Application of NMR-based metabolomics techniques to biological systems: a case study on bivalves

In the past years, metabolomics has progressed greatly, providing a reliable and high-throughput approach particularly feasible for the study of complex biological systems. Indeed, thanks to the development of powerful analytical methods capable of screening a large number of chemical compounds in a sample, nowadays metabolomics plays an important role in gaining biological insights toward the influence of internal (genetic and developmental) and external (environmental) factors on phenotypes. The work presented in this Ph.D. thesis shows examples of applications of NMR-based metabolomics to the study of bivalves, facing challenges of interest in both food and environmental sciences. The outcome of such studies yield insights, at molecular level, into several aspects concerning the impact of different storage conditions on shellfish quality and the effect of natural and anthropogenic environmental stressors on bivalves’ metabolic profiles. Firstly, the effect of different cold storage conditions on the hydrosoluble chemical components of Mytilus galloprovincialis (Lamarck, 1819) was investigated for the first time by NMR-based metabolomics. The data revealed substantial time-related changes in the metabolic profiles of mussels stored at 0 °C and 4 °C. The observed biochemical modifications were in good agreement with the microbiological quality of samples, reflecting changes in their microbial loads. These results confirmed the potential use of metabolomics as a reliable method to assess seafood freshness. Secondly, a metabolomic approach was also applied to study the effect of short-term exposure to heavy metals on two different clams’ species: Ruditapes decussatus (Linnaeus, 1758) and Ruditapes philippinarum (Adams & Reeve, 1850). Heavy metals are considered to be among the most harmful pollutants that can contaminate marine environments. The toxicity of trace metals gives rise from their persistent nature which lead to environmental accumulation. Given the high accumulation rates associated with their filter feeding attitude, bivalves mollusks are considered to be feasible monitoring organisms and are widely used in biomonitoring programs. R. decussatus and R. philippinarum, two bivalves’ species widely distributed along the Italian coasts, were selected in this Ph.D. project for assessing lead and zinc effects on their metabolic profiles. The results evidenced a sensible short-time metabolic response upon metal exposure, pointing out a main variability in the content of amino acids and organic osmolytes in relation to both metal nature and bivalve species. These findings show that NMR-based metabolomics has the required sensitivity and specificity to gain insights into the biochemical consequences arising upon heavy metals exposure, providing thus a useful tool for the identification of putative biomarkers as fast and sensitive indicators of contaminant-induced stress. Finally, 1H NMR-based metabolomics was applied with the aim to assess the effects of seasonal change on Ruditapes decussatus metabolic profile. The results of the present study demonstrated that the combined use of advanced multivariate statistical techniques with NMR spectroscopy is a feasible approach to discriminate specimens of R. decussatus according to the sampling season. Moreover, the sensitivity of this analytical tool allowed the individuation of those metabolites whose relative amount significantly varied according to seasonal change (alanine and glycine), paving the way for further investigations that would contribute to achieve additional insights on bivalves’ bio-ecological framework

Application of NMR-based metabolomics to the study of marine bivalves

This paper reviews the results of three studies on the application of high-field proton Nuclear Magnetic Resonance (1 H NMR) spectroscopy and metabolomics to the assessment of different issues related to marine bivalves. The outcomes of such applications yielded insights into multiple aspects concerning the impact of cold storage on shellfish quality and the effect of natural and anthropogenic stressors on bivalve metabolic profiles. In the first work, the effect of different cold storage conditions on the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated, revealing substantial time-related changes both in the metabolic profiles and microbial loads of stored mussels. In the second study, the seasonal change and reproductive cycle were demonstrated to be the leading causes behind the fluctuation in the amino acid and carbohydrate contents in the European clam Ruditapes decussatus (Linnaeus, 1758). Finally, NMR metabolomics was applied for assessing the effect of lead and zinc short-term exposure on the metabolic profiles of two clam species, Ruditapes decussatus and Ruditapes philippinarum (Adams & Reeve, 1850). The results evidenced a sensible metabolic response upon metal pollution, pointing out a main variability in the content of amino acids and organic osmolytes in relation to both metal nature and bivalve species

Effects of Water Stress, Defoliation and Crop Thinning on Vitis vinifera L. cv. Solaris Must and Wine Part II: 1HNMR Metabolomics

Proton nuclear magnetic resonance (1H NMR) metabolomics was employed to investigate the impact of water deficit, defoliation, and crop thinning on the chemical composition of must and wines from the cool-climate white grape variety Solaris. The obtained results show that viticultural practices (defoliation and crop thinning) affected the amino acid and sugar content of Solaris must and thereby the quality of the final wine—mainly in terms of compounds normally related to fruity aroma (i.e., isopentanol), non-sugar sweetness (i.e., proline and glycerol), and alcohol content. The content of tyrosol, a natural phenolic antioxidant with a high bioavailability, was increased in the final wine by a combination of defoliation and crop thinning. The results of the metabolomics analysis performed on the must and wine samples from the water stress experiment showed that short-term water deficit significantly affected the concentration of several flavor-related compounds, including glutamate, butyrate and propanol, of the organic acids lactate and fumarate, and of the phenolic compounds caffeic acid and p-coumaric acid. ANOVA simultaneous component analysis showed that the effect of water deficit accounted for 11% (p < 0.001) and 8% (p < 0.001) of the variability in the metabolite concentrations in must and wines, respectively, while viticultural practices accounted for 38% (p < 0.001) and 30% (p < 0.001) of the metabolite variability in must and wines, respectivelyinfo:eu-repo/semantics/publishedVersio

