1,721,006 research outputs found
The Isoquinolone Derived Prolyl Hydroxylase Inhibitor ICA Is a Potent Substrate of the Organic Anion Transporters 1 and 3
No full textObjective: Many cellular responses to hypoxia are mediated by the transcription factor complex hypoxia-inducible factor (HIF). HIF stability is governed by a family of dioxygenases called HIF prolyl hydroxylases (PHDs). Isoquinolone-derived PHD inhibitors, like 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), which stabilize the intracellular HIF-alpha have been suggested as a potentially beneficial therapeutic strategy for the treatment of disorders associated with ischemia. To stabilize HIF-alpha, ICA has to be taken up into proximal tubule cells (PCTs) across the basolateral membrane by one of the organic anion transporters 1, 2 or 3 (OAT1, OAT2 or OAT3). The release into the urine across the lumina] membrane may be mediated by OAT4. Method: To demonstrate interaction of ICA with human OAT1, OAT2, OAT3 and OAT4, ICA was tested on these transporters stably transfected in HEK293 cells by using p-aminohippurate (PAH), cGMP and estrone-3-sulfate (ES) as reference substrates, respectively. Results: Uptakes of PAH and ES in OAT1- and OAT3-transfected HEK293 cells were inhibited by ICA with half-maximal inhibition values of 0.29 +/- 0.05 and 2.58 +/- 0.16 mu m, respectively. OAT2 was less sensitive to ICA. Efflux experiments identified ICA as an OAT1 and OAT3 sub-strate. Preloading OAT4-transfected HEK293 cells with ICA stimulated ES uptake by 18.3 +/- 3.8%. Conclusion: The uptake of ICA across the basolateral membrane of PCTs occurs mainly by OAT1 and the efflux into the tubular lumen by OAT4. (C) 2015 S. Karger AG, Base
Inhibitors of prolyl hydroxlase are substrates of the organic anion transporter 1 (OAT1)
No full textInhibitors of prolyl hydroxlase are substrates of the organic anion transporter 1 (OAT1)
No full text
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
Aktivierung von HIF-Zielgenen durch CRISPR/dCas9-Aktivatoren
Full text linkBackground and Objectives:
Oxygen plays a fundamental role in the development, performance and survival of the human organism. Thus, humans maintain a cellular regulatory system for adapting to changes in oxygen levels. The key regulators of this system are hypoxia-inducible transcription factors (HIFs). HIFs are transcription factors that modulate the expression of specific genes in order to improve oxygen delivery under conditions of low oxygen tension (hypoxia). These HIF target genes are involved in a multitude of different processes such as angiogenesis, erythropoiesis and energy metabolism. As a dysregulation of HIFs shows implication in many pathologies such as cancer or stroke and the HIF system further provides new potential therapeutic approaches, it is of great interest to widen the knowledge about HIF-regulated gene expression and to influence expression of HIF target genes with protective effects directly. For therapeutic purposes, for instance, the modulation of the HIF system has already demonstrated to be a promising strategy in the treatment of renal anaemia in patients with chronic kidney disease.
The CRISPR/dCas9-system proved to be a tool for selective and specific gene activation. It is composed of an activator domain fused to an inactivated Cas9 enzyme. After a gene-specific single guide RNA directs the CRISPR/dCas9-activator to a certain gene of interest, it behaves as a transcriptional activator of the target gene.
Aim of this dissertation was to test different CRISPR/dCas9-activators for their application in gene activation. Furthermore, it should been investigated whether the HIF target genes Carboanhydrase IX (CA9) and Erythropoietin (EPO) can be activated independently of HIF by using CRISPR/dCas9-activators and specific single guide RNAs. In addition, the activation of EPO via different regulatory elements and cell specific differences in the regulation of the EPO gene was examined using a Chromatin Immunoprecipitation (ChIP) assay.
Material and Methods:
The used CRISPR/dCas9-activators CRISPR/dCas9-VPR, -SunTag and -p300 were obtained from the company Addgene in form of transformed bacteria.
A cloning protocol was used for generating single guide RNAs directing regulatory elements of the target genes. Therefore, oligonucleotides carrying gene specific promoter or enhancer sequences (Sigma-Aldrich) were inserted into the expression plasmid pSPgRNA (Addgene).
