Urinary volatile Organic compounds in non-alcoholic fatty liver disease (NAFLD), type two diabetes mellitus (T2DM) and NAFLD-T2DM coexistence.

INTRODUCTION: Accumulating evidence have shown a significant correlation between urinary volatile organic compounds (VOCs) profile and the manifestation of several physiological and pathological states, including liver diseases. Previous studies have investigated the urinary metabolic signature as a non-invasive tool for the early discrimination between non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), which nowadays represents one of the most important challenges in this context, feasible only by carrying out liver biopsy. OBJECTIVES: The aim of the study was to investigate the differences in the urinary VOCs profiles of non-alcoholic fatty liver disease (NAFLD) patients, diabetes mellitus (T2DM) subjects and NAFLD/T2DM patients. METHODS: Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was applied to profile the urinary VOCs. Urine samples were analysed both under acid and alkaline conditions, to obtain a range of urinary volatiles with different physicochemical properties. RESULTS: Urinary VOCs profiles of 13 NAFLD patients, 13 T2DM subjects and 13 NAFLD/T2DM patients were investigated by multivariate and univariate data analysis techniques which allowed to identify 21 volatiles under alkaline conditions able to describe the NAFLD/T2DM group concerning the other two groups. CONCLUSION: Our results suggest that VOCs signatures can improve the knowledge of the pathological condition where NAFLD coexists with T2DM and discovering new features that are not simply the sum of the two diseases. These preliminary findings may be considered as hypothesis-generating, to be clearly confirmed by larger prospective investigations.

Chemical Characterization and Biological Properties of Leguminous Honey.

Honey can beneficially act against different human diseases, helping our body to improve its health. The aim of the present study was first to increase knowledge of some biochemical characteristics (amount and composition of polyphenols and volatile organic compounds, vitamin C content) of five Italian legume honeys (alfalfa, astragalus, carob, indigo, and sainfoin). Furthermore, we evaluated their potential health properties by studying their antioxidant and in vitro anti-inflammatory activities and in vitro inhibitory effects on three enzymes involved in neurodegenerative diseases (acetylcholinesterase, butyrylcholinesterase, and tyrosinase). Alfalfa honey showed the highest total polyphenol content (TPC) (408 µg g-1 of product). Indigo honey showed the lowest TPC (110 µg g-1 of product). The antioxidant activity was noteworthy, especially in the case of sainfoin honey (IC50 = 6.08 mg), which also exhibited excellent inhibitory action against butyrylcholinesterase (74%). Finally, the correlation between the biochemical and functional results allowed us to identify classes of molecules, or even single molecules, present in these five honeys, which are capable of influencing the properties indicated above.

Antibacterial Activity and Prebiotic Properties of Six Types of Lamiaceae Honey.

Our work investigated the antimicrobial and prebiotic properties of basil, mint, oregano, rosemary, savory, and thyme honey. The potential antimicrobial action, assessed against the pathogens Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, evidenced the capacity of the honey to influence the pathogenic hydrophobicity and hemolytic activities. Honey inhibited pathogen biofilms, acting especially on the mature biofilms, with inhibition rates of up to 81.62% (caused by the presence of mint honey on L. monocytogenes). S. aureus biofilms were the most susceptible to the presence of honey, with inhibition rates up of to 67.38% in the immature form (caused by basil honey) and up to 80.32% in the mature form (caused by mint honey). In some cases, the amount of nuclear and proteic material, evaluated by spectrophotometric readings, if also related to the honey's biofilm inhibitory activity, let us hypothesize a defective capacity of building the biofilm scaffold or bacterial membrane damage or an incapability of producing them for the biofilm scaffold. The prebiotic potentiality of the honey was assessed on Lacticaseibacillus casei Shirota, Lactobacillus gasseri, Lacticaseibacillus paracasei subsp. paracasei, and Lacticaseibacillus rhamnosus and indicated their capacity to affect the whole probiotic growth and in vitro adhesive capacity, as well as the antioxidant and cytotoxic abilities, and to inhibit, mainly in the test performed with the L. casei Shirota, L. gasseri, and L. paracasei supernatants, the immature biofilm of the pathogens mentioned above.

In Vitro Prebiotic Effects and Antibacterial Activity of Five Leguminous Honeys.

