Serum Vascular Adhesion Protein-1 and Endothelial Dysfunction in Hepatic Cirrhosis: Searching for New Prognostic Markers.

Endothelial dysfunction plays a key role in the development of liver cirrhosis. Among the biomarkers of endothelial dysfunction, the soluble form of Vascular Adhesion Protein-1 (sVAP-1) is an unconventional and less known adhesion molecule endowed also with amine oxidase activity. The aim of this study was to explore and correlate the behavior of sVAP-1 with that of the soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) and with the severity of liver cirrhosis. A cross-sectional study was carried out by enrolling 28 controls, 59 cirrhotic patients without hepatocellular carcinoma, and 56 patients with hepatocellular carcinoma (HCC), mainly caused by alcohol abuse. The levels of adhesion molecules and of the pro-inflammatory cytokines (IL-6 and TNF-αα) were determined by immunoassay and the enzymatic activity of sVAP-1 by a fluorometric assay. In non-diabetic patients without HCC, a specific behavior of sVAP-1 was highlighted. Differently from sVCAM-1, sICAM-1, and cytokines, the sVAP-1 level was significantly increased only in the early stage of disease, and then, it decreased in the last stage (866 ± 390 ng/mL vs. 545 ± 316 ng/mL, in Child-Pugh class A vs. C, respectively, p < 0.05). Bivariate analysis correlates sVAP-1 to sVCAM-1, in the absence of HCC (Spearman's rho = 0.403, p < 0.01). Multiple linear regression analysis revealed that sVCAM-1 appears to be a predictor of sVAP-1 (ß coefficient = 0.374, p = 0.021). In conclusion, in non-diabetic and non-HCC cirrhotic patients, sVAP-1 may be a potential prognostic biomarker that, together with sVCAM-1 and pro-inflammatory cytokines, may provide information on the progression of sinusoidal liver endothelium damage.

Genome-wide screening in pluripotent cells identifies Mtf1 as a suppressor of mutant huntingtin toxicity.

Huntington's disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.

Cardiolipin drives the catalytic activity of GPX4 on membranes: Insights from the R152H mutant.

The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to "positive feedback" in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.

Acidic Shift of Optimum pH of Bovine Serum Amine Oxidase upon Immobilization onto Nanostructured Ferric Tannates.

Protein-nanoparticle hybrids represent entities characterized by emerging biological properties that can significantly differ from those of the parent components. Herein, bovine serum amine oxidase (i.e., BSAO) was immobilized onto a magnetic nanomaterial constituted of surface active maghemite nanoparticles (i.e., SAMNs, the core), surface-modified with tannic acid (i.e., TA, the shell), to produce a biologically active ternary hybrid (i.e., SAMN@TA@BSAO). In comparison with the native enzyme, the secondary structure of the immobilized BSAO responded to pH variations sensitively, resulting in a shift of its optimum activity from pH 7.2 to 5.0. Conversely, the native enzyme structure was not influenced by pH and its activity was affected at pH 5.0, i.e., in correspondence with the best performances of SAMN@TA@BSAO. Thus, an extensive NMR study was dedicated to the structure-function relationship of native BSAO, confirming that its low activity below pH 6.0 was ascribable to minimal structural modifications not detected by circular dichroism. The generation of cytotoxic products, such as aldehydes and H2O2, by the catalytic activity of SAMN@TA@BSAO on polyamine oxidation is envisaged as smart nanotherapy for tumor cells. The present study supports protein-nanoparticle conjugation as a key for the modulation of biological functions.

Bentonite does not affect in vitro ruminal gross fermentations but could modify ruminal metabolome and mineral content. A proof of concept.

Clay minerals, such as bentonite, are used as feed additives capable of adsorbing mycotoxins and heavy metals and have been related to many positive effects on animal health and productivity. However, these compounds seem to induce also side effects and to interact with the intestinal and ruminal microbiota. The present in vitro study is aimed at evaluating the effects of different doses of bentonite on ruminal fermentations, metabolome and mineral content. Five doses of bentonite (0, 2.5, 5, 10 and 50 mg in 150 mL total volume) were incubated (39 °C for 24 h) with a dairy cow Total Mixed Ratio (TMR) and the ruminal fluid obtained from one healthy Holstein lactating cow. The kinetics of gas production (GP) continuously monitored during the incubation evidenced no significant differences in either cumulative GP (mL/g DM) or GP rate (mL/g DM/h) between the treatment groups. After the incubation, metabolome and mineral content of treated ruminal fluids were studied in pooled replicate samples by 1H NMR spectroscopy and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), respectively. The NMR analysis led to the identification of 20 metabolites and suggested a clear metabolic differentiation among treatments. The ICP-OES analysis suggested that the addition of bentonite affected the concentration of Al, Ba, Ca, Cr, Mn, Mo and Sr. It is conceivable that bentonite administration does not affect gross ruminal fermentations, while it seems to modify the ruminal metabolome and the concentrations of few minerals in ruminal fluid.

