Integration of LC/MS-based molecular networking and classical phytochemical approach allows in-depth annotation of the metabolome of non-model organisms - The case study of the brown seaweed Taonia atomaria.

Untargeted LC-MS based metabolomics is a useful approach in many research areas such as medicine, systems biology, environmental sciences or even ecology. In such an approach, annotation of metabolomes of non-model organisms remains a significant challenge. In this study, an analytical workflow combining a classical phytochemical approach, using the isolation and the full characterization of the chemical structure of natural products, together with the use of MS/MS-based molecular networking with various levels of restrictiveness was developed. This protocol was applied to the marine brown seaweed Taonia atomaria, a cosmopolitan algal species, and allowed to annotate more than 200 metabolites. First, the algal organic crude extracts were fractionated by flash-chromatography and the chemical structure of eight of the main chemical constituents of this alga were fully characterized by means of spectroscopic methods (1D and 2D NMR, HRMS). These compounds were further used as chemical standards. In a second step, the main fractions of the algal extracts were analyzed by UHPLC-MS/MS and the resulting data were uploaded to the Global Natural Products Social Molecular Networking platform (GNPS) to create several molecular networks (MNs). A first MN (MN-1) was built with restrictive parameters and allowed the creation of clusters composed by nodes with highly similar MS/MS spectra. Then, using database hits and chemical standards as "seed" nodes and/or similarity between MS/MS fragmentation pattern, the main clusters were easily annotated as common glycerolipids and phospholipids, much rare lipids -such as acylglycerylhydroxymethyl-N,N,N-trimethyl-ß-alanines or fulvellic acid derivatives- but also new glycerolipids bearing a terpene moiety. Lastly, the use of less and less constrained MNs allowed to further increase the number of annotated metabolites.

Humic-like acids from hydrochars: Study of the metal complexation properties compared with humic acids from anthropogenic soils using PARAFAC and time-resolved fluorescence.

Humic acids (HA) play an important role in the distribution, toxicity, and bioavailability of metals in the environment. Humic-like acids (HLA) that simulate geochemical processes can be prepared by NaOH aqueous extraction from hydrochars produced by hydrothermal carbonization (HTC). HLA can exhibit properties such as those found in HA from soils, which are known for their ability to interact with inorganic and organic compounds. The molecular characteristics of HLA and HA help to explain the relationship between their molecular features and their interaction with metallic species. The aim of this study is to assess the molecular features of HA extracted from Terra Mulata (TM) and HLA from hydrochars as well as their interaction with metals by using Cu(II) ions as a model. The results from 13C NMR, elemental analysis, FTIR, and UV-Vis showed that HA are composed mostly of aromatic structures and oxygenated functional groups, whereas HLA showed a mutual contribution of aromatic and aliphatic structures as main constituents. The interactions of HA and HLA with Cu(II) ions were evaluated through fluorescence quenching, in which the density of complexing sites per gram of carbon for interaction was higher for HLA than for HA. Furthermore, the HLA showed similar values for stability constants, and higher than those found for other types of HA in the literature. In addition, the average lifetime in both humic extracts appeared to be independent of the copper addition, indicating that the main mechanism of interaction was static quenching with a non-fluorescent ground-state complex formation. Therefore, the HLA showed the ability to interact with Cu(II) ions, which suggests that their application can provide a new approach for remediation of contaminated areas.

Changes in fatty acid composition in the giant clam Tridacna maxima in response to thermal stress.

Temperature can modify membrane fluidity and thus affects cellular functions and physiological activities. This study examines lipid remodelling in the marine symbiotic organism, Tridacna maxima, during a time series of induced thermal stress, with an emphasis on the morphology of their symbiont Symbiodinium First, we show that the French Polynesian giant clams harbour an important proportion of saturated fatty acids (SFA), which reflects their tropical location. Second, in contrast to most marine organisms, the total lipid content in giant clams remained constant under stress, though some changes in their composition were shown. Third, the stress-induced changes in fatty acid (FA) diversity were accompanied by an upregulation of genes involved in lipids and ROS pathways. Finally, our microscopic analysis revealed that for the giant clam's symbiont, Symbiodinium, thermal stress led to two sequential cell death processes. Our data suggests that the degradation of Symbiodinium cells could provide an additional source of energy to T maxima in response to heat stress.

Optimization of hydrogen peroxide detection for a methyl mercaptan biosensor.

Several kinds of modified carbon screen printed electrodes (CSPEs) for amperometric detection of hydrogen peroxide (H2O2) are presented in order to propose a methyl mercaptan (MM) biosensor. Unmodified, carbon nanotubes (CNTs), cobalt phthalocyanine (CoPC), Prussian blue (PB), and Os-wired HRP modified CSPE sensors were fabricated and tested to detect H2O2, applying a potential of +0.6 V, +0.6 V, +0.4 V, -0.2 V and -0.1 V (versus Ag/AgCl), respectively. The limits of detection of these electrodes for H2O2 were 3.1 µM, 1.3 µM, 71 nM, 1.3 µM, 13.7 nM, respectively. The results demonstrated that the Os-wired HRP modified CSPEs gives the lowest limit of detection (LOD) for H2O2 at a working potential as low as -0.1 V. Os-wired HRP is the optimum choice for establishment of a MM biosensor and gives a detection limit of 0.5 µM.

Development of an electrochemical metal-ion biosensor using self-assembled peptide nanofibrils.

This article describes the combination of self-assembled peptide nanofibrils with metal electrodes for the development of an electrochemical metal-ion biosensor. The biological nanofibrils were immobilized on gold electrodes and used as biorecognition elements for the complexation with copper ions. These nanofibrils were obtained under aqueous conditions, at room temperature and outside the clean room. The functionalized gold electrode was evaluated by cyclic voltammetry, impedance spectroscopy, energy dispersive X-ray and atomic force microscopy. The obtained results displayed a layer of nanofibrils able to complex with copper ions in solution. The response of the obtained biosensor was linear up to 50 µM copper and presented a sensitivity of 0.68 µA cm⁻² µM⁻¹. Moreover, the fabricated sensor could be regenerated to a copper-free state allowing its reutilization.