Optical coherence tomography features of the repair tissue following RPE tear and their correlation with visual outcomes

To assess the optical coherence tomography (OCT) features of the repair tissue after retinal pigment epithelial (RPE) tear in neovascular age-related macular degeneration. Retrospective, observational study. Medical and imaging records of patients that developed tears after starting anti-VEGF treatment and with at least 12 months of follow-up were reviewed. OCT reflectivity of the RPE-subretinal hyperreflective tissue (SHT) complex was measured at 6, 12 and 18 months (when available). Reflectivity of the adjacent unaffected RPE-Bruch’s membrane was taken as internal reference. Other variables: grade and rip occurrence (early/late); number of intravitreal injections; type of macular neovascularization; sub-macular hemorrhage (SMH) at onset. Forty-nine eyes (age: 76.1 ± 7.0 years; VA: 0.54 ± 0.27 LogMAR) were included. Thirty-eight eyes had OCT signs of healing during the follow-up, with 21 showing SMH at baseline. Final VA positively correlated with the number of injections and negatively correlated with the RPE-SHT reflectivity and the presence of SMH (p < 0.001). Reflectivity of the RPE-SHT complex was positively associated with time and SMH at baseline (p < 0.05). In our study, most eyes showed signs of tissue repair after RPE tear. The reflectivity of repair tissue, the SMH presence and the number of anti-VEGF injections appeared to be major predictors of visual outcomes

Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects

Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 degrees C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions

Hairy garlic (Allium subhirsutum) from Sicily (Italy): LC-DAD-MSn analysis of secondary metabolites and in vitro biological properties

Allium subhirsutum, known as hairy garlic, is a bulbous plant widespread in the Mediterranean area and locally used as a food and spice. In the present study, the chemical profile of the ethanolic extracts from bulbs (BE) and aerial parts (APE) were analyzed by HPLC-ESI-MSn, and antioxidant properties were evaluated by DPPH, ABTS and TEAC assays. The traditional use in the diet, and the well documented biological activity of Allium species suggest a potential as a new nutraceutical. For this reason, the potential usefulness of this food can be considered in the treatment and prevention of degenerative Alzheimer disease. For this reason, acetylcholinesterase inhibitory property was investigated. Furthermore, due to the observed presence of sulfur-containing and phenolic constituents, the cytotoxicity on tumor cells line was investigated. Results revealed significant AChE inhibitory activity for BE and APE. Both extracts exhibited also moderate antioxidant properties in the in vitro assays. Finally, limited cytotoxic activity was observed towards Human colon carcinoma and adenocarcinoma cell line, with differences between the individual parts tested. HPLC-ESI-MSn analysis showed that hairy garlic is a good source of sulphur compounds, flavonoids and phenylpropanoids derivatives, thus being a valid alternative to the common garlic (A. sativum). This work opens new opportunities for the application of A. subhirsutum as a health-promoting food

Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-alpha were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-alpha, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F

Antioxidant and enzyme inhibitory properties of the polyphenolic-rich extract from an ancient apple variety of central Italy (Mela Rosa dei Monti Sibillini)

This study was undertaken to evaluate the nutraceutical potential of the Mela Rosa dei Monti Sibillini (MR), an ancient apple variety of the Sibillini Mountains, central Italy. The chemical profile of the apple''s polyphenolic-rich extract (MRE) obtained from first-and second-choice samples using the Amberlite® XAD7HP resin was analyzed by High Performance Liquid Chromatography with Diode-Array and Mass spectrometry (HPLC-DAD-MS) and 21 phytochemicals were quali–quantitatively determined. For comparative purposes, the polyphenol-rich extract of Annurca (ANE), a southern Italian variety, was analyzed. The antioxidant capacity of MREs was evaluated by Folin–Ciocalteu, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The inhibitory capacity of MREs for the enzymes a-glucosidase, lipase, monoamine oxidase A, tyrosinase, and acetylcholinesterase was also determined. The MREs showed higher polyphenolic and triterpene profiles than the ANE. Their radical scavenging activity was higher than that of ANE and comparable to the reference trolox. The MRE from the second-choice apples displayed higher contents of the 21 phytochemicals investigated. Either MRE from second-choice or first-choice samples showed enzymatic inhibition with IC50 values higher than those of reference inhibitors but worthy of nutraceutical consideration. Taken together, these results show the potential of MRE as a source of bioactive compounds to be used for pharmaceutical, nutraceutical, and cosmeceutical applications has been confirmed

The natural compound climacostol as a prodrug strategy based on pH activation for efficient delivery of cytotoxic small agents

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds

Novel, Meso-Substituted Cationic Porphyrin Molecule for Photo-Mediated Larval Control of the Dengue Vector Aedes aegypti

Dengue is a life-threatening viral disease of growing importance, transmitted by Aedes mosquito vectors. The control of mosquito larvae is crucial to contain or prevent disease outbreaks, and the discovery of new larvicides able to increase the efficacy and the flexibility of the vector control approach is highly desirable. Porphyrins are a class of molecules which generate reactive oxygen species if excited by visible light, thus inducing oxidative cell damage and cell death. In this study we aimed at assessing the potential of this photo-mediated cytotoxic mechanism to kill Aedes (Stegomyia) aegypti mosquito larvae. The selected porphyrin molecule, meso-tri(N-methylpyridyl),meso-mono(N-tetradecylpyridyl)porphine (C14 for simplicity), killed the larvae at doses lower than 1 µM, and at light intensities 50–100 times lower than those typical of natural sunlight, by damaging their intestinal tissues. The physicochemical properties of C14 make it easily adsorbed into organic material, and we exploited this feature to prepare an ‘insecticidal food’ which efficiently killed the larvae and remained active for at least 14 days after its dispersion in water. This study demonstrated that photo-sensitizing agents are promising tools for the development of new larvicides against mosquito vectors of dengue and other human and animal diseases