Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

Local-scale dynamics of plant-pesticide interactions in a northern Brittany agricultural landscape.

Soil pollution by anthropogenic chemicals is a major concern for sustainability of crop production and of ecosystem functions mediated by natural plant biodiversity. Understanding the complex effects of soil pollution requires multi-level and multi-scale approaches. Non-target and agri-environmental plant communities of field margins and vegetative filter strips are confronted with agricultural xenobiotics through soil contamination, drift, run-off and leaching events that result from chemical applications. Plant-pesticide dynamics in vegetative filter strips was studied at field scale in the agricultural landscape of a long-term ecological research network in northern Brittany (France). Vegetative filter strips effected significant pesticide abatement between the field and riparian compartments. However, comparison of pesticide usage modalities and soil chemical analysis revealed the extent and complexity of pesticide persistence in fields and vegetative filter strips, and suggested the contribution of multiple sources (yearly carry-over, interannual persistence, landscape-scale contamination). In order to determine the impact of such persistence, plant dynamics was followed in experimentally-designed vegetative filter strips of identical initial composition (Agrostis stolonifera, Anthemis tinctoria/Cota tinctoria, Centaurea cyanus, Fagopyrum esculentum, Festuca rubra, Lolium perenne, Lotus corniculatus, Phleum pratense, Trifolium pratense). After homogeneous vegetation establishment, experimental vegetative filter strips underwent rapid changes within the following two years, with Agrostis stolonifera, Festuca rubra, Lolium perenne and Phleum pratense becoming dominant and with the establishment of spontaneous vegetation. Co-inertia analysis showed that plant dynamics and soil residual pesticides could be significantly correlated, with the triazole fungicide epoxiconazole, the imidazole fungicide prochloraz and the neonicotinoid insecticide thiamethoxam as strong drivers of the correlation. However, the correlation was vegetative-filter-strip-specific, thus showing that correlation between plant dynamics and soil pesticides likely involved additional factors, such as threshold levels of residual pesticides. This situation of complex interactions between plants and soil contamination is further discussed in terms of agronomical, environmental and health issues.

Liquid chromatography-tandem mass spectrometry method for the analysis of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products.

A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC-MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5-150 µg kg-1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg-1 for precision. The limit of quantification (LOQ) was around 5-7 µg kg-1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg-1 in cheese.

Liquid chromatography-tandem mass spectrometry multiresidue method for the analysis of quaternary ammonium compounds in cheese and milk products: Development and validation using the total error approach.

Quaternary ammonium compounds (QACs) are both cationic surfactants and biocidal substances widely used as disinfectants in the food industry. A sensitive and reliable method for the analysis of benzalkonium chlorides (BACs) and dialkyldimethylammonium chlorides (DDACs) has been developed that enables the simultaneous quantitative determination of ten quaternary ammonium residues in dairy products below the provisional maximum residue level (MRL), set at 0.1mgkg-1. To the best of our knowledge, this method could be the one applicable to milk and to three major processed milk products selected, namely processed or hard pressed cheeses, and whole milk powder. The method comprises solvent extraction using a mixture of acetonitrile and ethyl acetate, without any further clean-up. Analyses were performed by liquid chromatography coupled with electrospray tandem mass spectrometry detection (LC-ESI-MS/MS) operating in positive mode. A C18 analytical column was used for chromatographic separation, with a mobile phase composed of acetonitrile and water both containing 0.3% formic acid; and methanol in the gradient mode. Five deuterated internal standards were added to obtain the most accurate quantification. Extraction recoveries were satisfactory and no matrix effects were observed. The method was validated using the total error approach in accordance with the NF V03-110 standard in order to characterize the trueness, repeatability, intermediate precision and analytical limits within the range of 5-150µgkg-1 for all matrices. These performance criteria, calculated by e.noval® 3.0 software, were satisfactory and in full accordance with the proposed provisional MRL and with the recommendations in the European Union SANTE/11945/2015 regulatory guidelines. The limit of detection (LOD) was low (<1.9µgkg-1) and the limit of quantification (LOQ) ranged from 5µgkg-1 to 35µgkg-1 for all matrices depending on the analytes. The validation results proved that the method is suitable for quantifying quaternary ammoniums in foodstuffs from dairy industries at residue levels, and could be used for biocide residues monitoring plans and to measure the exposition consumer to biocides products.