Effects of Water Stress, Defoliation and Crop Thinning on Vitis vinifera L. cv. Solaris: Part I: Plant Responses, Fruit Development and Fruit Quality

Viticultural practices and irrigation have a major impact on fruit development and yield, and ultimately on must quality. The effects of water deficit (WD), defoliation (Def), and crop-thinning (CT) on Solaris plants and fruit development, as well as on the chemical composition of grape juice were investigated. WD was induced at three periods during fruit development (pre-veraison, veraison, and ripening) in pot-grown plants, while Def and CT were carried out on field-grown plants. Environmental and vegetative parameters were monitored during the experiments. The bulk chemical composition of the fruits was determined in juice by Fourier Transform-Infrared (FT-IR) spectroscopy throughout fruit ripening and at final harvest. The results showed that WD reduced soil water content and leaf water status. CT significantly reduced yield per vine, but increased cluster size. Mid to late WD reduced soluble solids by 1%. CT increased sugar content in juice, while Def decreased sugar accumulation. Total acids were higher in the juice from the field vines. Yet, CT lowered malic and tartaric acids. Def increased tartaric acid. Ammonia and alpha amino nitrogen were higher in the juice from pot-grown vines, while pH was lowered by Def and raised by CT. It is concluded that Solaris has a remarkable ability to tolerate and recover from WD. CT and Def significantly affected the bulk chemical composition of grapes in terms of total acidity and sugar accumulation, with CT grapes having the highest sugar content and the lowest total acidity and Def the oppositeinfo:eu-repo/semantics/publishedVersio

Effects of functional ingredients on gut inflammation in Atlantic salmon (Salmo salar L)

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The Effect of Season on the Metabolic Profile of the European Clam Ruditapes decussatus as Studied by 1H-NMR Spectroscopy

In this study, the metabolome of Ruditapes decussatus, an economically and ecologically important marine bivalve species widely distributed in the Mediterranean region, was characterized by using proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy. Significant seasonal variations in the content of carbohydrates and free amino acids were observed. The relative amounts of alanine and glycine were found to exhibit the same seasonal pattern as the temperature and salinity at the harvesting site. Several putative sex-specific biomarkers were also discovered. Substantial differences were found for alanine and glycine, whose relative amounts were higher in males, while acetoacetate, choline and phosphocholine were more abundant in female clams. These findings reveal novel insights into the baseline metabolism of the European clam and represent a step forward towards a comprehensive metabolic characterization of the species. Besides providing a holistic view on the prominent nutritional components, the characterization of the metabolome of this bivalve represents an important prerequisite for elucidating the underlying metabolic pathways behind the environment-organism interactions

Effects of Water Stress, Defoliation and Crop Thinning on Vitis vinifera L. cv. Solaris: Part I: Plant Responses, Fruit Development and Fruit Quality

Viticultural practices and irrigation have a major impact on fruit development and yield, and ultimately on must quality. The effects of water deficit (WD), defoliation (Def), and crop-thinning (CT) on Solaris plants and fruit development, as well as on the chemical composition of grape juice were investigated. WD was induced at three periods during fruit development (pre-veraison, veraison, and ripening) in pot-grown plants, while Def and CT were carried out on field-grown plants. Environmental and vegetative parameters were monitored during the experiments. The bulk chemical composition of the fruits was determined in juice by Fourier Transform-Infrared (FT-IR) spectroscopy throughout fruit ripening and at final harvest. The results showed that WD reduced soil water content and leaf water status. CT significantly reduced yield per vine, but increased cluster size. Mid to late WD reduced soluble solids by 1%. CT increased sugar content in juice, while Def decreased sugar accumulation. Total acids were higher in the juice from the field vines. Yet, CT lowered malic and tartaric acids. Def increased tartaric acid. Ammonia and alpha amino nitrogen were higher in the juice from pot-grown vines, while pH was lowered by Def and raised by CT. It is concluded that Solaris has a remarkable ability to tolerate and recover from WD. CT and Def significantly affected the bulk chemical composition of grapes in terms of total acidity and sugar accumulation, with CT grapes having the highest sugar content and the lowest total acidity and Def the opposite

<i>Saccharomyces cerevisiae</i> does not undergo a quorum sensing-dependent switch of budding pattern

Saccharomyces cerevisiae can alter its morphology to a filamentous form associated with unipolar budding in response to environmental stressors. Induction of filamentous growth is suggested under nitrogen deficiency in response to alcoholic signalling molecules through quorum sensing. To investigate this further, we analysed the budding pattern of S. cerevisiae cells over time under low nitrogen conditions while concurrently measuring cell density and extracellular metabolite concentration. We found that the proportion of cells displaying unipolar budding increased between local cell densities of 4.8 × 10(6) and 5.3 × 10(7) cells/ml. This increase in unipolar budding was not reproduced with cells growing at the critical cell density and in conditioned media. Growth under high nitrogen conditions also resulted in increased unipolar budding between local cell densities of 5.2 × 10(6) and 8.2 × 10(7) cells/ml, but with differences in metabolite concentration compared to low nitrogen conditions. Neither cell density, metabolite concentration, nor nitrogen deficiency were therefore sufficient to increase unipolar budding. Therefore, by using the budding pattern as an early indicator of filamentous growth, our results suggest that quorum sensing may not control the switch of budding behaviour in S. cerevisiae. Only a high concentration of the putative signalling molecule, 2-phenylethanol, resulted in an increase in unipolar budding. However, this concentration was not physiologically relevant, suggesting toxicity rather than a known quorum sensing mechanism