Comparison of the Hemostatic Efficacy of Pathogen-Reduced Platelets vs Untreated Platelets in Patients With Thrombocytopenia and Malignant Hematologic Diseases: A Randomized Clinical Trial

Importance: Pathogen reduction of platelet concentrates may reduce transfusion-transmitted infections but is associated with qualitative impairment, which could have clinical significance with regard to platelet hemostatic capacity. Objective: To compare the effectiveness of platelets in additive solution treated with amotosalen-UV-A vs untreated platelets in plasma or in additive solution in patients with thrombocytopenia and hematologic malignancies. Design, Setting, and Participants: The Evaluation of the Efficacy of Platelets Treated With Pathogen Reduction Process (EFFIPAP) study was a randomized, noninferiority, 3-arm clinical trial performed from May 16, 2013, through January 21, 2016, at 13 French tertiary university hospitals. Clinical signs of bleeding were assessed daily until the end of aplasia, transfer to another department, need for a specific platelet product, or 30 days after enrollment. Consecutive adult patients with bone marrow aplasia, expected hospital stay of more than 10 days, and expected need of platelet transfusions were included. Interventions: At least 1 transfusion of platelets in additive solution with amotosalen-UV-A treatment, in plasma, or in additive solution. Main Outcomes and Measures: The proportion of patients with grade 2 or higher bleeding as defined by World Health Organization criteria. Results: Among 790 evaluable patients (mean [SD] age, 55 [13.4] years; 458 men [58.0%]), the primary end point was observed in 126 receiving pathogen-reduced platelets in additive solution (47.9%; 95% CI, 41.9%-54.0%), 114 receiving platelets in plasma (43.5%; 95% CI, 37.5%-49.5%), and 120 receiving platelets in additive solution (45.3%; 95% CI, 39.3%-51.3%). With a per-protocol population with a prespecified margin of 12.5%, noninferiority was not achieved when pathogen-reduced platelets in additive solution were compared with platelets in plasma (4.4%; 95% CI, -4.1% to 12.9%) but was achieved when the pathogen-reduced platelets were compared with platelets in additive solution (2.6%; 95% CI, -5.9% to 11.1%). The proportion of patients with grade 3 or 4 bleeding was not different among treatment arms. Conclusions and Relevance: Although the hemostatic efficacy of pathogen-reduced platelets in thrombopenic patients with hematologic malignancies was noninferior to platelets in additive solution, such noninferiority was not achieved when comparing pathogen-reduced platelets with platelets in plasma. Trial Registration: clinicaltrials.gov Identifier: NCT01789762

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Dynamique des Protéines par RMN à l’Etat Solide avec Rotation Ultra Rapide à l’Angle Magique : des Microcristaux aux Fibrilles Amyloïdes et Protéines Membranaires

