An Association of Vitamins A and E with Hyaluronic and Lactobionic Acids may Prevent Molecular Changes Associated with Keratocyte to Myofibroblast Transition

Inflammatory events in the corneal stroma may activate keratocytes and trigger their transition towards myofibroblasts, which now produce different extracellular matrix (ECM) proteins thus causing corneal opacification.Corneal haze is a frequent side effect after photorefractive keratectomy (PRK) to correct high myopia.Currently, a preventive treatment with mitomycin-c can be used to limit the occurrence of this phenomenon. However, mitomycin-c is a toxic drug, not devoid of side effects, which may occasionally involve the corneal endothelium. Therefore, we have searched for a less risky, natural way, to prevent keratocytes transition. To this purpose, we have used as markers of the phenotype switch the proteins lumican (highly expressed by keratocytes and much less by myofibroblasts) and smooth muscle actin (αSMA) (highly expressed by myofibroblasts and poorly found in keratocytes), beside Fibronectin (Fn), the expression of which is also increased by transforming growth factor-beta (TGFβ treatment. Treatment of human keratocytes with TGFβ was used to induce the protein shift. Among different possible candidates, we have found that vitamins A and E, hyaluronic and lactobionic acids may prevent, either alone, or much better in association, the shift in the ratio between lumican and αSMA and the increased Fn expression. In conclusion, it could be speculated that topic treatment of the ocular surface with an association of these four compounds could be able to prevent or at least limit the occurrence of post-PRK corneal haze, with the additional advantage of lubrication, hydration and antioxidant defense exerted by these molecules

An Innovative, Open, Interoperable Citizen Engagement Cloud Platform for Smart Government and Users' Interaction

This paper introduces an open, interoperable, and cloud-computing-based citizen engagement platform for the management of administrative processes of public administrations, which also increases the engagement of citizens. The citizen engagement platform is the outcome of a 3-year Italian national project called PRISMA (Interoperable cloud platforms for smart government). The aim of the project is to constitute a new model of digital ecosystem that can support and enable new methods of interaction among public administrations, citizens, companies, and other stakeholders surrounding cities. The platform has been defined by the media as a flexible (enable the addition of any kind of application or service) and open (enable access to open services) Italian "cloud" that allows public administrations to access to a vast knowledge base represented as linked open data to be reused by a stakeholder community with the aim of developing new applications ("Cloud Apps") tailored to the specific needs of citizens. The platform has been used by Catania and Syracuse municipalities, two of the main cities of southern Italy, located in the Sicilian region. The fully adoption of the platform is rapidly spreading around the whole region (local developers have already used available application programming interfaces (APIs) to create additional services for citizens and administrations) to such an extent that other provinces of Sicily and Italy in general expressed their interest for its usage. The platform is available online and, as mentioned above, is open source and provides APIs for full exploitation.Comment: 23 pages, 7 figures, journal pape

Carnosine Protects Macrophages against the Toxicity of Aβ1-42 Oligomers by Decreasing Oxidative Stress

Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aβ) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aβ have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aβ1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aβ1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aβ1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology

Cx26 partial loss causes accelerated presbycusis by redox imbalance and dysregulation of Nfr2 pathway

Mutations in GJB2, the gene that encodes connexin 26 (Cx26), are the most common cause of sensorineural hearing impairment. The truncating variant 35delG, which determines a complete loss of Cx26 protein function, is the prevalent GJB2 mutation in several populations. Here, we generated and analyzed Gjb2+/- mice as a model of heterozygous human carriers of 35delG. Compared to control mice, auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) worsened over time more rapidly in Gjb2+/- mice, indicating they were affected by accelerated age-related hearing loss (ARHL), or presbycusis. We linked causally the auditory phenotype of Gjb2+/- mice to apoptosis and oxidative damage in the cochlear duct, reduced release of glutathione from connexin hemichannels, decreased nutrient delivery to the sensory epithelium via cochlear gap junctions and deregulated expression of genes that are under transcriptional control of the nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal regulator of tolerance to redox stress. Moreover, a statistically significant genome-wide association with two genes (PRKCE and TGFB1) related to the Nrf2 pathway (p-value < 4\u202f 7 10-2) was detected in a very large cohort of 4091 individuals, originating from Europe, Caucasus and Central Asia, with hearing phenotype (including 1076 presbycusis patients and 1290 healthy matched controls). We conclude that (i) elements of the Nrf2 pathway are essential for hearing maintenance and (ii) their dysfunction may play an important role in the etiopathogenesis of human presbycusis

Genomic analysis in human solid tumours: Copper Homeostasis Genes in Colorectal cancer

1. The public database: Cancer Genome Atlas Network has been consulted in order to reveal the presence of somatic mutations in copper homeostasis genes (CHGs) in colorectal cancer, and such analysis, performed on 228 colorectal tumor samples, has revealed that inactivating mutations are extremely rare in CHGs. 2. The collection of whole transcriptome profiles by oligonucleotide microarrays represents the second aim of the thesis. CHGs mRNA levels have been measured in 37 colorectal carcinoma samples in comparison to matched normal colonic mucosae. The transcriptome analysis has been perfomed using the last Human Transcriptome Array (HTA 2.0, Affymetrix) which allows to analyze simultaneously 40.000 coding transcripts and 20.000 non-coding transcripts and to reveal variations of mRNA levels between in colorectal cancer samples respect to normal colonic mucosae. The gene expression analysis has showed an up-regulation of several transcripts involved in copper homeostasis pathway (such as SLC31A1, SLC31A2, COX11, SCO1, SOD1) in two different conditions. On the contrary, some genes did not show variations of expression between two tested conditions suggesting the idea that these genes, if experimental altered in their expression, could represent the good targets for specific drug treatments. The transcriptome analysis by the last generation on oligonucleotide microarrays gives a possibility to analyse gene expression levels and single exon expression levels. In the 37 human colorectal samples, the exon-level expression analysis has been revealed the presence of alternative transcripts prevalent in a condition respect to other. 3. In this part of thesis the experimental down-regulation, by short interfering, of a copper chaperone ATOX1, significantly reduced the tumor growth in a Caco2 colorectal cell line. It has been demonstrated that the copper addition increase the toxic effects of some copper binding compounds, in particular, the ionophore copper-ionophore 5-chloro-8-hydroxyquinoline (ClHQ) and the copper chelator (N,N,N'N',-tetrakis (2- pyridylmethyl)ethylenediamine (TPEN) in two human colorectal cancer cell lines (Caco-2 and HT29). Moreover, the Atox1 silencing has enhanced the toxic effects of copper-ClHQ complexes and TPEN in Caco-2 cells confirming that the inhibition of copper chaperone Atox1 could be a good strategy to attenuate the cancer cell proliferation and to increase the anticancer effects of some copper binding drugs

