1,720,964 research outputs found
Multi‐Color, Bleaching‐Resistant Super‐Resolution Optical Fluctuation Imaging with Oligonucleotide‐Based Exchangeable Fluorophores
No full textSuper-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI
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
Einzelmolekül-Fluoreszenzmikroskopie in lebenden und Modellmembranen
No full textDer eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt um der
Immunantwort eines Wirtes zu entkommen und eine persistente Infektion innerhalb dessen
aufrechtzuerhalten. Ein zentrales Element seiner Verteidigungsstrategie stützt sich auf die
Schutzfunktion seines Proteinmantels auf der Zelloberfläche. Dieser Mantel besteht aus
einer dichten Schicht aus identischen, Glykosylphosphatidylinositol (GPI)-verankerten
variablen Oberflächenglykoproteinen (VSG). Der VSG Mantel verhindert die Erkennung
der darunterliegenden, invarianten Epitope durch das Immunsystem. Obwohl es notwendig
ist die Funktionsweise des VSG Mantels zu verstehen, vor allem um ihn als mögliches Angriffsziel
gegen den Parasiten zu verwenden, sind seine biophysikalischen Eigenschaften
bisher nur unzureichend verstanden. Dies ist vor allem der Tatsache geschuldet, dass die
hohe Motilität der Parasiten mikroskopische Studien in lebenden Zellen bisher weitestgehend
verhinderten.
In der vorliegenden Arbeit wird nun hochmoderne Einzelmolekül-Fluoreszenzmikroskopie
(EMFM) als Möglichkeit für mikroskopische Untersuchungen im Forschungsbereich der
Trypanosomen vorgestellt. Die Arbeit umfasst Untersuchungen der VSG Dynamik unter
definierten Bedingungen künstlicher Membransysteme. Es wurde zuerst der Einfluss der
lateralen Proteindichte auf die VSG Diffusion untersucht. Experimente mittels Fluoreszenz-
Wiederkehr nach irreversiblem Photobleichen und komplementäre Einzelmolekül-
Verfolgungs Experimente offenbarten, dass ein molekularer Diffusionsschwellenwert
existiert. Über diesem Schwellenwert wurde eine dichteabhänige Reduzierung des
Diffusionskoeffizienten gemessen. Eine relative Quantifizierung der rekonstituierten VSGs
verdeutlichte, dass der Oberflächenmantel der Trypanosomen sehr nahe an diesem
Schwellenwert agiert. Der VSG Mantel ist optimiert um eine hohe Proteindichte bei gleichzeitiger
hoher Mobilität der VSGs zu gewährleisten. Des Weiteren wurde der Einfluss
der VSG N-Glykosylierung auf die Diffusion des Proteins quantitativ untersucht. Die
Messungen ergaben, dass die N-Glykosylierung dazu beiträgt eine hohe Mobilität bei
hohen Proteindichten aufrechtzuerhalten. Eine detaillierte Analyse von VSG Trajektorien
offenbarte, dass zwei unterschiedliche Populationen frei diffundierender VSGs in der
künstlichen Membran vorlagen. Kürzlich wurde entdeckt, dass VSGs zwei strukturell unterschiedliche Konformationen annehmen können. Die Messungen in der Arbeit stimmen
mit diesen Beschreibungen überein.
Die Ergebnisse der EMFM in künstlichen Membranen wurden durch VSG Einzelmolekül-
Verfolgungs Experimente auf lebenden Zellen ergänzt. Es wurde eine hohe Mobilität
und Dynamik einzelner VSGs gemessen, was die allgemein dynamische Natur des VSG
Mantels verdeutlicht. Dies führte zu der Schlussfolgerung, dass der VSG Mantel auf
lebenden Trypanosomen ein dichter und dennoch dynamischer Schutzmantel ist. Die
Fähigkeit der VSGs ihre Konformation flexibel anzupassen, unterstützt das Erhalten der Fluidität bei variablen Dichten. Diese Eigenschaften des VSG Mantels sind elementar für
die Aufrechterhaltung einer presistenden Infektion eines Wirtes.
In dieser Arbeit werden des Weiteren verschiedene, auf Hydrogel basierende Einbettungsmethoden
vorgestellt. Diese ermöglichten die Zellimmobilisierung und erlaubten
EMFM in lebenden Trypanosomen. Die Hydrogele wiesen eine hohe Zytokompatibilität
auf. Die Zellen überlebten in den Gelen für eine Stunde nach Beginn der Immobilisierung.