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization.

A "green" solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n.

Role of carboxylic group pattern on protein surface in the recognition of iron oxide nanoparticles: A key for protein corona formation.

The knowledge of protein-nanoparticle interplay is of crucial importance to predict the fate of nanomaterials in biological environments. Indeed, protein corona on nanomaterials is responsible for the physiological response of the organism, influencing cell processes, from transport to accumulation and toxicity. Herein, a comparison using four different proteins reveals the existence of patterned regions of carboxylic groups acting as recognition sites for naked iron oxide nanoparticles. Readily interacting proteins display a distinctive surface distribution of carboxylic groups, recalling the geometric shape of an ellipse. This is morphologically complementary to nanoparticles curvature and compatible with the topography of exposed FeIII sites laying on the nanomaterial surface. The recognition site, absent in non-interacting proteins, promotes the nanoparticle harboring and allows the formation of functional protein coronas. The present work envisages the possibility of predicting the composition and the biological properties of protein corona on metal oxide nanoparticles.

1H-NMR spectroscopy metabonomics of reactive, ovarian carcinoma and hepatocellular carcinoma ascites.

BACKGROUND: Metabolomic profiling of human malignant effusion remain a field poorly investigated. Proton nuclear magnetic resonance (1H-NMR) spectroscopy is a rapid relatively low cost technique, and effusion is an optimal biospecimen suitable for metabonomic investigations. With this study we addressed metabolomic profiling of malignant ascitic effusion (mAE) from patients with high grade serous ovarian carcinoma (HGSOC), Hepatocellular carcinoma (HCC), and benign AEs (bAEs) from patients with reactive peritonitis. METHODS: Metabolic profiling with 1H-NMR was performed on 72 AEs (31 HGSOC, 16 HCC and 25 bAE) prospectively collected in our cytology service. Histological confirmation was requested for all malignant case. Multivariate analysis comprising PCA and PLS-DA was applied to discover metabolites suitable to differentiate effusions among the investigated groups. RESULTS: 1H-NMR metabonomic analysis showed clearly different spectra for malignant and benign AEs, as well as for HGSOC vs. HCC effusion. When compared with HCC effusions, the HGSOC effusion were enriched, among all, in alanine, lipids, N-acetyl groups and phenylalanine and depleted in glutamine. CONCLUSIONS: Subject to validation in further larger studies, 1H-NMR metabonomics could be an effective and reliable ancillary tool for AE investigations and diagnosis particularly in acellular effusions.

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1.

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

Glutathione peroxidase 4-catalyzed reduction of lipid hydroperoxides in membranes: The polar head of membrane phospholipids binds the enzyme and addresses the fatty acid hydroperoxide group toward the redox center.

GPx4 is a monomeric glutathione peroxidase, unique in reducing the hydroperoxide group (-OOH) of fatty acids esterified in membrane phospholipids. This reaction inhibits lipid peroxidation and accounts for enzyme's vital role. Here we investigated the interaction of GPx4 with membrane phospholipids. A cationic surface near the GPx4 catalytic center interacts with phospholipid polar heads. Accordingly, SPR analysis indicates cardiolipin as the phospholipid with maximal affinity to GPx4. Consistent with the electrostatic nature of the interaction, KCl increases the KD. Molecular dynamic (MD) simulation shows that a -OOH posed in the core of the membrane as 13 - or 9 -OOH of tetra-linoleoyl cardiolipin or 15 -OOH stearoyl-arachidonoyl-phosphaphatidylcholine moves to the lipid-water interface. Thereby, the -OOH groups are addressed toward the GPx4 redox center. In this pose, however, the catalytic site facing the membrane would be inaccessible to GSH, but the consecutive redox processes facilitate access of GSH, which further primes undocking of the enzyme, because GSH competes for the binding residues implicated in docking. During the final phase of the catalytic cycle, while GSSG is produced, GPx4 is disconnected from the membrane. The observation that GSH depletion in cells induces GPx4 translocation to the membrane, is in agreement with this concept.

