Rose Bengal-photosensitized oxidation of 4-thiothymidine in aqueous medium: evidence for the reaction of the nucleoside with singlet state oxygen.

The photoreactivity of 4-thiothymidine (S(4)TdR) under visible light in the presence of Rose Bengal (RB), acting as a photosensitizer, was investigated in aqueous solutions at pH 7 and 12, using UV-vis, FTIR-ATR and (1)H-NMR spectroscopic techniques, time resolved absorption spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Evidence for the generation of thymidine (TdR) as the main product, after one hour of irradiation, was obtained from UV-Vis data, that suggested 4-thiothymidine photodegradation to be faster at basic pH, and confirmed by FTIR-ATR and (1)H-NMR data. Clues for the presence of a further product, likely corresponding to a dimeric form of S(4)TdR, were obtained from the latter techniques. Besides indicating the presence of thymidine, the ESI-MS and MS/MS spectra of the reaction mixtures enabled the identification of the additional product as a S-S bridged covalent dimer of 4-thiothymidine. The concentration of the dimeric species could be estimated with the aid of (1)H-NMR data and was found to be lower than that of thymidine in pH 7 reaction mixtures and almost negligible in the pH 12 ones. From a mechanistic point of view, time-resolved absorption spectroscopy measurements provided direct evidence that the formation of the two products cannot be ascribed to a photoinduced electron transfer involving S(4)TdR and the excited triplet state of RB. Rather, their generation can be interpreted as the result of a bimolecular reaction occurring between singlet state oxygen ((1)O2), photogenerated by RB, and S(4)TdR, as demonstrated by the direct detection of (1)O2 through IR luminescence spectroscopy. More specifically, a sequential reaction pathway, consisting in the generation of an electrophilic hydroxylated form of S(4)TdR and its subsequent, rapid reaction with S(4)TdR, was hypothesized to explain the presence of the S-S bridged covalent dimer of 4-thiothymidine in the reaction mixtures. The described processes make S(4)TdR an interesting candidate in the role of molecular probe for the detection of (1)O2 under different pH conditions.

Photosystem II based multilayers obtained by electrostatic layer-by-layer assembly on quartz substrates.

Photosystem II (PSII) proteins from spinach leaves were immobilized onto quartz substrates according to the Layer-by-Layer (LbL) procedure, alternating protein to polyethylenimine (PEI) layers by exploiting electrostatic interactions. The effects of several factors, such as storage conditions, ageing of the PSII-modified substrates, as well as PSII concentration in buffer, on the quality of the prepared multilayers, were investigated by UV-vis Absorption Spectroscopy and Atomic Force Microscopy (AFM). A number of 13 layers was found to be optimal to guarantee intense PSII optical signals with homogeneous morphological distributions of proteins. The multilayers resulted stable if stored in contact with air at 4 °C, as observed by UV-vis Absorption spectra recorded after 48 h. The best results in terms of AFM images and electron transfer efficiency (measured by Hill Reaction assays) were gained by using 5.6 × 10(-7) M chlorophyll concentration, obtaining multilayers with the most ordered protein distributions and the highest electron transfer efficiency, i.e. 85% of an iso-absorbing PSII suspension. The results highlight the possibility to successfully immobilize PSII proteins, without considerable loss of bioactivity, thanks to the mild nature of the electrostatic LbL technique, opening up possibilities of applications in the bioelectrochemical energy conversion and biosensoristic fields.

Use of vitrified MSWI bottom ashes for concrete production.

Bottom ashes from a north Italian municipal solid waste incinerator (MSWI) were vitrified at 1450 degrees C without adding any vitrifying agent, then ground and sieved to different granulometry (ranging from 50 microm to 20mm), and used as filler, sand, or aggregate for concrete. Samples were characterized via slump tests (UNI 9418), alkali-silica reactivity (UNI 8520/22 and ASTM C 298), and compression strength tests (UNI 6132, 6132/72, 6686/72), and compared to reference samples obtained without vitrified bottom ashes (VBA). Our results show that vitrified bottom ashes are unsuitable as a sand substitute; however, concrete containing up to 20 wt.% of VBA filler used as a substitute for cement and up to 75 vol.% of VBA as a substitute for natural aggregate retains the same mechanical properties as reference samples. Alkali-silica or other detrimental reactions were not observed in VBA-containing concrete samples after a period of two years. The results of this work demonstrate that vitrified bottom ashes from MSWI can be used instead of natural aggregates in mortar and concrete production.

Geographical origin discrimination of lentils (Lens culinaris Medik.) using 1H NMR fingerprinting and multivariate statistical analyses.

Lentil samples coming from two different countries, i.e. Italy and Canada, were analysed using untargeted 1H NMR fingerprinting in combination with chemometrics in order to build models able to classify them according to their geographical origin. For such aim, Soft Independent Modelling of Class Analogy (SIMCA), k-Nearest Neighbor (k-NN), Principal Component Analysis followed by Linear Discriminant Analysis (PCA-LDA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were applied to the NMR data and the results were compared. The best combination of average recognition (100%) and cross-validation prediction abilities (96.7%) was obtained for the PCA-LDA. All the statistical models were validated both by using a test set and by carrying out a Monte Carlo Cross Validation: the obtained performances were found to be satisfying for all the models, with prediction abilities higher than 95% demonstrating the suitability of the developed methods. Finally, the metabolites that mostly contributed to the lentil discrimination were indicated.

The effect of in-amphorae aging on oenological parameters, phenolic profile and volatile composition of Minutolo white wine.

A wine was obtained from cryomacerated Minutolo grapes under reductive conditions and aged for 12months in glass container and in 3 types of amphorae. After aging, wines in glass containers showed the highest alcohol content, volatile acidity, dissolved oxygen, concentrations of aromatics, alcohols, and esters and by the lowest contents of enols and terpenes. They also showed the highest decrease of flavonoids, hydroxycinnamoyl tartaric acids, and procyanidins. Wines in raw amphorae showed the dramatic decrease of flavonoids and flavans reactive with vanillin. The highest antioxidant activity was exhibited by wines in engobe amphorae, while the lowest values were showed by the wines in glass containers and glazed amphorae. Caftaric acid and procyanidin B3 decreased in wine aged under glass while epicatechin mainly reduced in raw amphorae. According to the Principal Component Analysis, the wines resulted homogeneously grouped as a function of the type of container in which were aged.

Effects of different vinification technologies on physical and chemical characteristics of Sauvignon blanc wines.

In order to investigate the effects of cryomaceration and reductive vinification on chemical and physical indices and on antioxidant compounds of Sauvignon blanc wines, four wine-making procedures were applied: traditional white vinification, skin cryomaceration, vinification in a reductive environment, and a combination of the last two procedures. Significant differences were highlighted by both conventional analyses and NMR spectroscopy. The strongest changes were for organic acid concentrations (tartaric, in particular) and phenolic content. Cryomaceration caused a strong precipitation of tartaric acid, which may be desired if grapes have high acidity values. Cryomaceration protected those flavans reactive with vanillin from the action of oxidative enzymes. Vinification in a reductive environment, alone or combined with a cryomaceration step, gave wines with the highest solids content and caused a greater extraction of phenolic compounds from skins compared to traditional winemaking or cryomaceration alone, due to SO(2) solubilisation. Grape oenological expression can be strongly affected by the application of the investigated wine-making procedures.

Biomaterials based on photosynthetic membranes as potential sensors for herbicides.