Different cell lines were transfected with CRISPR/dCas9-activators and single guide RNAs using Lipofectamine® 3000 (Invitrogen). After RNA isolation and reverse transcription into complementary DNA gene expression was measured by quantitative real-time PCR using a SYBR Green® Assay (ThermoFisher Scientific).
For H3K27ac ChIP experiments, cells were treated with DMOG [1 mM] for 16 hours in order to stabilize HIF. After crosslinking proteins to the DNA with formaldehyde and the sonication of the DNA, bound DNA fragments were isolated using specific antibodies. Then the DNA was extracted with phenol-chloroform and amplified via qPCR.
Results:
After transfecting HEK293T, Hep3B and MCF-7 with the three different CRISPR/dCas9-activators and single guide RNAs targeting the promoters of IL1RN and OCT4, CRISPR/dCas9-VPR showed the most robust and effective gene activation under the used experimental conditions. Furthermore, the application of CRISPR/dCas9-VPR proved to be efficient in activating the HIF target genes CA9 and erythropoietin in Hep3B, HepG2 and Kelly cell lines. Additionally, the expression of the erythropoietin gene was inducible not only in cells known to produce erythropoietin, but also in the MCF-7 breast cancer cell line, which has not been described to be capable of producing EPO. The H3K27ac ChIP confirmed the activation of EPO via the different regulatory elements tested (EPO5’-enhancer, EPO3’-enhancer and EPO-promotor) and validated the published cell specific differences in EPO-regulation in Hep3B and Kelly cells.
Conclusion:
This work demonstrates that CRISPR/dCas9-VPR is a suitable tool for specific and selective activation of HIF target genes and confirms the usability of the CRISPR/Cas9-technique. Moreover, this work reveals potential applications of CRISPR/Cas9 for HIF-based research and provides insight into potential therapeutic approaches. Additionally, ChIP-experiments confirmed regulatory elements that are fundamental for HIF-induced expression of the EPO gene as well as cell specific differences in the regulation of this gene in Kelly and Hep3B cells lines.Hintergrund und Ziele:
Sauerstoffhomöostase spielt eine zentrale Rolle für Entwicklung, Überleben und Funktion des menschlichen Organismus. Daher besitzt der Mensch ein wichtiges Regulationssystem zur Adaptation an das wechselnde Sauerstoffangebot. Dies wird von den Hypoxie-induzierbaren Faktoren (HIFs) begründet. Dabei handelt es sich um Transkriptionsfaktoren, welche unter Sauerstoffmangel (Hypoxie) die Expression spezifischer Gene zugunsten einer verbesserten Sauerstoffversorgung beeinflussen. Diese HIF-Zielgene codieren unter anderem für Proteine, die in der Neubildung von Blutgefäßen (Angiogenese), Erythrozyten (Erythropoese) sowie im Energiehaushalt involviert sind.
Aufgrund der pathologischen Konsequenzen unter HIF-Dysregulation und potentiellen Therapiemöglichkeiten durch Beeinflussung des HIF-Systems ist es von großem Interesse, die HIF-vermittelte Gentranskription näher zu erforschen und die Aktivität von HIF-Zielgenen beeinflussen zu können. Die Modulation des HIF-Systems zu therapeutischen Zwecken erwies sich beispielsweise bereits als erfolgsversprechendes Konzept für die Therapie der renalen Anämie.
Als eine geeignete Technik zur selektiven und spezifischen Beeinflussung von Genen zeigte sich das CRISPR/dCas9-System. Hierbei handelt es sich um eine an das deaktivierte CRISPR/Cas9-Enzym gekoppelte Aktivatordomäne, die durch eine der DNA-Zielsequenz komplementäre single guide RNA zum ausgewählten Genlocus dirigiert werden kann und eine Aktivierung des Zielgens vermittelt. Für diese Anwendung wurden bereits verschiedene CRISPR/dCas9-Aktivatoren etabliert.
Ziel dieser Abhandlung war es daher, einige CRISPR/dCas9-Aktivatoren auf ihre Effizienz in Beeinflussung von Genexpression zu testen sowie mithilfe von CRISPR/dCas9-Aktivatoren und genspezifischen single guide RNAs eine HIF-unabhängige Aktivierung der HIF-Zielgene Carboanhydrase IX (CA9) und Erythropoetin (EPO) zu erreichen. Vorab sollten die Aktivierung von EPO über einzelne regulatorische Elemente des EPO-Gens und publizierte zelltypspezifische Unterschiede der EPO-Regulation mithilfe von Chromatinimmunpräzipitation (ChIP) untersucht werden.