Metabonomics by proton nuclear magnetic resonance in human pleural effusions: A route to discriminate between benign and malignant pleural effusions and to target small molecules as potential cancer biomarkers.

BACKGROUND: Cytopathology is a noninvasive and cost-effective method for detecting cancer cells in pleural effusions (PEs), although in many cases, the diagnostic performance is hindered by the paucity of significant cells or the lack of clear morphological criteria. This study presents the results of an omics approach to improving the diagnostic performance of PE cytology. METHODS: Metabolic profiling with proton nuclear magnetic resonance (1 H-NMR) was performed for 92 PEs (44 malignant cases of 8 different cancers and 48 benign cases of 7 nonneoplastic conditions). Light's criteria were used to further classify PEs as transudates or exudates, and 1 H-NMR spectroscopy was used to differentiate malignant pleural effusions (mPEs) from benign pleural effusions (bPEs). RESULTS: 1 H-NMR metabolic analysis showed clearly different spectra for mPEs and bPEs in the regions of the signals due to lipids, branched amino acids, and lactate, which were increased in mPEs. Transudates and exudates in bPEs were differentiated as well on the basis of the 1 H-NMR signals from lipids and lipoproteins, which were increased in exudates. CONCLUSIONS: Subject to validation in further larger studies, 1 H-NMR metabonomics could be an effective and reliable ancillary tool for PE investigations and diagnoses. Cancer Cytopathol 2017;125:341-348. © 2017 American Cancer Society.

Reaction rates of α-tocopheroxyl radicals confined in micelles and in human plasma lipoproteins.

α-Tocopherol, the main component of vitamin E, traps highly reactive radicals which otherwise might react with lipids present in plasmatic lipoproteins or in cell membranes. The α-tocopheroxyl radicals generated by this process have also a pro-oxidant action which is contrasted by their reaction with ascorbate or by bimolecular self-reaction (dismutation). The kinetics of this bimolecular self-reaction were explored in solution such as ethanol, and in heterogeneous systems such as deoxycholic acid micelles and in human plasma. According to ESR measurements, the kinetic rate constant (2k(d)) of the bimolecular self-reaction of α-tocopheroxyl radicals in micelles and in human plasma was calculated to be of the order of 10(5) M(-1) s(-1) at 37 °C. This value was obtained considering that the reactive radicals are confined into the micellar pseudophase and is one to two orders of magnitude higher than the value we found in homogeneous phase. The physiological significance of this high value is discussed considering the competition between bimolecular self-reaction and the α-tocopheroxyl radical recycling by ascorbate.

Stable aqueous solutions of naked titanate nanotubes.

Aqueous solutions of naked nanotubes with Ti concentration up to 10 mM are obtained by hydrothermal synthesis followed by extensive ultrasound treatment. The morphology, surface characteristics, and solution behavior of the solubilized nanotubes are investigated. The time course of the solubilization process driven by ultrasound follows a first-order kinetic law and is mediated by the competition between Na(+) and H(+) for surface sites. The dynamics of interaction with small cations (i.e. the sodium ion) is studied by nuclear magnetic resonance spectroscopy and is demonstrated to be a multifaced process, since Na(+) is in part free to exchange between the binding sites on nanotubes and the bulk and in part is confined to slowly exchanging nanotube sites. The aqueous titanate nanotube solutions are stable for months, thus opening new perspectives for the use of this material in drug delivery and in homogeneous photocatalysis.

Fast and simple method for the simultaneous evaluation of the capacity and efficiency of food antioxidants in trapping peroxyl radicals in an intestinal model system.

A simple oxygraphic method, for which the theoretical and experimental bases have been recently revised, has been successfully applied to evaluate the peroxyl radical chain-breaking characteristics of some typical food antioxidants in micelle systems, among which is a system that reproduces conditions present in the upper part of the digestive tract, where the absorption and digestion of lipids occur. This method permits one to obtain from a single experimental run the peroxyl radical trapping capacity (PRTC, that is, the number of moles of peroxyl radicals trapped by a given amount of food), the peroxyl radical trapping efficiency (PRTE, that is, the reciprocal of the amount of food that reduces to half the steady-state concentration of peroxyl radicals), and the half-life of the antioxidant ( t(1/2)) when only a small fraction of peroxyl radicals reacts with the antioxidants present in foods. Examples of application of the method to various types of foodstuffs have been reported, assessing the general validity of the method in the simple and fast evaluation of the above-reported fundamental antioxidant characteristics of foods.

