Trophic ecology of Angolan cold-water coral reefs (SE Atlantic) based on stable isotope analyses.

Cold-water coral (CWC) reefs of the Angolan margin (SE Atlantic) are dominated by Desmophyllum pertusum and support a diverse community of associated fauna, despite hypoxic conditions. In this study, we use carbon and nitrogen stable isotope analyses (δ13C and δ15N) to decipher the trophic network of this relatively unknown CWC province. Although fresh phytodetritus is available to the reef, δ15N signatures indicate that CWCs (12.90 ± 1.00 ‰) sit two trophic levels above Suspended Particulate Organic Matter (SPOM) (4.23 ± 1.64 ‰) suggesting that CWCs are highly reliant on an intermediate food source, which may be zooplankton. Echinoderms and the polychaete Eunice norvegica occupy the same trophic guild, with high δ13C signatures (-14.00 ± 1.08 ‰) pointing to a predatory feeding behavior on CWCs and sponges, although detrital feeding on 13C enriched particles might also be important for this group. Sponges presented the highest δ15N values (20.20 ± 1.87 ‰), which could be due to the role of the sponge holobiont and bacterial food in driving intense nitrogen cycling processes in sponges' tissue, helping to cope with the hypoxic conditions of the reef. Our study provides first insights to understand trophic interactions of CWC reefs under low-oxygen conditions.

New Evidence on the Reliable Use of Stable Isotopes of Bitumen Fractions in Archaeological Research.

One of the goals of archaeological studies is to determine how material goods and ideas moved among human populations, and bitumen is a worthy proxy because it has been used since prehistory. As a result, when bitumen is excavated from archaeological sites, determining its provenance is important because it sheds light on the trade and communication of populations at a given time. During the study of archaeological bitumen from coastal sites in central and southern Puglia (Italy), we observed that stable isotope ratios of saturated and aromatic fractions were incompatible with those obtained from asphaltenes, supporting the absorption of a foreign substance. Experiments showed that lipids are absorbed by bitumen and, in the case of oils, are distributed mainly in the saturated and aromatic fractions as their isotopic ratios change. The same experiments showed that the isotopic ratios of the asphaltenes do not change. Lipid absorption on the archaeological bitumen may have occurred before the bitumen was applied to the pottery, during the use of the pottery or while underground, before being excavated. These hypotheses are discussed, and it is concluded that the isotopic ratio of asphaltenes is a reliable proxy for provenance, whereas those of the saturated and aromatic fractions should be considered with caution due to possible lipid absorption. Nevertheless, they provide new information on pottery use that can be used in archaeological chemistry.

HPLC-MS/MS method applied to an untargeted metabolomics approach for the diagnosis of "olive quick decline syndrome".

Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants.

Optimization of a new multi-reagent procedure for quantitative mussel digestion in microplastic analysis.

Over the last few years, different digestion protocols have been proposed to extract microplastics from mussels, an important product from aquaculture and a relevant economic resource, always scrutinized as a potential pollutant concentrator. In this study, a full factorial experimental design technique has been employed to achieve efficiency in removing biological materials while maximizing the recoveries of five common microplastics (polyethylene, polystyrene, polyethylene terephthalate, polypropylene and polyamide). A robust setpoint was calculated, 2.5% potassium hydroxide at 60 °C for 3 h with 5% hydrogen peroxide and 2.7% of methanol, permitting the quantitative digestion of mussel tissues and recovery of microplastics. These experimental conditions were successfully used to digest whole mussels bought from a local market, which possess high levels of microplastic contamination (41 items/g dry weight). The results highlight the importance of optimizing protocols to develop robust, easy to use and cheap quantitative approaches for analysing microplastic accumulation in edible organisms.

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

Decanoic Acid and Not Octanoic Acid Stimulates Fatty Acid Synthesis in U87MG Glioblastoma Cells: A Metabolomics Study.

Medium-chain fatty acids (MCFA) are dietary components with a chain length ranging from 6 to 12 carbon atoms. MCFA can cross the blood-brain barrier and in the brain can be oxidized through mitochondrial ß-oxidation. As components of ketogenic diets, MCFA have demonstrated beneficial effects on different brain diseases, such as traumatic brain injury, Alzheimer's disease, drug-resistant epilepsy, diabetes, and cancer. Despite the interest in MCFA effects, not much information is available about MCFA metabolism in the brain. In this study, with a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach, coupled with multivariate data analyses, we followed the metabolic changes of U87MG glioblastoma cells after the addition of octanoic (C8), or decanoic (C10) acids for 24 h. Our analysis highlighted significant differences in the metabolism of U87MG cells after the addition of C8 or C10 and identified several metabolites whose amount changed between the two groups of treated cells. Overall, metabolic pathway analyses suggested the citric acid cycle, Warburg effect, glutamine/glutamate metabolism, and ketone body metabolism as pathways influenced by C8 or C10 addition to U87MG cells. Our data demonstrated that, while C8 affected mitochondrial metabolism resulting in increased ketone body production, C10 mainly influenced cytosolic pathways by stimulating fatty acid synthesis. Moreover, glutamine might be the main substrate to support fatty acids synthesis in C10-treated cells. In conclusion, we identified a metabolic signature associated with C8 or C10 addition to U87MG cells that can be used to decipher metabolic responses of glioblastoma cells to MCFA treatment.

Bioactive composition and sensory evaluation of innovative spaghetti supplemented with free or α-cyclodextrin chlatrated pumpkin oil extracted by supercritical CO2.

The feasibility of producing durum wheat pasta enriched with a lipophilic phytocomplex, extracted using supercritical carbon dioxide (SC-CO2), from ripe pumpkin, as free oil or as ready-to-mix oil/α-cyclodextrins (α-CDs) powder, was explored. Four types of pasta were prepared: (i) control spaghetti (S-CTRL); (ii) spaghetti supplemented with α-CDs (S-α-CD); (iii) spaghetti supplemented with pumpkin oil (S-Oil) and (iv) spaghetti supplemented with the pumpkin oil/α-CD powder (S-Oil/α-CD). The chemical, antioxidant, textural and sensory attributes of the different pasta were evaluated and compared. S-Oil and S-Oil/α-CD spaghetti were significantly enriched with phytosterols, squalene, carotenoids, tocochromanols and unsaturated fatty acids. Spaghetti containing α-CDs were slightly improved in terms of fiber content. Oil chlatration increased the stability of some bioactives during pasta production and ameliorated poor textural and sensory characteristics of the cooked spaghetti compared with S-Oil sample. S-Oil/α-CD spaghetti might be accepted by customers, if the potential health benefits were also explained.

Volatolomics approach by HS-SPME-GC-MS and multivariate analysis to discriminate olive tree varieties infected by Xylella fastidiosa.

INTRODUCTION: Xylella fastidiosa (Xf) is a pathogenic bacterium that causes diseases in olive trees. Therefore, analytical methods for both the characterisation of the host/pathogen interaction and infection monitoring are needed. Volatile organic compounds (VOCs) are emitted by plants relate to their physiological state, therefore VOCs monitoring can assist in detecting stress or infection states before visible signs are present. OBJECTIVE: In this work, the headspace-solid phase microextraction-gaschromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the first time to highlight VOCs differences between healthy and Xf-infected olive trees. METHODOLOGY: VOCs from olive tree twig samples were extracted and analysed by HS-SPME-GC-MS, and hence identified by comparing the experimental linear retention indexes with the reference values and by MS data obtained from NIST library. Data were processed by principal component analysis (PCA) and analysis of variance (ANOVA). RESULTS: The HS-SPME step was optimised in terms of adsorbent phase and extraction time. HS-SPME-GC-MS technique was applied to the extraction and analysis of VOCs of healthy and Xf-infected olive trees. More than 100 compounds were identified and the differences between samples were evidenced by the multivariate analysis approach. The results showed the marked presence of methyl esters in Xf-infected samples, suggesting their probable involvement in the mechanism of diffusible signal factor. CONCLUSION: The proposed approach represents an easy and solvent-free method to evaluate the presence of Xf in olive trees, and to evidence volatiles produced by host/pathogen interactions that could be involved in the defensive mechanism of the olive tree and/or in the infective action of Xf.

Stimulatory Effects of Methyl-ß-cyclodextrin on Spiramycin Production and Physical-Chemical Characterization of Nonhost@Guest Complexes.

Spiramycin is a macrolide antibiotic and antiparasitic that is used to treat toxoplasmosis and various other infections of soft tissues. In the current study, we evaluated the effects of α-cyclodextrin, ß-cyclodextrin, or methyl-ß-cyclodextrin supplementation to a synthetic culture medium on biomass and spiramycin production by Streptomyces ambofaciens ATCC 23877. We found a high stimulatory effect on spiramycin production when the culture medium was supplemented with 0.5% (w/v) methyl-ß-cyclodextrin, whereas α-cyclodextrin or ß-cyclodextrin weakly enhanced antibiotic yields. As the stimulation of antibiotic production could be because of spiramycin complexation with cyclodextrins with effects on antibiotic stability and/or efflux, we analyzed the possible formation of complexes by physical-chemical methods. The results of Job plot experiment highlighted the formation of a nonhost@guest complex methyl-ß-cyclodextrin@spiramycin I in the stoichiometric ratio of 3:1 while they excluded the formation of complex between spiramycin I and α- or ß-cyclodextrin. Fourier-transform infrared spectroscopy measurements were then carried out to characterize the methyl-ß-cyclodextrin@spiramycin I complex and individuate the chemical groups involved in the binding mechanism. These findings may help to improve the spiramycin fermentation process, providing at the same time a new device for better delivery of the antibiotic at the site of infection by methyl-ß-cyclodextrin complexation, as it has been well-documented for other bioactive molecules.

Time-Resolved Transcriptomics and Constraint-Based Modeling Identify System-Level Metabolic Features and Overexpression Targets to Increase Spiramycin Production in Streptomyces ambofaciens.

In this study we have applied an integrated system biology approach to characterize the metabolic landscape of Streptomyces ambofaciens and to identify a list of potential metabolic engineering targets for the overproduction of the secondary metabolites in this microorganism. We focused on an often overlooked growth period (i.e., post-first rapid growth phase) and, by integrating constraint-based metabolic modeling with time resolved RNA-seq data, we depicted the main effects of changes in gene expression on the overall metabolic reprogramming occurring in S. ambofaciens. Moreover, through metabolic modeling, we unraveled a set of candidate overexpression gene targets hypothetically leading to spiramycin overproduction. Model predictions were experimentally validated by genetic manipulation of the recently described ethylmalonyl-CoA metabolic node, providing evidence that spiramycin productivity may be increased by enhancing the carbon flow through this pathway. The goal was achieved by over-expressing the ccr paralog srm4 in an ad hoc engineered plasmid. This work embeds the first metabolic reconstruction of S. ambofaciens and the successful experimental validation of model predictions and demonstrates the validity and the importance of in silico modeling tools for the overproduction of molecules with a biotechnological interest. Finally, the proposed metabolic reconstruction, which includes manually refined pathways for several secondary metabolites with antimicrobial activity, represents a solid platform for the future exploitation of S. ambofaciens biotechnological potential.

Accumulation of endocrine disrupting chemicals in the liver of Diplodus sargus sargus in Torre Guaceto Natural Reserve.

Studies on the quality of marine environments in Italy have traditionally favoured heavily impacted areas, such as harbours and industrial areas, while there are few investigations aimed at the evaluation of the presence of organic pollutants in the areas of marine reserve. The aim of this study was to determine endocrine-disrupting compounds (EDCs) in the liver of white seabreams (Diplodus sargus sargus) from the Natural Reserve of Torre Guaceto, Italy. Among EDCs, alkyl-phenols 2,6-DTBP, 2,4-DTBP, OP, and BPA were identified and quantified. The mean concentration of 2,6-DTBP, 2,4-DTBP, OP, and BPA were 2.2, 2.0, 1.2 and 3.6ng/g of liver, respectively, confirming the occurrence and transfer of these organic pollutants in the food web of marine organisms in marine reserves.

Bioanalytical Application of Amino Acid Detection by Capillary Electrophoresis.

This chapter illustrates the usefulness of capillary electrophoresis (CE) for the analysis of amino acids, and both normal and chiral separations are covered. In order to provide a general description of the main results and challenges in the biomedical field, some relevant applications and reviews on CE of amino acids are tabulated. Furthermore, some detailed experimental procedures are shown, regarding the CE analysis of amino acids in body fluids, in microdialysate, and released upon hydrolysis of proteins. In particular, the protocols will deal with the following compounds: (1) underivatized aminoacids in blood; (2) γ-Aminobutyric acid, glutamate, and L-Aspartate derivatized with Naphthalene-2,3-dicarboxaldehyde; (3) hydrolysate from bovine serum albumine derivatized with phenylisothiocyanate. By examining these applications on real matrices, the capillary electrophoresis efficiency as tool for Amino Acid analysis can be ascertained.

New evidences on efficacy of boronic acid-based derivatization method to identify sugars in plant material by gas chromatography-mass spectrometry.

This work presents an analytical procedure based on gas chromatography-mass spectrometry which allows the determination of aldoses (glucose, mannose, galactose, arabinose, xylose, fucose, rhamnose) and chetoses (fructose) in plant material. One peak for each target carbohydrate was obtained by using an efficient derivatization employing methylboronic acid and acetic anhydride sequentially, whereas the baseline separation of the analytes was accomplished using an ionic liquid capillary column. First, the proposed method was optimized and validated. Successively, it was applied to identify the carbohydrates present in plant material. Finally, the procedure was successfully applied to samples from a XVII century painting, thus highlighting the occurrence of starch glue and fruit tree gum as polysaccharide materials.

