Joëlle Widart

Detection of Oligonucleotide Gas-Phase Conformers: H/D Exchange and Ion Mobility as Complementary Techniques

Gas-phase hydrogen/deuterium exchange of small oligonucleotides (dTG, dC6 and C6) with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. Ion activation experiments were conducted by accelerating the ions at the entrance of the H/D exchange cell under conditions promoting exclusively collisional isomerization. These experiments allowed us to assess the presence of several conformers, and to probe the height of the isomerization barrier separating these conformers. Ion mobility experiments were also performed. Their results were consistent with the H/D exchange data. A model accounting for the competing isomerization and H/D exchange reactions is proposed. Comparing the ion acceleration experiments for H/D exchange and for ion mobility reveals that the most compact conformer displays the fastest H/D exchange. This observation shows that H/D exchange and ion mobility provide us with complementary information because hydrogen accessibility and macromolecule compactness are not univocally associated.

Adverse effects of enrofloxacin when associated with environmental stress in Tra catfish (Pangasianodon hypophthalmus).

The aim of this study was to assess the adverse effects of enrofloxacin (EF) on Tra catfish, Pangasianodon hypophthalmus, in relation with density stress. Fish were held at 40, 80 or 120 fish m(-3) and fed with pellets containing either 1 g kg(-1) EF or no EF. Antibiotic exposure lasted 7d and all fish were fed without EF for another 7-d recovery period. Fish were sampled at 3, 7, 8, 10 and 14 d after the beginning of EF exposure. Lipid peroxidation (LPO) and total glutathione (GSH) levels, catalase (CAT), glutathione-s-transferase (GST) and acetylcholine-esterase (AChE) activities were assessed in gill, brain, liver and muscle. At day 7, LPO levels in gills of EF-fish reared at low or high density were significantly more than 5-fold higher than their respective control. On the contrary, LPO in gills of EF-fish reared at medium density was significantly 3-fold lower than the control fish. Similarly, CAT activities in gills of EF-fish reared under low or high density were higher than in their control groups, while this activity was lower in EF-fish of the medium density group. AChE activities in muscles of EF-fish reared at low or high density were lower than controls at days 3 and 7, respectively. These results suggest that EF exposure may lead to disorders like lipid peroxidation and neural dysfunction in fish. However, when reared under lower stress condition (medium density), they may cope better with EF-induced stress than chronically stressed fish (low or high density).

Determination and kinetics of enrofloxacin and ciprofloxacin in Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a liquid chromatography/mass spectrometry method

Determination and kinetics of enrofloxacin and ciprofloxacin in Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a liquid chromatography/mass spectrometry method. J. vet. Pharmacol. Therap. 34, 142-152. The fluoroquinolones enrofloxacin (EF) and ciprofloxacin (CF) residues were investigated in the edible tissues of two important Asian aquacultured species such as Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry method. Fish and prawn were treated with medicated feed with multiple doses of EF, in field conditions. A validation study of the analytical method was realized in terms of linearity, specificity, precision (repeatability and within-laboratory reproducibility), recovery and decision limit (CCα). The time needed before the antibiotic disappears from animal tissues or reach the maximum residue limit (MRL, 100μg/kg) was assessed. The concentration values of EF detected in Tra catfish tissue were between the MRL and 2×MRL concentrations, according to the fish density, 7days following the end of the enrofloxacin treatment (20mg/kg body weight per day, for seven consecutive days). The concentration value of ER in prawn tissue was lower than the MRL and the limit of quantification (LOQ, 14μg/kg) 5 and 7days after the stop of the EF treatment (50mg/kg body weight per day, for five consecutive days), respectively. The mean detected levels of CF was much lower in comparison with that of EF, indicating that only a small part of EF is metabolized into CF (<5%) in both Tra catfish and prawn.

Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics.

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.