A method to evaluate capacity and efficiency of water soluble antioxidants as peroxyl radical scavengers.

In this paper, we report on a method to evaluate the activity of water soluble and H-atom donor antioxidants as peroxyl radical scavengers in a micelle system reproducing the conditions occurring in the upper small intestine in humans, during digestion and absorption of lipids. This method, which overcomes some of the problems of the total radical trapping antioxidant parameter (TRAP) assays, measures the peroxyl radical trapping capacity (n) and the peroxyl radical trapping efficiency IC50(-1) of antioxidants, that is the number "n" of peroxyl radicals trapped by one molecule of the studied antioxidant and the reciprocal of the antioxidant concentration that halves the steady-state concentration of peroxyl radicals, respectively. These two fundamental parameters characterizing the radical chain breaking of many water soluble antioxidants, among which dietary polyphenols, can be obtained with relatively good precision from a single experiment, on the basis of a rigorous treatment of the kinetic data.

A coulometric biosensor to determine hydrogen peroxide using a monomolecular layer of horseradish peroxidase immobilized on a glass surface.

A biosensor to detect hydrogen peroxide, by coulometry, down to submicromolar concentration using a monomolecular layer of horseradish peroxidase was developed. In this device 0.3 pmol of the enzyme were covalently immobilized on the glass surface of the biosensor and the enzyme layer was characterized by atomic force microscopy and activity measurements. The glass surface bearing the peroxidase was faced to a carbon electrode in a cell of 1 microl of active volume. The polarization of the working electrode at -100 mV versus Ag/AgCl, in the presence of 1,4-hydroquinone as mediator, allowed the fast reduction of the injected hydrogen peroxide via the hydroquinone-peroxidase system. This device permitted to measure the total number of H(2)O(2) molecules present in the cell in the concentration range of 0.3-100 microM H(2)O(2), with a sensitivity of 196 nC/microM H(2)O(2), which is close to the theoretical value (193 nC/microM).

Stable free radicals and peroxyl radical trapping capacity in red wines.

Thirty-two experimental red wines, obtained from eight cultivars and aged in bottles for 2 and 7 years, were examined for the presence of stable free radicals (SFR), for the peroxyl radical trapping capacity (PRTC), and for the concentrations of some important polyphenol families. Aging significantly increases SFR, polyphenol polymers with n > or = 5 (HMWP), and PRTC and is accompanied by a strong decrease of free anthocyanins. Multivariate regression analyses show that HMWP and SFR are independently associated with PRTC while HMWP and anthocyanins are independently associated with the formation of SFR. These results indicate that polymeric polyphenols generated from anthocyanins and proanthocyanidins during wine aging are able to convert highly reactive free radicals into nonreactive radicals through electron delocalization. The strict correlation between SFR and antioxidant activity that we found suggests that these characteristics are related to the functional properties of food.

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element.

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.

Surface characterisation of bags for total parenteral nutrition by tensiometry and atomic force microscopy.

Bags made of poly-ethylene and poly-vinylchloride and of the copolymer ethylene-vinylacetate were used as containers of perfusion solutions for total parenteral nutrition. The bags were characterised by tensiometry (free energy and its polar and dispersed components) and atomic force microscopy (AFM) before and after various periods of storage of solutions for total parenteral nutrition containing L-aminoacids, electrolytes or glucose. In most of the cases, after storage of these solutions, tensiometric characterisation and atomic force microscopy analysis of the internal surface of bags showed deep modifications which highlight the adsorption of the solutes. The changes of surface characteristics were found to depend on the time of contact, the wettability of the polymer and the compounds present into the solutions, while their concentration has a negligible effect. Generally, the aminoacid solutions produced a higher increase in the polar component even after short storage times. Poly-ethylene and the copolymer ethylene-vinylacetate showed a greater inertia if compared with the poly-vinylchloride bags.

Determination of glucose oxidase immobilised as monolayer onto a flat surface.

An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.