Determination of estrogen presence in water by SPR using estrogen receptor dimerization

International audienceEstrogenic compounds are a class of pharmaceutical products harmful to animals and a cause of environmental damage. The biological activity of these compounds is high since they have been designed to act at low concentrations. Thus, even at the low concentrations found in the environment, they may produce deleterious effects on aquatic organisms as well as on humans, who might be contaminated in a number of ways (via drinking water or contaminated food, for example). We used the property of these compounds to bind a specific protein (estrogen receptor, ER) to develop a quantification method of these chemical entities. Estrogenic compound detection was performed using ER dimerization properties monitored by surface plasmon resonance (SPR). The ligand-activated ER dimer was detected by its interaction with a specific DNA consensus sequence estrogen response element. The concentration and the nature of the estrogenic compounds modified the SPR signal and were characteristic of the ligand-dependent homodimerization of ER. For 17β-estradiol, dimerization of ER was experimentally determined at an ER to 17β-estradiol ratio near 1:1. Estrogenic compounds (17β-estradiol, estriol, estrone, ethynyl estradiol) activated the dimerization process at different concentration levels, while some others (tamoxiphen, resveratrol, genistein, bisphenol A) did not seem to have any effects on it. We demonstrated that this method allows the direct detection of 17β-estradiol at concentrations above 1.4 μg/L (5 nM)

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.

International audienceNumerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions

Experimental separation steps influence the protein content of corona around mesoporous silica nanoparticles

International audienc

Interaction of ultraintense radially-polarized laser pulses with plasma mirrors

International audienceWe present experimental results of vacuum laser acceleration (VLA) of electrons using radially polarized laser pulses interacting with a plasma mirror. Tightly focused, radially polarized laser pulses have been proposed for electron acceleration because of their strong longitudinal electric field, making them ideal for VLA. However, experimental results have been limited until now because injecting electrons into the laser field has remained a considerable challenge. Here, we demonstrate experimentally that using a plasma mirror as an injector solves this problem and permits us to inject electrons at the ideal phase of the laser, resulting in the acceleration of electrons along the laser propagation direction while reducing the electron beam divergence compared to the linear polarization case. We obtain electron bunches with few-MeV energies and a 200-pC charge, thus demonstrating, for the first time, electron acceleration to relativistic energies using a radially polarized laser. High-harmonic generation from the plasma surface is also measured, and it provides additional insight into the injection of electrons into the laser field upon its reflection on the plasma mirror. Detailed comparisons between experimental results and full 3D simulations unravel the complex physics of electron injection and acceleration in this new regime: We find that electrons are injected into the radially polarized pulse in the form of two spatially separated bunches emitted from the p-polarized regions of the focus. Finally, we leverage on the insight brought by this study to propose and validate a more optimal experimental configuration that can lead to extremely peaked electron angular distributions and higher energy beams