Honey is a natural remedy for various health conditions. It exhibits a prebiotic effect on the gut microbiome, including lactobacilli, essential for maintaining gut health and regulating the im-mune system. In addition, monofloral honey can show peculiar therapeutic properties. We in-vestigated some legumes honey's prebiotic properties and potential antimicrobial action against different pathogens. We assessed the prebiotic potentiality of honey by evaluating the antioxidant activity, the growth, and the in vitro adhesion of Lacticaseibacillus casei, Lactobacillus gasseri, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus intact cells. We also tested the honey's capacity to inhibit or limit the biofilm produced by five pathogenic strains. Finally, we assessed the anti-biofilm activity of the growth medium of probiotics cultured with honey as an energy source. Most probiotics increased their growth or the in vitro adhesion ability to 84.13% and 48.67%, respectively. Overall, alfalfa honey best influenced the probiotic strains' growth and in vitro adhesion properties. Their radical-scavenging activity arrived at 83.7%. All types of honey increased the antioxidant activity of the probiotic cells, except for the less sensitive L. plantarum. Except for a few cases, we observed a bio-film-inhibitory action of all legumes' honey, with percentages up to 81.71%. Carob honey was the most effective in inhibiting the biofilm of Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus; it retained almost entirely the ability to act against the bio-film of E. coli, L. monocytogenes, and S. aureus also when added to the bacterial growth medium instead of glucose. On the other hand, alfalfa and astragalus honey exhibited greater efficacy in acting against the biofilm of Acinetobacter baumannii. Indigo honey, whose biofilm-inhibitory action was fragile per se, was very effective when we added it to the culture broth of L. casei, whose supernatant exhibited an anti-biofilm activity against all the pathogenic strains tested. Conclusions: the five kinds of honey in different ways can improve some prebiotic properties and have an inhibitory biofilm effect when consumed.

Profiles of Volatile and Phenolic Compounds as Markers of Ripening Stage in Candonga Strawberries.

Volatile compounds, quality traits (total phenols and antioxidant capacity) and High-performance liquid chromatography (HPLC)-isolated polyphenols of strawberries, variety Sabrosa, commercially referred to as "Candonga", harvested at three different times (H1, H2 and H3) and at two different ripening stages, namely half-red (Half-red-H1, Half-red-H2 and Half-red-H3) and red (Red-H1, Red-H2 and Red-H3) were evaluated. Dominant anthocyanins, namely cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside, as well as p-coumaryl hexoside increased during harvesting, differently from flavonoids, such as quercetin-3-O-glucoside, kaempferol-3-O-glucoronide and quercetin 3-O-glucoronide, that declined. Samples clustered in different quadrants of the principal component analysis (PCA) performed on volatiles, quality traits and phenolic compounds, highlighting that only the red samples were directly correlated to volatile components, as volatiles clearly increased both in number and amount during ripening. In particular, volatiles with a positive impact on the consumers' acceptance, including butyl butyrate, ethyl hexanoate, hexyl acetate, nonanal, terpenes and lactones, were positively associated with the Red-H1 and Red-H2 strawberries, while volatiles with negative coefficients related to consumer liking, including isopropyl butyrate, isoamyl butyrate and mesifurane directly correlated with the Red-H3 samples. Accordingly, strawberries harvested at Red-H1 and Red-H2 ripening stages could be preferred by the consumers compared to the Red-H3 fruit. Altogether, these results could help to individuate quality traits as putative markers of the ripening stage, and optimize the process of post-harvesting ripening to preserve or improve the desirable aromatic characteristics of strawberries.

Changes in visual quality, physiological and biochemical parameters assessed during the postharvest storage at chilling or non-chilling temperatures of three sweet basil (Ocimum basilicum L.) cultivars.

Leaves of three different sweet basil (Ocimum basilicum L.) cultivars (Italico a foglia larga, Cammeo, and Italiano classico) packed in macro-perforated polyethylene bags were stored at chilling (4°C) or non-chilling temperature (12°C) for 9days. During storage, visual quality, physiological (respiration rate, ethylene production, ammonium content) and chemical (antioxidant activity, total polyphenols and polyphenol profile) parameters were measured. Detached leaves stored at chilling temperature showed visual symptoms related to chilling injury, while ethylene production and ammonium content resulted associated to cultivar sensibility to damage at low temperature. Storage at 4°C caused a depletion in polyphenols content and antioxidant capability, which was preserved at 12°C. Regarding the polyphenols profile, stressful storage conditions did not enhance the phenolic metabolism. However, leaves stored at 12°C did not loss a significant amount of metabolites respect to fresh leaves, suggesting the possibility to extend the storability after the expiration date, for a possible recovery of bioactive compounds.

Urinary volatile organic compounds in overweight compared to normal-weight children: results from the Italian I.Family cohort.

Accumulating evidence shows that urinary volatile organic compounds (VOCs) could be perturbed in many physiological and pathological states, including several diseases and different dietary exposures. Few studies investigated the urinary metabolic signature associated to excess body weight and obesity in adult populations, while a different VOCs profile was found in exhaled breath in obese as compared to lean children. Aim of this study was to evaluate the VOCs profile in the urine of 21 overweight/obese (OW/Ob) and 28 normal-weight (NW) children belonging to the Italian cohort of the I. Family study. Urine samples were analysed by Solid Phase Micro-Extraction (SPME) GC-MS under both acidic and alkaline conditions, in order to profile a wider range of urinary volatiles with different physicochemical properties. Multivariate statistics techniques were applied to bioanalytical data to visualize clusters of cases and detect the VOCs able to differentiate OW/Ob from NW children. Under alkaline conditions, fourteen VOCs were identified, distinguishing OW/Ob from NW children. Our results suggest that VOCs signatures differ between OW/Ob and NW children. However, the biological and pathophysiological meaning of the observed differences needs to be elucidated, in order to better understand the potential of urinary VOCs as early metabolic biomarkers of obesity.