Solid-state NMR with magic angle spinning (MAS) has emerged as a powerful technique for investigating structure and dynamics of insoluble or poorly soluble biomolecules. A number of approaches has been designed for reconstructing molecular structures from the accurate measurement of internuclear proximities, and for probing motions at atomic resolution over timescales spanning several orders of magnitude. Despite this impressive progress, however, MAS NMR studies are still far from routine. Complete determinations, which are often demonstrated on model microcrystalline preparations, are still rare when it comes to more complex systems such as non-crystalline amyloid fibrils or transmembrane proteins in lipid bilayers. My work aimed at extending the possibilities of MAS NMR for applications on complex biomolecular systems in different aggregation states. For this, I exploited the unique possibilities provided by high magnetic fields (700, 800 and 1000 MHz 1H Larmor frequency) in combination with the newest MAS probes capable of spinning rates exceeding 60 kHz. These experimental conditions al- low to boost the sensitivity of MAS NMR through 1H detection at high resolution and to enrich the palette of probes for protein dynamics. The first part of the thesis reports on my contribution to the development of new strategies for backbone resonance assignment, for structure elucidation, and for investigation of backbone and side-chain dynamics. These methodologies significantly reduce the requirements in terms of experimental time, sample quantities and isotopic labeling, and enlarge the molecular size of systems amenable to NMR analysis. The second part describes the application of 1H detected MAS NMR to evaluate the role of protein dynamics in problems such as amyloid fibril formation and membrane protein function. I first addressed the amyloid fibril formation propensity of human beta-2 microglobulin, the light chain of the major histocompatibility complex I. I performed comparative studies of backbone dynamics of the wild type protein as well as a D76N mutant in crystals, and determined some of the structural features of the fibrillar form. This allowed to identify the presence of pathological folding intermediates and to formulate hypotheses on the mechanism of fibrils formation. Finally, I studied the local and global dynamics of membrane proteins in lipid bilayers. In particular, I investigated the mechanism of action of the alkane trans- porter AlkL from P. putida in lipid bilayers. The measurement of parameters for fast (ps-ns) and slow (μs-ms) backbone dynamics of the protein in presence or in absence of a substrate highlights possible routes for molecular uptake and lays the basis for a more detailed mechanistic understanding of the process.La Résonance Magnétique Nucléaire (RMN) à l’état solide avec rotation à l’angle magique (MAS) est une technique de choix pour l’étude de la structure et de la dynamique de molécules biologiques peu ou non solubles. Un grand nombre d’approches ont été développées pour la reconstitution de structures tridimensionelles à partir de mesures précises de proximités internucléaires, ainsi que pour la détection de mouvements moléculaires avec une résolution atomique sur des échelles de temps couvrant plusieurs ordres de grandeur. Malgré d’impressionnants progrès, les études par RMN MAS sont cependant loin d’être réalisées en routine. Les déterminations structurelles et de dynamique sont souvent démontrées sur des préparations microcristallines modèles, mais sont encore rares pour des systèmes plus complexes tels que les fibrilles amyloïdes non cristallines ou les protéines trans-membranaires insérées dans des bi- couches lipidiques. Mon travail a pour objectif d’étendre les possibilités de la RMN MAS pour l’étude de systèmes biomoléculaires complexes dans différents états d’agrégation. Pour cela, j’ai exploité les possibilités uniques offertes par les hauts champs magnétiques (fréquence de Larmor du 1H 700, 800 et 1000 MHz) combinés avec les sondes MAS de dernières générations capables d’atteindre des vitesses de rotations supérieures à 60 kHz. Ces conditions expérimentales per- mettent d’augmenter la sensibilité de la RMN MAS à l’aide de la détection 1H à haute résolution et d’enrichir la palette de paramètres RMN rapporteurs de la dynamique des protéines. La première partie de cette thèse décrit le développement de nouvelles stratégies pour l’attribution des résonances du squelette de protéines, pour l’élucidation de structures, et pour l’étude de la dynamique du squelette peptidique et des chaînes latérales. Les méthodes présentées réduisent significative- ment les besoins en termes de temps expérimental, de quantités d’échantillon et de marquage isotopique, et permettent d’analyser par RMN des systèmes de plus hauts poids moléculaire. La seconde partie décrit l’application de la RMN MAS avec détection en 1H pour l’évaluation du rôle de la dynamique des protéines dans des processus tels que la formation de fibrilles amyloïdes et le fonctionnement de protéines membranaires. Une première application est l’étude de la tendance de la β-2 microglobuline humaine à former des fibrilles amyloïdes. Une comparaison de la dynamique du squelette peptidique de la protéine sauvage et du mutant D76N dans leur forme cristalline, ainsi que la détermination de propriétés structurales de la forme fibrillaire m’ont permis d’identifier la présence de repliements pathologiques et de formuler des hypothèses sur le mécanisme de formation des fibrilles. Finalement, la dynamique locale et globale de protéines membranaires dans des bicouches lipidiques a été étudiée. En particulier, le mécanisme d’action d’un transporteur d’alkanes, AlkL, de P. putida a été examiné dans un environnement lipidique. La détermination de paramètres pour la dynamique rapide (ps-ns) et lente (μs-ms) du squelette peptidique de la protéine en présence ou en absence de substrat met en évidence des acheminements possibles pour le transfert de molécules vers la membrane et jette les bases pour une meilleure compréhension du processus

Protein structural dynamics by Magic-Angle Spinning NMR

International audienceMagic-Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) is a fastdeveloping technique, capable of complementing solution NMR, X-ray crystallography and electron microscopy for the biophysical characterization of microcrystalline, poorly crystalline or disordered protein samples, such as enzymes, biomolecular assemblies, membraneembedded systems or fibrils. Beyond structures, MAS NMR is an ideal tool for investigation of dynamics, since it is unique in its ability to distinguish static and dynamic disorder, and to characterize not only amplitudes, but also timescales of motion. Building on seminal work on model proteins, the technique is now ripe for widespread application in structural biology. This review briefly summarizes the recent evolutions in biomolecular MAS NMR and accounts for the growing number of systems where this spectroscopy has provided a description of conformational dynamics over the very last few years

1 H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins

International audienceBuilding on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the 1 H-1 H dipolar couplings, so that direct detection of the larger magnetic moment available from 1 H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid " fingerprinting " of samples with a tenfold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca. 300 residues), and the determination of inter-nuclear proximities, relative orientations of secondary structural elements, protein-cofactor interactions, local and global dynamics. Fast MAS and 1 H detection techniques have nowadays been shown to be applicable to membrane-bound systems. This paper reviews the strategies underlying this recent leap forward in sensitivity and resolution, describing its potential for the detailed characterization of membrane proteins. Graphical TO

Insights into ion channel structure and function by ultrafast magic angle spinning NMR

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Conformational Stability and Dynamics in Crystals Recapitulate Protein Behavior in Solution

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