A conserved role for the mitochondrial citrate transporter Sea/SLC25A1 in the maintenance of chromosome integrity.

Histone acetylation plays essential roles in cell cycle progression, DNA repair, gene expression and silencing. Although the knowledge regarding the roles of acetylation of histone lysine residues is rapidly growing, very little is known about the biochemical pathways providing the nucleus with metabolites necessary for physiological chromatin acetylation. Here, we show that mutations in the scheggia (sea)-encoded Sea protein, the Drosophila ortholog of the human mitochondrial citrate carrier Solute carrier 25 A1 (SLC25A1), impair citrate transport from mitochondria to the cytosol. Interestingly, inhibition of sea expression results in extensive chromosome breakage in mitotic cells and induces an ATR-dependent cell cycle arrest associated with a dramatic reduction of global histone acetylation. Notably, loss of SLC25A1 in short interfering RNA (siRNA)-treated human primary fibroblasts also leads to chromosome breaks and histone acetylation defects, suggesting an evolutionary conserved role for Sea/SLC25A1 in the regulation of chromosome integrity. This study therefore provides an intriguing and unexpected link between intermediary metabolism and epigenetic control of genome stability.

A new CE-ESI-MS method for the detection of stable hemoglobin acetaldehyde adducts, potential biomarkers of alcohol abuse.

A new CE-ESI-MS method was developed to provide a simple way to study changes to hemoglobin (HbA) induced by acetaldehyde (Ach) in vitro. Instrumental parameters were univariately optimized in order to maximize the sensitivity of the CE-ESI-MS method. The electrophoretic separations were carried out in poly-E323-coated capillaries using 60 mM formic acid raised to pH 3.0 with ammonia and containing 5% 2-propanol while the sheath liquid, 2-propanol/water (30:70) with 0.1% formic acid, was delivered at 1.0 microL/min through a coaxial sheath flow electrospray interface. The HbA was incubated with Ach for intervals up to 24 h at concentration varying in the window 0.2-20 mM. Four stable Ach-hemoglobin adducts in the hemoglobin tryptic digest were observed at the submillimolar Ach concentration and characterized by MS/MS experiments: although the alpha and beta N-amino terminal modifications were expected, the two internal ones arising, respectively, from the condensation of Ach molecules on the histidine residue in position 4 in alpha4 (i.e. the fourth peptide after tryptic digestion of alpha chain starting from amino terminal) and on the asparagine residue in position 2 in beta3, were identified for the first time. During the in vitro experiments higher concentrations of Ach were also used; however, it was not possible to identify any other stable modification of hemoglobin. Interestingly, those stable modifications are the only ones in vivo identified in the hemoglobin of moderate alcohol drinkers.

Biomedical applications of amino acid detection by capillary electrophoresis.

Capillary electrophoresis (CE) is an efficient tool for amino acid (AA) analysis. However, its role can be fully accounted for only by examining the applications on real matrices. Methods must be successfully transferred into working environments for use by non-CE experts before their power can be realized. This transfer of technology is rapidly increasing. In this chapter, some applications to real samples are presented with the precise intent to illustrate the great capabilities of CE to AA analysis in clinical applications.

Solid-phase microextraction-gas chromatography mass spectrometry and multivariate analysis for the characterization of roasted coffees.

HS-SPME-GC-MS in combination with PCA was employed to discriminate different arabica/robusta blends having different geographical origins. HS-SPME confirmed to be an effective and reproducible sampling technique for routine characterization of coffees. In addition, the chemometric approach permitted to find parameters suitable for the differentiation of the different blends and the determination of the real quality of the products. Finally, the proposed methods have been successfully applied to some commercial coffee blends.

Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection.

A method was developed for the quantitative determination of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), using free solution capillary zone electrophoresis in uncoated fused-silica capillaries with laser-induced fluorescence (LIF) detection. Various factors influencing the separation and detection of flavin vitamers were investigated, including pH (5.5-10.5), concentration and nature of the run buffer (phosphate, borate and carbonate), applied voltage (15-30 kV), temperature (15-30 degrees C) and injection time. Optimal resolution and detection were obtained with a pH 9.8, 30 mM aqueous phosphate buffer at 15 degrees C and 30 kV of applied voltage. LIF detection was obtained with a He-Cd laser source using an excitation wavelength at 442 nm and lambda(em) > or = 515 nm. Riboflavin could be determined in the concentration ranges 0.5-350 microg/l with a rather low detection limit (LOD) down to 50 amol. The LODs of FAD and FMN were slightly higher, 300 and 350 amol, respectively. Combined with a simple clean-up procedure, the practical utility of this method is illustrated by the measurements of flavin derivates in foods and beverages, such as wines, milk, yoghurt and raw eggs.