Die Hydrogele erfüllten die Anforderungen der Superresolution Mikroskopie (SRM) da
sie eine geringe Autofluoreszenz im Spektralbereich der verwendeten Fluorophore besaßen.
Mittels SRM konnte nachgewiesen werden, dass die Hydrogele die Zellen effizient
immobilisierten. Als erstes Anwendungsbeispiel der Methode wurde die Organisation
der Plasmamembran in lebenden Trypanosomen untersucht. Die Untersuchung eines
fluoreszenten Tracers in der inneren Membranschicht ergab, dass dessen Verteilung nicht
homogen war. Es wurden spezifische Membrandomänen gefunden, in denen das Molekül
entweder vermehrt oder vermindert auftrat. Dies führte zu der Schlussfolgerung, dass diese
Verteilung durch eine Interaktion des Tracers mit Proteinen des zellulären Zytoskeletts
zustande kam.
Die in dieser Arbeit präsentierten Ergebnisse zeigen, dass EMFM erfolgreich für verschiedene
biologische Untersuchungen im Forschungsfeld der Trypanosomen angewendet
werden kann. Dies gilt zum Beispiel für die Untersuchung von der VSG Dynamik in künstlichen
Membransystemen, aber auch für Studien in lebenden Zellen unter Verwendung
der auf Hydrogelen basierenden Zelleinbettung.The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to escape
the host immune response and maintain a persistent infection inside a host. One central
feature of the parasite’s defense mechanism relies on the shielding function of their surface
protein coat. This coat is composed of a dense arrangement of one type of glycosylphosphatidylinositol
(GPI)-anchored variant surface glycoproteins (VSGs) which impair the
identification of epitopes of invariant surface proteins by the immune system. In addition
to the importance of understanding the function of the VSG coat and use it as a potential
target to efficiently fight the parasite, it is also crucial to study its biophysical properties as it is not yet understood sufficiently. This is due to the fact that microscopic investigations
on living trypanosomes are limited to a great extent by the intrinsic motility of the parasite.
In the present study, state-of-the-art single-molecule fluorescence microscopy (SMFM)
is introduced as a tool for biophysical investigations in the field of trypanosome research.
The work encompasses studies of VSG dynamics under the defined conditions of an
artificial supported lipid bilayer (SLB). First, the impact of the lateral protein density on
VSG diffusion was systematically studied in SLBs. Ensemble fluorescence after photobleaching
(FRAP) and complementary single-particle tracking experiments revealed that a
molecular crowding threshold (MCT) exists, above which a density dependent decrease
of the diffusion coefficient is measured. A relative quantification of reconstituted VSGs
illustrated that the VSG coat of living trypanosomes operates very close to its MCT and
is optimized for high density while maintaining fluidity. Second, the impact of VSG
N-glycosylation on VSG diffusion was quantitatively investigated. N-glycosylation was
shown to contribute to preserving protein mobility at high protein concentrations. Third,
a detailed analysis of VSG trajectories revealed that two distinct populations of freely
diffusing VSGs were present in a SLB, which is in agreement with the recent finding, that
VSGs are able to adopt two main structurally distinct conformations. The results from
SLBs were further complemented by single-particle tracking experiments of surface VSGs
on living trypanosomes. A high mobility and free diffusion were measured on the cell
surface, illustrating the overall dynamic nature of the VSG coat. It was concluded that
the VSG coat on living trypanosomes is a protective structure that combines density and
mobility, which is supported by the conformational flexibility of VSGs. These features are
elementary for the persistence of a stable infection in the host.
Different hydrogel embedding methods are presented, that facilitated SMFM in immobilized,
living trypanosomes. The hydrogels were found to be highly cytocompatible for one
hour after cross-linking. They exhibited low autofluorescence properties in the spectral
range of the investigations, making them suitable for super-resolution microscopy (SRM).
Exemplary SRM on living trypanosomes illustrated that the hydrogels efficiently immobilized
the cells on the nanometer lever. Furthermore, the plasma membrane organization was studied in living trypanosomes. A statistical analysis of a tracer molecule inside the
inner leaflet of the plasma membrane revealed that specific membrane domains exist, in
which the tracer appeared accumulated or diluted. It was suggested that this distribution
was caused by the interaction with proteins of the underlying cytoskeleton.
In conclusion, SMFM has been successfully introduced as a tool in the field of trypanosome
research. Measurements in model membranes facilitated systematic studies of VSG dynamics
on the single-molecule level. The implementation of hydrogel immobilization
allowed for the study of static structures and dynamic processes with high spatial and
temporal resolution in living, embedded trypanosomes for the first time
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
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