Assessment of volatile profile as potential marker of chilling injury of basil leaves during postharvest storage.

The volatile profile of three sweet basil cultivars, "Italico a foglia larga", "Cammeo" and "Italiano classico", packaged in air at 4 or 12°C until 9days, was monitored by solid phase microextraction with GC-MS. Chilling injury (CI) score and electrolyte leakage were also assessed. In total, 71 volatile organic compounds (VOCs) were identified in the headspace of basil samples. A preliminary principal component analysis highlighted the dominant effect of the cultivar on VOCs profiles. Data analysis by post-transformation of projection to latent structures regression (ptPLS2) clarified the role played by time and temperature of storage. Temperature influenced the emission of volatiles during storage, with much lower total volatile emissions at 4°C compared to 12°C. Finally, a ptPLS2 regression model performed on VOCs and the two CI parameters allowed selection of 10 metabolites inversely correlated to both CI parameters, which can be considered potential markers of CI in basil leaves.

Characterisation of volatile profile and sensory analysis of fresh-cut "Radicchio di Chioggia" stored in air or modified atmosphere.

The volatile profile of two hybrids of "Radicchio di Chioggia", Corelli and Botticelli, stored in air or passive modified atmosphere (MAP) during 12 days of cold storage, was monitored by solid phase micro-extraction (SPME) GC-MS. Botticelli samples were also subjected to sensory analysis. Totally, 61 volatile organic compounds (VOCs) were identified in the headspace of radicchio samples. Principal component analysis (PCA) showed that fresh product possessed a metabolic content similar to that of the MAP samples after 5 and 8 days of storage. Projection to latent structures by partial least squares (PLS) regression analysis showed the volatiles content of the samples varied depending only on the packaging conditions. Specifically, 12 metabolites describing the time evolution and explaining the effects of the different storage conditions were highlighted. Finally, a PCA analysis revealed that VOCs profile significantly correlated with sensory attributes.

Determination of volatile organic compounds in the dried leaves of Salvia species by solid-phase microextraction coupled to gas chromatography mass spectrometry.

Salvia spp. are used throughout the world both for food and pharmaceutical purposes. In this study, a method involving headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was developed, to establish the volatiles profile of dried leaves of four Iranian Salvia spp.: Salvia officinalis L., Salvia leriifolia Benth, Salvia macrosiphon Boiss. and two ecotypes of Salvia reuterana Boiss. A total of 95 volatiles were identified from the dried leaves of the five selected samples. Specifically, α-thujone was the main component of S. officinalis L. and S. macrosiphon Boiss. (34.40 and 17.84%, respectively) dried leaves, S. leriifolia Benth was dominated by ß-pinene (27.03%), whereas α-terpinene was the major constituent of the two ecotypes of S. reuterana Boiss. (21.67 and 13.84%, respectively). These results suggested that the proposed method can be considered as a reliable technique for isolating volatiles from aromatic plants, and for plant differentiation based on the volatile metabolomic profile.

Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies.

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.

Bovine lactoferrin-derived peptides as novel broad-spectrum inhibitors of influenza virus.

Bovine lactoferrin (bLf) is a multifunctional glycoprotein that plays an important role in innate immunity against infections, including influenza. Here we have dissected bLf into its C- and N-lobes and show that inhibition of influenza virus hemagglutination and cell infection is entirely attributable to the C-lobe and that all major virus subtypes, including H1N1 and H3N2, are inhibited. By far-western blotting and sequencing studies, we demonstrate that bLf C-lobe strongly binds to the HA(2) region of viral hemagglutinin, precisely the highly conserved region containing the fusion peptide. By molecular docking studies, three C-lobe fragments were identified which inhibited virus hemagglutination and infection at fentomolar concentration range. Besides contributing to explain the broad anti-influenza activity of bLf, our findings lay the foundations for exploiting bLf fragments as source of potential anti-influenza therapeutics.

Gliadin regulates the NK-dendritic cell cross-talk by HLA-E surface stabilization.

We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-gamma production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The alpha and omega fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin alpha increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.

Ion trap mass spectrometry in the structural analysis of haemoglobin peptides modified by epichlorohydrin and diepoxybutane.

Ion trap mass spectrometry has been shown to be particularly suitable for the structural analysis of high molecular weight peptides directly fragmented in the mass analyser without needing further sub-digestion reactions. Here we report the advantages of using multi-stage ion trap mass spectrometry in the structural characterisation of haemoglobin alkylated with epichlorohydrin and diepoxybutane. Alkylated globins were digested with trypsin and the peptide mixtures were analysed by MS(3). This technique allows the sequential fragmentation of peptides under analysis, giving rise to MS(3) product ion spectra with additional information with respect to MS(2) mass spectra. The results obtained complete the previously reported structural characterisation of alkylated haemoglobin, demonstrating the potential of ion trap mass spectrometry.

Proteolytic activity of bovine lactoferrin.

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.