A revisited history of cacao domestication in pre-Columbian times revealed by archaeogenomic approaches.

Humans have a long history of transporting and trading plants, contributing to the evolution of domesticated plants. Theobroma cacao originated in the Neotropics from South America. However, little is known about its domestication and use in these regions. In this study, ceramic residues from a large sample of pre-Columbian cultures from South and Central America were analyzed using archaeogenomic and biochemical approaches. Here we show, for the first time, the widespread use of cacao in South America out of its native Amazonian area of origin, extending back 5000 years, likely supported by cultural interactions between the Amazon and the Pacific coast. We observed that strong genetic mixing between geographically distant cacao populations occurred as early as the middle Holocene, in South America, driven by humans, favoring the adaptation of T. cacao to new environments. This complex history of cacao domestication is the basis of today's cacao tree populations and its knowledge can help us better manage their genetic resources.

Analysis of polar primary metabolites in biological samples using targeted metabolomics and LC-MS.

Primary metabolites are molecules of essential biochemical reactions that define the biological phenotype. All primary metabolites cannot be measured in a single analysis. In this protocol, we outline the multiplexed and quantitative measurement of 106 metabolites that cover the central part of primary metabolism. The protocol includes several sample preparation techniques and one liquid chromatography-mass spectrometry method. Then, we describe the steps of the bioinformatic data analysis to better understand the metabolic perturbations that may occur in a biological system. For complete details on the use and execution of this protocol, please refer to: Costanza et al.,1 Blomme et al.,2 Blomme et al.,3 Guillon et al.,4 Stuani et al.5.

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry.

Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.

Aggregation of magnetic nanoparticles functionalized with trans-resveratrol in aqueous solution.

In the framework of a protein-ligand-fishing strategy to identify proteins that bind to trans-resveratrol, a natural phenolic compound with pharmacological benefits, we have developed magnetic nanoparticles covalently linked to trans-resveratrol through three different derivatives and examined their aggregation behavior in aqueous solution. The monodispersed magnetic core (18 nm diameter) with its mesoporous silica shell (93 nm diameter) exhibited a notable superparamagnetic behavior useful for magnetic bioseparation. The hydrodynamic diameter, deduced from dynamic light scattering analysis, of the nanoparticle increased from 100 to 800 nm when the aqueous buffer changed from pH 10.0-3.0. A size polydispersion occurred from pH 7.0-3.0. In parallel, the value of the extinction cross section increased according to a negative power law of the UV wavelength. This was mainly due to light scattering by mesoporous silica, whereas the absorbance cross section remained very low in the 230-400 nm domain. The three types of resveratrol-grafted magnetic nanoparticles exhibited similar scattering properties, but their absorbance spectrum was consistent with the presence of trans-resveratrol. Their functionalization increased their negative zeta potential when pH increased from 3.0 to 10.0. The mesoporous nanoparticles were monodispersed in alkaline conditions, where their anionic surface strongly repulsed each other but aggregated progressively under van der Waals forces and hydrogen bonding when negative zeta potential decreased. The characterized results of nanoparticle behavior in aqueous solution provide critical insight for further study of nanoparticles with proteins in biological environment.

Reg-1α, a New Substrate of Calpain-2 Depending on Its Glycosylation Status.

Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer's disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln4 and Thr5, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg11-Ile12 peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr5. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.

Primary flavonoids in marigold dye: extraction, structure and involvement in the dyeing process.

Flavonoids extracted from marigold flowers were investigated for their dyeing potential. Patulitrin (1) and patuletin (2) were isolated and their structures established using NMR and HPLC-MS. These compounds were identified as the main flavonoids present in the dyeing bath. Following the dyeing process, it was demonstrated that aglycone 2 bound more strongly to wool fibres than its glucoside 1. Moreover, analysis focused on 1 and 2 dynamics during plant growth revealed that these components were only found in flowers during and after flowering. The influence of growing location was also investigated and it appeared that cultivation under Mediterranean conditions enhanced biosynthesis of 1 and 2 . Finally, several solvents were tested for their potential to extract the flavonoids: the use of a water-ethanol mixture gave a high extraction efficiency and allowed selective extraction of 1 and 2. The implications of these results are discussed in relation to the development of marigold as a potential dyeing plant.