Material und Methoden:
Die CRISPR/dCas9-Aktivatoren CRISPR/dCas9-VPR, -SunTag und-p300 wurden in Form von transformierten Bakterien von der Firma Addgene bezogen.
Zur Herstellung der single guide RNAs gegen die genregulatorischen Elemente von ausgewählten Zielgenen wurden die genspezifischen Promotor- beziehungsweise Enhancersequenzen in Form von doppelsträngigen Oligonukleotiden (Sigma-Aldrich) in das pSPgRNA-Expressions-Plasmid (Addgene) eingefügt und anschließend in kompetente Escherichia coli Bakterien transformiert.
Die Transfektion verschiedener Zelllinien mit CRISPR/dCas9-Aktivatoren und den genspezifischen single guide RNAs erfolgte mittels Lipofectamine® 3000 (Invitrogen). Im Anschluss konnte die Gesamt-RNA isoliert und in komplementäre DNA übersetzt werden. Die Auswertung der Genexpression erfolgte mittels quantitativer Real-Time-PCR unter Einsatz eines SYBR Green® Assays (ThermoFisher Scientific).
Für die Chromatinimmunpräzipitation gegen den Histonmarker H3K27ac erfolgte eine HIF-Stabilisierung mittels DMOG [1 mM] für 16 Stunden. Nach Fixierung von Proteinen an die DNA durch Formaldehyd und Fragmentierung der DNA mit Hilfe von Ultraschall, wurden die H3K27ac gebundenen DNA-Fragmente durch spezifische Antikörper isoliert. Anschließend wurde die mittels Phenol-Chloroform extrahierte DNA mithilfe von qPCR amplifiziert.
Ergebnisse:
Nach Transfektion der Zelllinien HEK293T, Hep3B und MCF-7 mit den drei verschiedenen CRISPR/dCas9-Aktivatoren (CRISPR/dCas9-SunTag, -p300 und -VPR) und single guide RNAs gegen die Promotoren der Gene IL1RN und OCT4 konnte CRISPR/dCas9-VPR als der effektivste Aktivator unter den angewendeten Versuchsbedingungen identifiziert werden. Die Testung dieses Aktivators für die HIF-Zielgene CA9 und Erythropoetin zeigte ebenfalls eine Aktivierung in den EPO-produzierenden Zelllinien Hep3B, HepG2 und Kelly. Des Weiteren konnte sogar eine Induktion von Erythropoetin in der Brustkrebszelllinie MCF-7 erreicht werden, welche bekanntlich keine substantielle EPO-Produktion aufweist.
In den ChIP-Experimenten konnten die Aktivierung des EPO-Gens über verschiedene Regulationselemente (EPO5’-Enhancer, EPO3’-Enhancer und EPO-Promotor) sowie zelltypspezifische Unterschiede in der EPO-Regulation in den Zelllinien Hep3B und Kelly bestätigt werden.
Schlussfolgerungen:
In dieser Abhandlung wurde gezeigt, dass der Einsatz von CRISPR/dCas9-VPR eine selektive und spezifische HIF-Zielgenaktivierung unabhängig von Hypoxie erlaubt. Des Weiteren konnte die Spezifität der hier angewendeten CRISPR/Cas9-Technik bestätigt werden. Möglichkeiten zur genaueren Erforschung der HIF-abhängigen Genregulation und einen potentiellen Grundstein für therapeutische Ansätze werden durch die CRISPR/Cas9-Methode geboten und wurden in dieser Arbeit verdeutlicht. Zudem zeigten ChIP-Experimente gegen H3K27ac zelltypspezifische Unterschiede der EPO-Regulation in den Zellen Hep3B und Kelly
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
α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4)
Full text link2-Oxoglutarate or alpha-ketoglutarate (alpha KG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1 alpha (HIF-1 alpha) in the presence of oxygen. alpha KG analogs are applied to stabilize HIF-1 alpha even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of alpha KG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these alpha KG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1 alpha revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1 alpha. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.German Research Council [BU998/5-1
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
- …
