-cut produce are Listeria monocytogenes, pathogenic Escherichia coli mainly O157:H7, and Salmonella spp. This article describes the quality of fresh-cut produce, factors affecting quality, and various techniques for evaluating quality. In addition, the microbiological safety of fresh-cut produce and factors affecting pathogen survival and growth on fresh-cut produce are discussed in detail.

FAO and WHO guidelines define as 'probiotics' those microorganisms which confer a health benefit to the host. Most of the currently used probiotics belong to the genera of Lactobacillus and Bifidobacterium. Lactobacillus plantarum (Lp) is a lactic acid bacterium (LAB) which grows on various substrates, including animal and vegetable foods (either as autochthonous contamination or being added as starter culture), soil and mammalian intestine. We have generated Lp WCFS1 mutant strains which are defective for different stress response genes. Preliminary works suggest that some of these strains might present distinctive cell surface physico-chemical and morphological features. Because bacterial cell envelope is primarily implicated in cell-host interaction, with the aim to evaluate their potential probiotic properties, we have compared the effects of wild type and mutant L. plantarum cells on the expression pattern of some immune-related genes in human intestinal epithelial cells. Caco-2 monolayers were incubated with bacterial cells at defined titre; transcriptional profiling of human immune genes was assessed by real time quantitative RT-PCR

Dietary probiotics should reach the intestine viable and in high numbers; therefore, they should tolerate the stress associated to the gastro-intestinal (GI) environment. Indeed, all along the different GI sections, probiotics are challenged by several sources of stress, including low pH, bile and digestive enzymes. Bacterial cells are equipped with various defense mechanisms to allow survival in hostile environments. The food matrix used to deliver beneficial bacteria may contribute to their probiotic action, e.g. by enhancing survival to stress and gut colonization. In this study, the survival of the lactic acid bacterium Lactobacillus plantarum WCFS1, a model probiotic strain, was examined in a human oro-gastric-intestinal (OGI) in vitro system, using different carrier matrices to compare protective and buffering properties. Higher survival was observed in complex and/or nutrient-rich matrices, and when potential prebiotics were added. The molecular response of L. plantarum to the OGI transit was analyzed by studying the transcriptional levels of genes involved in stress response and probiosis. The OGI steps of higher mortality corresponded to greater induction of stress genes, thus implying their involvement in adaptation to the gut environment. Plantaricins were significantly upregulated all along the different OGI sections; adhesion genes were mainly induced by gastric environment.

Screening and characterization of novel tyrosinase inhibitors is useful for applications in food technology, cosmetics and medicine. The hydrodistilled essential oils from lavender Lavandula spica L. and peppermint Mentha x piperita L. were investigated for tyrosinase inhibitory activity. Their composition was assessed by gas chromatography–mass spectrometry. Both oils inhibited mushroom tyrosinase in a dose dependent manner. The IC50 values were estimated, kinetics were analysed and Ki values were determined. Our results indicate that lavender and peppermint essential oils may be promising herbal ingredients for developing depigmenting agents in clinical, cosmetic and industrial processes.

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes distinct embryonic origin. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyer’s patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature (1) adversely affects the quality of fish products and is a problem for the aquaculture industry. Moreover, a close relationship between nutrition products and human health has been widely demonstrated. Liver, spleen, kidney and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes (2). In teleosts, melanin-containing cells, named melano-macrophages, occur in the reticulo-endothelial system and are mainly located in kidney and spleen. In these organs, the pigment content may increase in different physiological conditions and as a response to pathological and inflammatory states (2). With the aim to reveal novel functions and behaviour of the poorly understood teleost extracutaneous pigment system, we undertook the investigation of aspects of the melanogenic systems of Atlantic salmon tissues as well as of leukocyte salmon head-kidney cells (SHK-1 cells). SHK-1 cells is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney (3). Using a radiometric method we demonstrated that a tyrosinase activity is present in SHK-1 cells and salmon tissues. Western blotting showed the presence of a tyrosinase protein. Moreover differing melanin-producing activities in salmon tissues were demonstrated suggesting that pigmented cells in the organs undergo a tissue-specific differentiation. These results were confirmed by a tyrosinase protein expression in lymphoid organs, also highlighted by immunohistochemical analysis. As teleosts lack lymph nodes and Peyer’s patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs (4). The detection of tyrosinase activity in lymphoid organs indicates that a link may exist between the extracutaneous pigmentary system and the immune system in salmon. The extracutaneous melanogenetic system could be used as a biomarker for the health status of farmed fishes. References: 1. Koppang, E.O., Haugarvoll, E., Hordvik, I., Aune, L., Poppe, T. (2005) Journal of Fish Diseases 28: 13-22. 2. Agius, C., Roberts, R.J. (2003) Journal of Fish Diseases 26: 499-509. 3. Dannevig, B., Falk, K., Narmork, E. (1995) Journal of General Virology 76: 1353-1359. 4. Zapata, A., Amemiya, CT. (2000) Curr Top Microbiol Immunol 248: 67-107.

