Getting to grips with resilience: Toward large-scale phenotyping of this complex trait.

The capacity of animals to cope with environmental perturbations, hereafter called resilience, is an increasingly important trait. Resilience at the level of the animal is an emergent property of multiple underlying mechanisms (physiological, immunological, behavioral). This means that there is no direct measure of resilience, no easy key traits.

Established and emerging players in phospholipid scrambling: A structural perspective.

The maintenance of a diverse and non-homogeneous lipid composition in cell membranes is crucial for a multitude of cellular processes. One important example is transbilayer lipid asymmetry, which refers to a difference in lipid composition between the two leaflets of a cellular membrane. Transbilayer asymmetry is especially pronounced at the plasma membrane, where at resting state, negatively-charged phospholipids such as phosphatidylserine (PS) are almost exclusively restricted to the cytosolic leaflet, whereas sphingolipids are mostly found in the exoplasmic leaflet.

Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins.

Activation and substrate specificity of the human P4-ATPase ATP8B1.

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.

Disentangling the dynamics of energy allocation to develop a proxy for robustness of fattening pigs.

Background: There is a growing need to improve robustness of fattening pigs, but this trait is difficult to phenotype. Our first objective was to develop a proxy for robustness of fattening pigs by modelling the longitudinal energy allocation coefficient to growth, with the resulting environmental variance of this allocation coefficient considered as a proxy for robustness. The second objective was to estimate its genetic parameters and correlations with traits under selection and with phenotypes that are routinely collected.

Delayed-onset eptifibatide-induced thrombocytopenia.

Purpose: We present a unique case of delayed-onset, profound eptifibatide-induced thrombocytopenia that occurred 5 days after initiation of the drug.

Summary: Eptifibatide is a platelet glycoprotein IIb/IIIa receptor inhibitor with indications for use in patients with acute coronary syndromes. Eptifibatide-induced thrombocytopenia is uncommon but well studied and typically occurs within 24 hours of initiation of the drug.

[Facilitators and barriers to shared decision-making in France in 2021: National survey in cancer].

Introduction: Shared-decision making (SDM) combines clinical expertise of the healthcare professional with patient's knowledge, values and preferences. This survey explores from a patient perspective, the implementation, facilitators and barriers of SDM in oncology in France in 2021.

Patients And Methods: From August to October 2021, the digital platform Cancer contribution conducted an online survey relayed by 11 patient associations.

Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase.

Membrane proteins (MPs) are challenging to study from a biochemical standpoint owing to the difficulties associated with the isolation of these proteins from the membranes they are embedded in. Even for the expression of closely-related homologues, protocols often require to be adjusted. Prominently, the solubilization step and the stabilization of recombinant proteins during the purification process are key issues, and remain a serious bottleneck.

Robustness scores in fattening pigs based on routinely collected phenotypes: determination and genetic parameters.

The objective was to determine operational proxies for robustness based on data collected routinely on farm that allow phenotyping of these traits in fattening pigs, and to estimate their genetic parameters. A total of 7,256 pigs, from two Piétrain paternal lines (Pie and Pie NN), were tested at the AXIOM boar testing station (Azay-sur-Indre, France) from 2019 to 2021. During the fattening period (from 75 to 150 d of age), individual performance indicators were recorded (growth, backfat, loin depth, feed intake, and feed conversion ratio [FCR]) together with indicators such as insufficient growth, observable defect, symptoms of diseases, and antibiotic and anti-inflammatory injections.

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 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.

Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine.

Phosphatidylserine (PS) is a negatively charged phospholipid that displays a highly uneven distribution within cellular membranes, essential for establishment of cell polarity and other processes. In this review, we discuss how combined action of PS biosynthesis enzymes in the endoplasmic reticulum (ER), lipid transfer proteins (LTPs) acting within membrane contact sites (MCS) between the ER and other compartments, and lipid flippases and scramblases that mediate PS flip-flop between membrane leaflets controls the cellular distribution of PS. Enrichment of PS in specific compartments, in particular in the cytosolic leaflet of the plasma membrane (PM), requires input of energy, which can be supplied in the form of ATP or by phosphoinositides.

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit.

Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a -encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates.

The SERCA residue Glu340 mediates interdomain communication that guides Ca transport.

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is a P-type ATPase that transports Ca from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A.

Genetic parameters of resistance to pasteurellosis using novel response traits in rabbits.

Background: Pasteurellosis (Pasteurella infection) is one of the most common bacterial infections in rabbits on commercial farms and in laboratory facilities. Curative treatments using antibiotics are only partly efficient, with frequent relapses. Breeding rabbits for improved genetic resistance to pasteurellosis is a sustainable alternative approach.

P4-ATPases: how an old dog learnt new tricks - structure and mechanism of lipid flippases.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active, inward directed translocation (flip) of lipids in eukaryotic membranes. The resulting lipid asymmetry potentiates the membrane and is essential for a wide range of cellular processes such as vesicle biogenesis and trafficking and membrane protein regulation, whereas dissipation of lipid asymmetry is required in blood coagulation and apoptosis. Through recent advances in cryo-electron microscopy, several landmark structures of yeast and human lipid flippases have been reported, highlighting the similarities and differences they share with the cation transporting P-type ATPases.

Interaction of detergents with biological membranes: Comparison of fluorescence assays with filtration protocols and implications for the rates of detergent association, dissociation and flip-flop.

The present study mainly consists of a re-evaluation of the rate at which C12E8, a typical non-ionic detergent used for membrane studies, is able to dissociate from biological membranes, with sarcoplasmic reticulum membrane vesicles being used as an example. Utilizing a brominated derivative of C12E8 and now stopped-flow fluorescence instead of rapid filtration, we found that the rate of dissociation of this detergent from these membranes, merely perturbed with non-solubilizing concentrations of detergent, was significantly faster (t1/2 < 10 ms) than what had previously been determined (t1/2 ~300-400 ms) from experiments based on a rapid filtration protocol using 14C-labeled C12E8 and glass fiber filters (Binding of a non-ionic detergent to membranes: flip-flop rate and location on the bilayer, by Marc le Maire, Jesper Møller and Philippe Champeil, Biochemistry (1987) Vol 26, pages 4803-4810). We here pinpoint a methodological problem of the earlier rapid filtration experiments, and we suggest that the true overall dissociation rate of C12E8 is indeed much faster than previously thought.

Screening of Detergents for Stabilization of Functional Membrane Proteins.

Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions.

A prospective evaluation of plasma phospholipid fatty acids and breast cancer risk in the EPIC study.

Background: Intakes of specific fatty acids have been postulated to impact breast cancer risk but epidemiological data based on dietary questionnaires remain conflicting.

Materials And Methods: We assessed the association between plasma phospholipid fatty acids and breast cancer risk in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition study. Sixty fatty acids were measured by gas chromatography in pre-diagnostic plasma phospholipids from 2982 incident breast cancer cases matched to 2982 controls.

High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes.

A robust method to screen detergents for membrane protein stabilization, revisited.

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents.