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H2O2 dynamics in the malaria parasite Plasmodium falciparum.
No full textHydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity
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
Etablierung von genetisch kodierten H2O2-Sonden und dynamischen Messungen von H2O2-Leveln im Malaria-Parasiten Plasmodium falciparum
Full text linkMalaria, caused by the apicomplexan parasite Plasmodium falciparum (P. falciparum), is still one of the world s most severe human infectious diseases and threatens the health and life of millions of people, mainly those living in tropical and subtropical regions, despite strong eradication efforts. P. falciparum depends on its complex antioxidative system based on thioredoxin and glutathione for survival. The intraerythrocytic parasite lives in a pro-oxidant environment, and the host immune response increases the oxidative burden on the parasite, which can cause major oxidative damage. Interfering with the parasites essential redox system is a promising target for malaria-eradicating drugs. Several antimalarial drugs are supposed to mediate their effects at least partially by increasing reactive oxygen species (ROS) in the parasite. ROS are highly reactive and damaging towards DNA, lipids and proteins. Hydrogen peroxide (H2O2) is one of the most important cellular ROS. Until now, neither molecular targets nor regulatory mechanisms and dynamic changes of H2O2-mediated signaling in P. falciparum are known. The development of the genetically encoded H2O2 sensors roGFP2-Orp1 and HyPer paved the way for non-disruptive, ratiometric, real-time, dynamic, and specific measurements of changes in H2O2 concentrations within a living cell. Studying the dynamics of H2O2 signaling in parasites exposed to antimalarial drugs can lead to a better comprehension of their molecular mode of action and give further insights into the development of future chemotherapeutic agents. In this work, the ratiometric H2O2 redox sensors roGFP2-Orp1 and HyPer-3 and the pH-insensitive version of HyPer (SypHer) were successfully transiently expressed in the cytosol of blood stage P. falciparum parasites and their functionality was systematically characterized in vitro and in cell culture. Both redox probes showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in cell culture. Due to the pH sensitivity of HyPer-3, parasites expressing roGFP2-Orp1 were used for evaluating the short, medium, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium in combination with confocal live-cell imaging. None of the quinolines or artemisinins tested had significant effects on H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. Based on the data, both roGFP2-Orp1 and HyPer-3 probes are reliable and valuable tools for studying H2O2 metabolism in living malaria parasites. However, the necessity to use a pH probe in parallel makes utilizing HyPer-3 more challenging and time consuming.Determination of the effects of oxidative and pharmacological stress on the H2O2 homeostasis was optimized by stably integrating the redox sensor roGFP2-Orp1 into the genome of P. falciparum using the attB/attP integration method. Stable genomic integration overcomes limitations of transient transfection of the probes, allowing more detailed in-cell studies. For the first time H2O2 dynamics in the mitochondrial subcellular compartment could be determined using the stably integrated Mito-roGFP2-Orp1 redox probe. In both cytosol and mitochondrion, the sensors showed reproducible sensitivity towards H2O2 in the low micromolar range and towards antimalarial compounds at pharmacologically relevant concentrations. Upon short-term exposure (4 h), artemisinin derivatives, quinine and mefloquine impacted H2O2 levels in mitochondria, whereas chloroquine and G6PD inhibitors affected the cytosol; 24 h exposure to an arylmethylamino steroid and G6PD inhibitors revealed oxidation of mitochondria and cytosol, respectively. Furthermore, the redox sensors hGrx1-roGFP2 (glutathione sensor) and sfroGFP2 were expressed in the cytosol of NF54-attB blood-stage P. falciparum parasites. Prior to stable integration, studies with the episomal transfected hGrx1-roGFP2 strain were performed. Both sensors were evaluated with regard to their sensitivity towards oxidative stress in cell culture. The results showed that G6PD inhibitors and the arylmethylamino steroid disturb GSH (reduced glutathione) redox ratio in either 4 h or 24 h incubations. The redox sensors hGrx1-roGFP2 and sfroGFP2 are both reliable tools for studying redox metabolism in malaria parasites with comparable oxidation/reduction sensitivities, at which sfroGFP2 appeared to exhibit a more pronounced fluorescence intensity. Microscopic and plate reader-based detection methods were directly compared in order to evaluate plate reader-based measurement of bulk cell cultures as an alternative for single live-cell imaging. It is now possible to investigate quickly and efficiently with high reproducibility direct responses and long-term effects of compounds on H2O2/GSH homeostasis in cell populations.Tropische Malaria, verursacht durch den einzelligen Parasiten Plasmodium falciparum (P. falciparum), stellt noch immer eine der weltweit schwerwiegendsten Infektionskrankheiten dar. Trotz intensiver Bemühungen die Malaria auszurotten, bedroht sie