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.

A new analytical method for the determination of ochratoxin A (OTA) in red wine has been developed by using a double-extract cleanup and a fluorometric measurement after spectral deconvolution. Wine samples were diluted with a solution containing 1% polyethylene glycol and 5% sodium hydrogencarbonate, filtered, and purified by immunoaffinity and aminopropyl solid-phase column. OTA contents in the purified extract were determined by a spectrofluorometer (excitation wavelength, 330 nm; emission wavelength, 470 nm) after deconvolution of fluorescence spectra. Average recoveries from wine samples spiked with OTA at levels ranging from 0.5 to 3.0 ng/mL were 94.5–105.4% with relative standard deviations (RSD) of <15% (n = 4). The limit of detection (LOD) was 0.2 ng/mL, and the total time of analysis was 30 min. The developed method was tested on 18 red wine samples (naturally contaminated and spiked with OTA at levels ranging from 0.4 to 3.0 ng/mL) and compared with AOAC Official Method 2001.01, based on immunoaffinity column cleanup and HPLC with fluorescence detector. A good correlation (r2 = 0.9765) was observed between OTA levels obtained with the two methods, highlighting the reliability of the proposed method, the main advantage of which is the simple OTA determination by a benchtop fluorometer with evident reductions of cost and time of analysis.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30 W and 20 kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32 min in detergent micelles, ranged from 3.8 to 6.5 min in the different proteoliposomes formulations, and 5.5 min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to, the sonication-induced denaturation, in agreement with the scattering increase observed in solution. (C) 2016 Elsevier B.V. All rights reserved.

Sweet cherries from two Italian regions, Apulia and Emilia Romagna, were analysed using electronic nose (EN) and isotope ratio mass spectrometry (IRMS), with the aim of distinguishing them according to their geographic origin. The data were elaborated by statistical techniques, examining the EN and IRMS datasets both separately and in combination. Preliminary exploratory overviews were performed and then linear discriminant analyses (LDA) were used for classification. Regarding EN, different approaches for variable selection were tested, and the most suitable strategies were highlighted. The LDA classification results were expressed in terms of recognition and prediction abilities and it was found that both EN and IRMS performed well, with IRMS showing better cross-validated prediction ability (91.0%); the EN–IRMS combination gave slightly better results (92.3%). In order to validate the final results, the models were tested using an external set of samples with excellent results.

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 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 results, 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.

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.

In this paper, virgin olive oils (VOOs) coming from three different geographic origins of Apulia, were analysed for free acidity, peroxide value. spectrophotometric indexes, chlorophyll content. sterol, fatty acid, and triacylglycerol compositions. In order to predict the geographical origin of VOOs, different multivariate approaches were applied. By performing principal component analysis (PCA) a modest natural grouping of the VOOs was observed on the basis of their origin, and consequently three supervised techniques, i.e., general discriminant analysis (GDA), partial least squares-discriminant analysis (PLS-DA) and soft independent modelling of class analogy (SIMCA) were used and the results were compared. In particular, the best prediction ability was produced by applying GDA (average prediction ability of 82.5%), even if interesting results were obtained also by applying the other two classification techniques, i.e., 77.2% and 75.5% for PLS-DA and SIMCA, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

T-2 and HT-2 toxins are mycotoxins produced by several Fusarium species that are commonly found in various cereal grains, including oats, barley, wheat and maize. Intake estimates indicate that the presence of these mycotoxins in the diet can be of concern for public health. In this work, the inclusion processes occurring between fluorescent anthracene-derivatives of T-2 and HT-2 toxins and different cyclodextrin (CD) molecules were investigated in aqueous solutions by means of UV–Vis absorption, fluorescence emission and dynamic light scattering. Binding constant values and chemico-physical parameters were calculated. It was found that b-CDs give stronger inclusion reactions with both T-2 and HT-2 derivatives, as stated by important emission intensity increments. Such interactions were found to be fundamentally enthalpy-driven. Among b-CDs, the effect of the methylation at hydroxyl groups was tested: as a result, the di-methyl form of b-CD was found to induce the best fluorescence intensity enhancements.

