1,720,955 research outputs found
Origami d'ADN pour la spectrométrie de masse spatio-temporelle
No full textL'endocytose de molécules de la membrane plasmique est essentielle pour des fonctions physiologiques telles que la signalisation, le renouvellement des membranes, la neurotransmission, le métabolisme et l'invasion de pathogènes. Ce processus d'internalisation est fortement régulé par l'interaction entre des protéines, lipides et glycanes spécifiques. Des mécanismes d'endocytose distincts existent et fonctionnent en parallèle, chacun associé à une composition moléculaire des structures endocytiques différente.Notre laboratoire a récemment découvert que la glycosylation des protéines et des lipides est essentielle pour certains événements d'endocytose non médiés par la clathrine, mais dépendant des glyco-lipides et les lectines (GL-Lect). Les galectines sont une famille de 15 lectines qui reconnaissent les glycanes sur les glycoprotéines de la membrane plasmique (cargos) et en contrôlent l'endocytose. Elles constituent donc l'outil idéal pour étudier les événements endocytiques dépendant de la glycosylation des protéines cargo. De plus, les glycolipides étant nécessaires à la déformation membranaire induite lors de l'endocytose GL-Lect, nous souhaitons également déterminer l'enrichissement en glycolipides de ces structures d'endocytose. Notre hypothèse est que les différentes structures endocytiques induites par les différentes galectines ont un glycocode protéique et lipidique unique, déterminant pour le destin intracellulaire des molécules internalisées.Pour déterminer la composition en protéines, lipides et glycanes de ces structures endocytiques, il est essentiel de les purifier spécifiquement par rapport à la lectine qui leur est associée. Bien que les protocoles d'isolement (immuno-isolation ou centrifugation différentielle) des compartiments subcellulaires pour la protéomique soient optimisés, ils ne peuvent pas être utilisés pour obtenir une spécificité de voie ou de cargo puisque la plupart des ligands et lectines sont localisés dans la partie luminale des structures endocytiques. En outre, l’approche APEX de biotinylation proximale peut être appliquée pour déterminer le protéome spécifique de la voie, mais pas le lipidome.C'est pourquoi nous avons développé une méthode nouvelle et universelle basée sur des particules superparamagnétiques enfermées dans des origamis d'ADN, fonctionnalisées avec différentes galectines. L'origami d'ADN a été construit sur la base d’un ADN simple brin (ss) dérivé de l'ADN du phage m13mp18. Ce brin d'échafaudage constitue une base d'agrafage d’ADN ss pour former un nanotube d'ADN monofonctionnalisé par une galectine d’intérêt. La lumière du nanotube d'ADN est chargée soit de nanoparticules magnétiques (NP) pour des applications de pulldown, soit d'APEX pour la microscopie électronique, ou de quantum dots pour le suivi de particule isolée. Les galectines couplées au nanotube d'ADN agissent ainsi comme des vecteurs pour délivrer le nanotube d'ADN dans les structures endocytiques précoces qu'elles induisent. Pour les études de validation de concept, nous avons choisi la galectine-8 (Gal8), en raison de son rôle de récepteur de pathogènes et de sa dérégulation dans des contextes tumoraux. Des expériences d'immunofluorescence ont montré que les nanotubes d'ADN couplés à Gal8 se lient et s'internalisent avec succès dans les cellules cancéreuses de la prostate PC3, en fonction du ligand. Nous avons ensuite mis en place un protocole d’internalisation de ces nanotubes et de lyse mécanique des cellules permettant la purification de populations de vésicules magnétiques remplies de fer, spécifiques des différentes galectines, pour une analyse multi-omique du contenu protéique et lipidique de ces structures endocytiques. Dans une première application, cette nouvelle approche nous a ainsi permis d'identifier pour la première fois l'anatomie moléculaire (cargos intégrines et protéines Rab) des structures d'endocytose qui sont spécifiques de la Gal8.The endocytosis of extracellular and plasma membrane-localised molecules is essential for different physiological functions like signaling, membrane turnover, neurotransmission, metabolism, and invasion of pathogens. This internalisation process is highly regulated by the interplay of specific proteins, lipids and glycans. Distinct endocytic mechanisms exist and operate in parallel, hence the molecular composition of endocytic uptake structures may be different in each case.Our lab recently discovered that glycosylation patterns on proteins and lipids are critical for certain non-clathrin-mediated endocytic events, termed GlycoLipid-Lectin (GL-Lect) driven endocytosis. The extracellular parts of almost all membrane proteins are N- or O-glycosylated. Galectins are a 15-member family of lectins that recognise glycans on plasma glycoproteins (cargoes) and initiate endocytosis. Hence, galectins were the perfect tool to study glycosylation-specific endocytic events of cargo proteins. In addition, since glycolipids are required for the membrane deformation in GL-Lect driven endocytosis, we are interested to also determine glycolipid enrichment in endocytic structures. Our hypothesis is that different endocytic carrier populations induced by individual galectins have a unique protein and lipid glycocode, which might then also be critical for the final intracellular fate of the internalized molecules.To determine the protein, lipid, and glycan composition of endocytic carriers, it is critical to purify them in a ligand-specific manner.Although protocols for isolation of subcellular compartments for proteomics based on differential velocity or density gradient centrifugation have been excellently optimized, they cannot be used to achieve pathway or cargo specificity since most endocytic ligands and lectins are luminally localized. Immuno-isolation cannot be applied to luminal lectins, proximal biotinylation approach using APEX can be applied to determine the pathway-specific proteome, but not the lipidome. In addition, APEX does not directly target proteins on the cytosolic leaflet of endocytic vesicles.Therefore, we have developed a novel and universal method based on DNA origami-enclosed superparamagnetic particles, monofunctionalized by design with different galectins. The DNA origami was built based on a 14-helix model using a single stranded DNA derived from m13mp18 phage DNA as the backbone. This scaffold strand was shaped by a cocktail of ss-DNA staple oligonucleotides to form a DNA nanotube that is mono-functionalized on the outer wall with a chosen galectin (or any other endocytic protein of interest). Via the biotin-streptavidin bond, the lumen of the DNA nanotube is filled with magnetic nanoparticles (NP) for pulldown, APEX for electron microscopy, or a quantum dot for single particle tracking. The galectins, which are coupled to the DNA nanotube by an inverted protein coupling strategy, act as drivers to deliver the DNA nanotube into early endocytic uptake carriers that they themselves induce. For proof-of-concept studies, we have chosen galectin-8 (Gal8), based on its role as a pathogen receptor and its documented dysregulation in cancer. Immunofluorescence experiments showed successful binding and internalization of the Gal8-coupled DNA nanotubes in PC3 prostate cancer cells in a ligand dependent manner.For multi-omics, the Gal8-coupled DNA nanotubes are internalized for 3 to 10 min into PC3 cells. After removal of cell surface exposed DNA nanotubes, cells are mechanically lysed. The individual galectin-specific populations of iron-filled vesicles are purified by a magnetic-enhancement based method. The resulting carrier-specific protein and lipid fractions are analyzed by proteomics, lipidomics, and glycomics. In a first application, this new approach has allowed me to identify for the first time the molecular anatomy (e.g., integrin cargoes and Rab proteins) of endocytic structures that are specific to Gal8
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
- …