hauptsächlich in tropischen und subtropischen Regionen der Erde die Gesundheit und das Leben von Millionen von Menschen.P. falciparum ist in hohem Maße von seinen komplexen antioxidativen Systemen, insbesondere dem Thioredoxin- und dem Glutathionsystem, abhängig. Der intraerythrozytäre Parasit lebt in einer pro-oxidativen Umgebung und die Immunantwort des Wirtes erhöht zusätzlich den oxidativen Stress auf den Erreger. Das Eingreifen in den Redoxstoffwechsel des Parasiten stellt daher ein vielversprechendes Ziel für neue Wirkstoffe gegen Malaria dar. Mehrere Malariamedikamente vermitteln bereits jetzt ihre Wirkungen zumindest teilweise durch das Erhöhen der Konzentrationen von reaktiven Sauerstoffspezies (ROS) im Parasiten, wodurch DNA, Lipide und Proteine geschädigt werden können. Wasserstoffperoxid (H2O2) ist eine der wichtigsten zellulären ROS. Bislang waren molekulare Targets von H2O2, H2O2-vermittelte Signaltransduktion sowie dynamische Veränderungen der H2O2-Konzentration in P. falciparum wenig untersucht. Die Entwicklung der genetisch kodierten H2O2-Sensoren roGFP2-Orp1 und HyPer haben den Weg für ratiometrische, dynamische und hochspezifische Echtzeitmessungen der H2O2-Konzentrationen in lebenden Zellen geebnet. Das Erforschen der H2O2-Dynamik in Parasiten, die Malariamedikamenten ausgesetzt sind, kann zu einem besseren Verständnis ihrer molekularen Wirkungsweise und zu weiteren Erkenntnissen für die Entwicklung von zukünftigen Chemotherapeutika beitragen.In der vorliegenden Arbeit wurden die ratiometrischen H2O2-Redoxsensoren roGFP2-Orp1 und das pH-sensitive HyPer-3 sowie die pH-insensitive Version von HyPer (SypHer) im Zytosol von P. falciparum Blutstadien erfolgreich transient exprimiert und ihre Funktionalität systematisch in vitro und in Zellkultur charakterisiert. Beide Redoxsonden haben eine reproduzierbare Sensitivität in Bezug auf H2O2 im unteren mikromolaren Bereich in vitro und in Zellkultur. Aufgrund der pH-Sensitivität von HyPer-3, wurden nur in roGFP2-Orp1 exprimierenden Parasiten mittels konfokalem live-cell imaging Kurz-, Mittel- und Langzeiteffekte von Malariamedikamenten auf H2O2-Konzentrationen und Entgiftung analysiert. Keines der getesteten Quinoline oder Artemisinine zeigte in pharmakologisch relevanten Konzentrationen eine signifikante Wirkung auf die H2O2-Homöostase. Allerdings führte die Vorbehandlung der Zellen mit Malariamedikamenten oder Hitzeschock zu einer höheren Toleranz gegenüber exogenem H2O2. Auf diesen Daten basierend, sind beide Sonden, roGFP2-Orp1 und HyPer-3, zuverlässige und wertvolle Instrumente, um den H2O2-Metabolismus in lebenden Malariaparasiten zu erforschen. Die Notwendigkeit, zeitgleich mit HyPer-3 eine pH-Sonde als Kontrolle einsetzen zu müssen, macht die Verwendung von roGFP2-Orp1 allerdings deutlich leichter. In einem zweiten Schritt wurde die Bestimmung der Effekte von oxidativem und pharmakologischem Stress auf die H2O2-Homöostase weiter optimiert. Dies gelang durch die stabile Integration des Redoxsensors roGFP2-Orp1 in das Genom von P. falciparum mittels der attB/attP-Methode. Stabile genomische Integration hebt die multiplen Einschränkungen einer transienten Transfektion der Sonden auf, was deutlich detailliertere Zellstudien erlaubt. Mittels des stabil integrierten Mito-roGFP2-Orp1 Redoxsensors konnten somit zum ersten Mal H2O2-Dynamiken in einem subzellulären Kompartiment, hier dem Mitochondrium, bestimmt werden. Sowohl im Zytosol als auch im Mitochondrion zeigten die Sensoren reproduzierbare Sensitivität gegenüber H2O2 im unteren mikromolaren Bereich und gegenüber Malariamedikamenten in pharmakologisch relevanten Konzentrationen. Bei der Kurzzeit-Inkubation (4 h) zeigten Artemisinin-Derivate, Chinin und Mefloquin deutliche Effekte auf H2O2-Konzentrationen in Mitochondrien, wogegen Chloroquin und Glucose-6-Phosphat-Dehydrogenase Inhibitoren besonders das Zytosol beeinflussten. Eine 24 h Inkubation mit einem Arylmethylaminosteroid und mit G6PD Inhibitoren zeigten eine deutliche Oxidation von Mitochondrien und Zytosol.Parallel zu den H2O2-Sonden wurden die Redoxsensoren hGrx1-roGFP2 (Glutathionsensor) und sfroGFP2 stabil in das Genom von P. falciparum integriert und im Zytosol von NF54-attB Blutstadienparasiten exprimiert. Vorausgegangen waren auch hier Untersuchungen mit episomal transfizierten Zellen. Beide Sensoren messen das intrazelluläre glutathionabhängige Redoxpotenzial und wurden hinsichtlich ihrer Sensitivität auf oxidativen Stress in der Zellkultur evaluiert. In 4 h und 24 h Inkubationen wurden deutliche Effekte von G6PD Inhibitoren und dem Arylmethylaminosteroid auf das Redoxpotenzial gemessen. Dabei erwiesen sich beide stabil integrierten Sensoren, hGrx1-roGFP2 und sfroGFP2, als zuverlässige Instrumente mit vergleichbaren Oxidation-/Reduktion-Sensitivitäten, wobei sich sfroGFP2 durch eine stärkere Fluoreszenzintensität auszeichnete. Mikroskopische und Plattenleser-basierte Detektionsmethoden wurden für die Sonden direkt miteinander verglichen, um Plattenleser-basierte Messungen von Zellpopulationen als Alternative zur Einzelzell-Visualisierung zu evaluieren. Auf Basis dieser Arbeiten ist es nun möglich, die H2O2/GSH-Homöostase von Zellpopulationen schnell und effizient mit einer hohen Reproduzierbarkeit zu untersuchen
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
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