In this study, non-targeted 1H NMR fingerprinting was used in combination with multivariate statistical techniques for the classification of Italian sweet cherries based on their different geographical origins (Emilia Romagna and Puglia). As classification techniques, Soft Independent Modelling of Class Analogy (SIMCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Linear Discriminant Analysis (LDA) were carried out and the results were compared. For LDA, before performing a refined selection of the number/combination of variables, two different strategies for a preliminary reduction of the variable number were tested. The best average recognition and CV prediction abilities (both 100.0%) were obtained for all the LDA models, although PLS-DA also showed remarkable performances (94.6%). All the statistical models were validated by observing the prediction abilities with respect to an external set of cherry samples. The best result (94.9%) was obtained with LDA by performing a best subset selection procedure on a set of 30 principal components previously selected by a stepwise decorrelation. The metabolites that mostly contributed to the classification performances of such LDA model, were found to be malate, glucose, fructose, glutamine and succinate.

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60±2 to 93±3 mol cyt c s−1 (mol total cyt c)−1. Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc1 complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc1 complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc1 complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc1 complex.

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 degrees 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 x 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.

The interaction of ochratoxin A (OTA) with heptakis-2,6-di-O-methyl-β-cyclodextrin (DIMEB) in aqueous solutions at two different pHs (3.5 and 9.5) was studied by means of spectroscopic, calorimetric and electrochemical techniques. DIMEB affected the physico-chemical properties of OTA. A stronger interaction was observed at pH = 3.5 where the neutral form of OTA prevails. The spectroscopic information indicated that the OTA/DIMEB inclusion process occurred through the insertion of the phenylalanine group into the cyclodextrin (CD) cavity; moreover, voltammetry experiments showed that the isocoumarinic phenolic group was also involved, probably by non-inclusion interactions. At pH = 3.5, binding constants and thermodynamic parameters of the OTA/DIMEB complex were determined by means of the modified Benesi-Hildebrand equation and van’t Hoff plot. A 1:1 stoichiometry for the OTA/DIMEB complex was observed with a binding constant equal to 530 ± 50 L·mol−1 at 25 °C. The calculated thermodynamic parameters indicate that the interaction is highly endothermic and that the complex formation is driven by entropy. The results provide useful information for potential applications of cyclodextrins in the analysis of mycotoxins and in the field of mycotoxin sequestering agents

Valinomycin (VLM, 1) is a K+ ionophore cyclodepsipeptide capable of depolarizing mitochondria and inducing apoptosis to several mammalian cell types, including a number of tumor cell lines. With the aim of creating VLM-based ligand-targeted anticancer drugs that may selectively convey VLM to pathological cells, we have previously introduced derivatizable hydroxyl handles into the VLM structure, allowing to access a three-entity library of monohydroxyl VLMs (HyVLMs) bearing the OH group at the isopropyl side chain of a d-Hyi, d-Val, or l-Val residue (analogs 2-4, respectively). Herein, the levels of bioactivity retained by the conjugable HyVLMs have been assessed on the basis of their ability to alter the functionality of isolated rat-liver mitochondria. Experiments run with HyVLMs in the range 1-10nM and in 20 or 125mM KCl medium show that the hydroxyl group reduces the potency of HyVLMs relative to VLM to an extent that depends upon the molecular site involved in the hydroxylation. On the other hand, estimation of the stability constants of complexes (in methanol at 25 degrees C) of each analog with Na+, K+, and Cs+ reveals that HyVLMs nicely retain the VLM binding features, except for a moderate increase in the stability of Na+ complexes. These findings, along with pertinent structural considerations, suggest that the incorporation of OH into the VLM structure might actually have altered its K+ transporting ability across mitochondrial membranes. Besides facing new aspects of VLM structure-activity relationship, these studies set the basis for the rational design of ligand-HyVLMs conjugates through derivatization of hanging OH group. Copyright (c) 2013 European Peptide Society and John Wiley & Sons, Ltd.