In this study, ultrathin film multilayers of Photosystem II-enriched photosynthetic membranes (BBY) were prepared and immobilized on quartz substrates by means of a Layer by Layer procedure exploiting electrostatic interactions with poly(ethylenimine) as polyelectrolyte. The biomaterials thus obtained were characterized by means of optical techniques and Atomic Force Microscopy, highlighting the fact that the Layer by Layer approach allowed the BBYs to be immobilized with satisfactory results. The activity of these hybrid materials was evaluated by means of optical assays based on the Hill Reaction, indicating that the biosamples, which preserved about 65% of their original activity even ten weeks after preparation, were both stable and active. Furthermore, an investigation of the biochips' sensitivity to the herbicide terbutryn, as a model analyte, gave interesting results: inhibition of photosynthetic activity was observed at terbutryn concentrations higher than 10(-7)M, thus evidencing the potential of such biomaterials in the environmental biosensor field.

Herbicides affect fluorescence and electron transfer activity of spinach chloroplasts, thylakoid membranes and isolated Photosystem II.

In this work, studies on the effects produced by atrazine, terbutryn or diuron onto spinach photosynthetic materials were performed by observing changes in fluorescence emission and in electron transfer activities of the bio-samples in the presence of such herbicides; chloroplasts, thylakoids, Photosystem II-enriched thylakoids (BBYs) and isolated Photosystem II (PSII) were employed. This approach evidenced differences in the herbicide-photosynthetic material interactions going up-down from chloroplasts to proteins. Rapid emission increments were detected for chloroplasts and thylakoids, in particular in the presence of terbutryn; no remarkable emission increment was recorded when BBYs or PSII were used for this assay. The dependences of the chloroplast and thylakoid emission intensities upon herbicide concentration were investigated with responses even at concentrations below 10(-7)M. The influence of lowering the temperature was also tested, and the stabilizing effects on the resistances of the bio-samples against herbicides were recorded. Furthermore, Hill Reaction-based colorimetric assays were performed to monitor the electron transfer activities of the bio-samples in the presence of herbicides, after brief incubations. As a result, chloroplasts and thylakoids resulted to be sensitive tools in responding to concentrations even lower than 10(-7)M of most herbicides; nevertheless, an interesting sensitivity to herbicides was also observed for PSII.

Interactions between heavy metals and photosynthetic materials studied by optical techniques.

In this work studies on rapid inhibitory interactions between heavy metals and photosynthetic materials at different organization levels were carried out by optical assay techniques, investigating the possibility of applications in the heavy metal detection field. Spinach chloroplasts, thylakoids and Photosystem II proteins were employed as biotools in combination with colorimetric assays based on dichlorophenol indophenole (DCIP) photoreduction and on fluorescence emission techniques. It was found that copper and mercury demonstrated a strong and rapid photosynthetic activity inhibition, that varied from proteins to membranes, while other metals like nickel, cobalt and manganese produced only slight inhibition effects on all tested photosynthetic materials. By emission measurements, only copper was found to rapidly influence the photosynthetic material signals. These findings give interesting information about the rapid effects of heavy metals on isolated photosynthetic samples, and are in addition to the literature data concerning the effects of growth in heavy metal enriched media.

Cardiolipin increases in chromatophores isolated from Rhodobacter sphaeroides after osmotic stress: structural and functional roles.

Chromatophores isolated from cells of Rhodobacter sphaeroides exposed to hypertonic solutions were enriched in cardiolipin (CL). Because CL levels are raised by increasing the incubation time of R. sphaeroides in hypertonic solutions, it was possible to isolate chromatophores containing different CL amounts by starting from cells incubated in hypertonic solutions for different times. The functionality and stability of the photosynthetic proteins in chromatophore membranes having different CL levels were investigated. Reaction center (RC) stabilization with respect to thermal denaturation and photoxidative damage was observed by flash photolysis and fluorescence emission experiments in CL-enriched chromatophores. To gain detailed information about the structures of endogenous CLs, this lipid family was isolated and purified by preparative TLC, and characterized by high-resolution mass spectrometry. We conclude that osmotic shock can be used as a tool to modulate CL levels in isolated chromatophores and to change the composition of the RC lipid annulus, avoiding membrane artifacts introduced by the use of detergents.