Bright Field Microscopy and Atomic Force Microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of L-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100 nm wide and high, and granular aggregates, ~50 nm high and 100 nm wide, are found on both substrates, whereas point-like deposits less than 10 nm high and less than 50 nm wide are found on mica substrate. Dynamic Light Scattering measurements and Atomic Force Microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface.

Determination of phenolic derivatives is very important in medical, food and environmental samples because of their relevant significance in health care and pollution monitoring. Tyrosinase-based biosensors are promising tools for this purpose because of several advantages with respect to currently used detection methods. A key aspect in the development of a biosensor is the effective immobilization of the enzyme. In this work, ordered tyrosinase films on an optical transparent support were immobilized by a “layer-by-layer” (LbL) assembly, alternating the enzyme with the polycation polymer poly(dimethyldiallylammonium chloride). As confirmed by UV–vis spectroscopy, the LbL deposition allowed a high loading of enzyme. The immobilized tyrosinase functionality was proven and its kinetic parameters were spectrophotometrically determined. The prepared biosensor was used to optically detect the o-diphenolic compound l-3,4-dihydroxyphenyl-alanine (l-DOPA) and exhibited good repeatability and time stability. The sensing properties of the system were studied by means of both absorption and fluorescence spectroscopy. The bioassay based on the absorbance measurements gave a LOD of 23M and a linear response up to 350M. The bioassay based on the fluorescence measurements gave a LOD of 3Mand a linear response in the range of tens of micromolar (the exact value depends on the number of mushroom tyrosinase layers). Biosensor sensitivity could be modulated varying the number of the immobilized enzyme layers.

In 1975, Holliday and Pugh as well as Riggs independently hypothesized that DNA methylation in eukaryotes could act as a hereditary regulation mechanism that influences gene expression and cell differentiation. Interest in the study of epigenetic processes has been inspired by their reversibility as well as their potentially preventable or treatable consequences. Recently, we have begun to understand that the features of DNA methylation are not the same for all cells. Major differences have been found between differentiated cells and stem cells. Methylation influences various pathologies, and it is very important to improve the understanding of the pathogenic mechanisms. Epigenetic modifications may take place throughout life and have been related to cancer, brain aging, memory disturbances, changes in synaptic plasticity, and neurodegenerative diseases, such as Parkinson's disease and Huntington's disease. DNA methylation also has a very important role in tumor biology. Many oncogenes are activated by mutations in carcinogenesis. However, many genes with tumor-suppressor functions are "silenced" by the methylation of CpG sites in some of their regions. Moreover, the role of epigenetic alterations has been demonstrated in neurological diseases. In neuronal precursors, many genes associated with development and differentiation are silenced by CpG methylation. In addition, recent studies show that DNA methylation can also influence diseases that do not appear to be related to the environment, such as IgA nephropathy, thus affecting the expression of some genes involved in the T-cell receptor signaling. In conclusion, DNA methylation provides a whole series of fundamental information for the cell to regulate gene expression, including how and when the genes are read, and it does not depend on the DNA sequence.

In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat-inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host-probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.