Guillaume Clavé

An histidine covalent receptor and butenolide complex mediates strigolactone perception

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D-ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases and is known to hydrolyze the bond between the ABC lactone and the D-ring. Here we characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Efficient covalent functionalisation of carbon nanotubes: the use of “click chemistry”

Amongst the different classes of nanomaterials, carbon nanotubes (CNTs) are extremely promising for applications in electronics, solar energy conversion, materials science and medicinal chemistry. However in order to use them for those applications an accurate control of the functionalisation of the nanotubes is required. Many reactions can be performed on the π-conjugated framework of CNTs (i.e. cycloadditions, radical additions or halogenations). These reactions, although very efficient, require, in general, experimental conditions which are weakly compatible with sensitive organic or biological compounds. The emergence of new or re-actualised synthetic methods (known under the term of “click chemistry”) has the potential to provide an elegant protocol to prepare carbon nanotube-based functional materials. This review will gather the recent results described in the literature using “click chemistry” to functionalise carbon nanotubes.

A highly sensitive competitive enzyme immunoassay of broad specificity quantifying microcystins and nodularins in water samples

Microcystins (MCs) form a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae) and cause both acute and chronic toxicity. For immunization purposes, an amino derivative of MC-LR was prepared before coupling to BSA. Among the different monoclonal antibodies produced, mAb MC159 was selected due to its broad specificity to develop a sensitive enzyme immunoassay (EIA). This method measures MC-LR, MC-YR, MC-LA, nodularins in a similar way and exhibits an important recognition (cross reactivity up to 69%) for Adda analogues. Using MC-LR as standard, the present EIA proved to be very sensitive with a limit of detection close to 10 fmol/ml, largely below the provisional guideline level for drinking water proposed by the WHO (1 pmol/ml for MC-LR). This assay showed a high accuracy (CV% < 12) and a high recovery rate for MC-LR in spiked surface water (up to 96.5%). Moreover due to its broad spectrum of recognition, this method allows a real quantification of the sum of MCs in water bloom and cyanobacteria culture samples. Indeed, in parallel analysis of these samples using HPLC, EIA shows a good relationship between both measurements while LC-MS/MS demonstrates the presence of different variants of MCs whose heterogeneity did not impair EIA measurement.

Synthesis of a multibranched porphyrin–oligonucleotide scaffold for the construction of DNA-based nano-architectures

The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.