Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies

Alteration of the in vivo nicotinic receptor density in ADNFLE patients: a PET study

Nicotinic acetylcholine receptors (nAChRs) are involved in a familial form of frontal lobe epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In several ADNFLE families, mutations were identified in the nAChR α4 or β2 subunit, which together compose the main cerebral nAChR. Electrophysiological assessment using in vitro expression systems indicated a gain of function of the mutant receptors. However the precise mechanisms by which they contribute to the pathogenesis of a focal epilepsy remain obscure, especially since α4β2 nAChRs are known to be widely distributed within the entire brain. PET study using [18F]-F-A-85380, a high affinity agonist at the α4β2 nAChRs, allows the determination of the regional distribution and density of the nAChRs in healthy volunteers and in ADNFLE patients, thus offering a unique opportunity to investigate some in vivo consequences of the molecular defect. We have assessed nAChR distribution in eight non-smoking ADNFLE patients (from five families) bearing an identified mutation in nAChRs and in seven age-matched non-smoking healthy volunteers using PET and [18F]-F-A-85380. Parametric images of volume of distribution (Vd) were generated as the ratio of tissue to plasma radioactivities. The images showed a clear difference in the pattern of the nAChR density in the brains of the patients compared to the healthy volunteers. Vd values revealed a significant increase (between 12 and 21%, P < 0.05) in the ADNFLE patients in the mesencephalon, the pons and the cerebellum when compared to control subjects. Statistical parametric mapping (SPM) was then used to better analyse subtle regional differences. This analysis confirmed clear regional differences between patients and controls: patients had increased nAChR density in the epithalamus, ventral mesencephalon and cerebellum, but decreased nAChR density in the right dorsolateral prefrontal region. In five patients who underwent an additional [18F]-fluorodeoxyglucose (FDG) PET experiment, hypometabolism was observed in the neighbouring area of the right orbitofrontal cortex. The demonstration of a regional nAChR density decrease in the prefrontal cortex, despite the known distribution of these receptors throughout the cerebral cortex, is consistent with a focal epilepsy involving the frontal lobe. We also propose that the nAChR density increase in mesencephalon is involved in the pathophysiology of ADNFLE through the role of brainstem ascending cholinergic systems in arousa

The governance of justice and internal security in Scotland: Between the Scottish independence referendum and British decisions on the EU

This article examines how the governance of justice and internal security in Scotland could be affected by the outcome of the Scottish independence referendum in September 2014. The article argues that it is currently impossible to equate a specific result in the referendum with a given outcome for the governance of justice and internal security in Scotland. This is because of the complexities of the current arrangements in that policy area and the existence of several changes that presently affect them and are outside the control of the government and of the people of Scotland. This article also identifies an important paradox. In the policy domain of justice and internal security, a ‘no’ vote could, in a specific set of circumstances, actually lead to more changes than a victory of the ‘yes’ camp

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

Identification de cibles d’insecticides spécifiques du puceron du pois Acyrthosiphon piusm par analyse des relations structure-fonction des récepteurs nicotiniques

Session 1 - Thérapies et résistancesNational audienc

Taming trilogues: the EU's law-making process in a comparative perspective.

Trilogues have become the modus operandi of EU decision-making. They are an informal but institutionalised mechanism providing for in camera discussions of legislative texts between the three main EU decision-making institutions, with a view to securing legislative compromises. Trilogues present risks to an organ of parliamentary representation through their potential to depoliticise conflict and by reducing the accountability and transparency of the decision-making process. We examine how the European Parliament (EP) has responded to trilogues and what this response tells us about the development of the EP as an institutionalised organ of representative democracy. We compare these with arrangements for bicameral conflict resolution in the United States, where similar issues are presented by informal mechanisms of decision-making. We assess the institutionalisation of trilogues from a democratic perspective, highlighting achievements and future challenges, and the value of these findings for the ongoing reflection on the EP as a normal parliament and the role of informal institutions in EU law-making

A prolonged run-in period of standard subcutaneous microdialysis ameliorates quality of interstitial glucose signal in patients after major cardiac surgery

We evaluated a standard subcutaneous microdialysis technique for glucose monitoring in two critically ill patient populations and tested whether a prolonged run-in period improves the quality of the interstitial glucose signal. 20 surgical patients after major cardiac surgery (APACHE II score: 10.1 ± 3.2) and 10 medical patients with severe sepsis (APACHE II score: 31.1 ± 4.3) were included in this investigation. A microdialysis catheter was inserted in the subcutaneous adipose tissue of the abdominal region. Interstitial fluid and arterial blood were sampled in hourly intervals to analyse glucose concentrations. Subcutaneous adipose tissue glucose was prospectively calibrated to reference arterial blood either at hour 1 or at hour 6. Median absolute relative difference of glucose (MARD), calibrated at hour 6 (6.2 (2.6; 12.4) %) versus hour 1 (9.9 (4.2; 17.9) %) after catheter insertion indicated a significant improvement in signal quality in patients after major cardiac surgery (p < 0.001). Prolonged run-in period revealed no significant improvement in patients with severe sepsis, but the number of extreme deviations from the blood plasma values could be reduced. Improved concurrence of glucose readings via a 6-hour run-in period could only be achieved in patients after major cardiac surgery

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

10.1371/journal.pone.0048112PLoS ONE710