Comparative Efficiency of Lutein and Astaxanthin in the Protection of Human Corneal Epithelial Cells In Vitro from Blue-Violet Light Photo-Oxidative Damage

The aim of this study was to compare in vitro the protective and antioxidant properties of lutein and astaxanthin on human primary corneal epithelial cells (HCE-F). To this purpose, HCE-F cells were irradiated with a blue-violet light lamp (415–420 nm) at different energies (20 to 80 J/cm2). Lutein and astaxanthin (50 to 250 μM) were added to HCE-F right before blue-violet light irradiation at 50 J/cm2. Viability was evaluated by the CKK-8 assay while the production of reactive oxygen species (ROS) by the H2DCF-DA assay. Results have shown that the viability of HCE-F cells decreased at light energies from 20 J/cm2 to 80 J/cm2, while ROS production increased at 50 and 80 J/cm2. The presence of lutein or astaxanthin protected the cells from phototoxicity, with lutein slightly more efficient than astaxanthin also on the blunting of ROS, prevention of apoptotic cell death and modulation of the Nrf-2 pathway. The association of lutein and astaxanthin did not give a significant advantage over the use of lutein alone. Taken together, these results suggest that the association of lutein and astaxanthin might be useful to protect cells of the ocular surface from short (lutein) and longer (astaxanthin) wavelengths, as these are the most damaging radiations hitting the eye from many different LED screens and solar light

Gene expression profiles in genome instability-based classes of colorectal cancer

Abstract Background Broad copy number aberrations (BCNAs) represent a common form of genome instability in colorectal cancer (CRC). CRCs show large variations in their level of aneuploidy: microsatellite-instable (MSI) tumors are known to have a near-diploid karyotype while microsatellite-stable (MSS) tumors show high level of chromosomal instability. However, MSS tumors have great heterogeneity in the number of BCNAs, with a minor percentage of samples showing an almost normal karyotype. In the present work we subdivided MSS CRCs according to a “BCNA score” and characterized their transcriptome profiles, considered as a proxy to their phenotypic features. Methods Microsatellite testing, genome-wide DNA copy number and whole-transcript expression analysis (HTA) were performed on 33 tumor samples and 25 normal colonic tissue samples from 32 CRC patients. 15.1% of the samples were MSI tumors (n = 5), whereas 84.9% were MSS tumors (n = 28). Gene expression data of 34 additional MSI tumors was retrieved from a public functional genomics data repository. Results Using as a threshold the first quartile of the BCNA score distribution, MSS samples were classified as low-BCNA (LB, n = 7) or high-BCNA (HB, n = 21). LB tumors were enriched for mucinous CRCs and their gene-expression profile resembled that of MSI samples for what concerns a subset of genes involved in secretory processes, mucosal protection, and extracellular matrix remodeling. HB tumors were predominantly non-mucinous adenocarcinomas and showed overexpression of a subset of genes typical of surface colonocytes and EGF signaling. A large percentage of unclassified samples according to the consensus molecular subtypes (CMS) classifier was found in the LB group (43%), whereas 76% HB tumors belonged to CMS2. Conclusions A classification of colorectal tumors based on the number of BCNAs identifies two groups of MSS tumors which differ for histopathology and gene expression profile. Such information can be exploited for its translational relevance in different aspects of CRC clinical management

Synthesis of Bisphenol Neolignans Inspired by Honokiol as Antiproliferative Agents

Honokiol (2) is a natural bisphenol neolignan showing a variety of biological properties, including antitumor activity. Some studies pointed out 2 as a potential anticancer agent in view of its antiproliferative and pro-apoptotic activity towards tumor cells. As a further contribution to these studies, we report here the synthesis of a small library of bisphenol neolignans inspired by honokiol and the evaluation of their antiproliferative activity. The natural lead was hence subjected to simple chemical modifications to obtain the derivatives 3&ndash;9; further neolignans (12a-c, 13a-c, 14a-c, and 15a) were synthesized employing the Suzuki&ndash;Miyaura reaction, thus obtaining bisphenols with a substitution pattern different from honokiol. These compounds and the natural lead were subjected to antiproliferative assay towards HCT-116, HT-29, and PC3 tumor cell lines. Six of the neolignans show GI50 values lower than those of 2 towards all cell lines. Compounds 14a, 14c, and 15a are the most effective antiproliferative agents, with GI50 in the range of 3.6&ndash;19.1 &micro;M, in some cases it is lower than those of the anticancer drug 5-fluorouracil. Flow cytometry experiments performed on these neolignans showed that the inhibition of proliferation is mainly due to an apoptotic process. These results indicate that the structural modification of honokiol may open the way to obtaining antitumor neolignans more potent than the natural lead