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  • 1
    Keywords: RECEPTOR ; KINASE ; PROTEIN ; DOWN-REGULATION ; fibroblasts ; BONE-MARROW ; STROMAL CELLS ; INTERNATIONAL-SOCIETY ; THERAPY POSITION STATEMENT ; GENOME-WIDE RNAI
    Abstract: Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies ranging from tissue repair in regenerative medicine to immunomodulation in graft versus host disease after allogeneic transplantation or in autoimmune diseases. Nonetheless, progress has been hampered by their enormous phenotypic as well as functional heterogeneity and the lack of uniform standards and guidelines for quality control. In this study, we describe a method to perform cellular phenotyping by high-throughput RNA interference in primary human bone marrow MSCs. We have shown that despite heterogeneity of MSC populations, robust functional assays can be established that are suitable for high-throughput and high-content screening. We profiled primary human MSCs against human fibroblasts. Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines. In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.
    Type of Publication: Journal article published
    PubMed ID: 26120366
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  • 2
    Keywords: EXPRESSION ; MICE ; DOWN-REGULATION ; METHYLATION ; METHYLTRANSFERASE ; NEPHROGENIC DIABETES-INSIPIDUS ; AQUAPORIN-2 TRAFFICKING ; WATER CHANNELS ; ENAC-ALPHA ; SET DOMAIN
    Abstract: Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.
    Type of Publication: Journal article published
    PubMed ID: 23326416
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  • 3
    Keywords: CANCER ; PATHWAY ; TOXICITY ; NUCLEUS ; INHIBITORS ; DYSFUNCTION ; SYNTHETIC LETHALITY ; MUTANT HUNTINGTIN ; HUNTINGTONS-DISEASE MUTATION
    Abstract: Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7 +/- 2.3%-58.7 +/- 14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost
    Type of Publication: Journal article published
    PubMed ID: 22162763
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  • 4
    Keywords: IN-VITRO ; PATHWAY ; TOXICITY ; GENE-TRANSFER ; MAMMALIAN-CELLS ; SMALL INTERFERING RNAS ; SHORT HAIRPIN RNAS ; MICRORNA EXPRESSION ; DICER ; VIRUS-REPLICATION
    Abstract: As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
    Type of Publication: Journal article published
    PubMed ID: 24049077
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  • 5
    Keywords: EXPRESSION ; PATHWAY ; PROTEIN ; COMPONENTS ; PHOSPHORYLATION ; IDENTIFICATION ; CANCER-CELLS ; INTERFERENCE ; AUTOMATED MICROSCOPY ; GENOME-WIDE RNAI
    Abstract: Background: Discovering functional relationships of genes through cell-based phenotyping has become an important approach in functional genomics. High-throughput imaging offers the ability to quantitatively assess complex phenotypes after perturbation by RNA interference (RNAi). Such image-based high-throughput RNAi screening studies have facilitated the discovery of novel components of gene networks and their interactions. Images generated by automated microscopy are typically analyzed by extracting quantitative features of individual cells, resulting in large multidimensional data sets. Robust and sensitive methods to interpret these data sets and to derive biologically relevant information in a high-throughput and unbiased manner remain to be developed. Results: Here we propose a new analysis method, PhenoDissim, which computes the phenotypic dissimilarity between cell populations via Support Vector Machine classification and cross validation. Applying this method to a kinome RNAi screening data set, we demonstrate that the proposed method shows a good replicate reproducibility, separation of controls and clustering quality, and we are able to identify siRNA phenotypes and discover potential functional links between genes. Conclusions: PhenoDissim is a novel analysis method for image-based high-throughput screen, relying on two parameters which can be automatically optimized without a priori knowledge. PhenoDissim is freely available as an R package.
    Type of Publication: Journal article published
    PubMed ID: 24256072
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  • 6
    ISSN: 1042-7147
    Keywords: Polymer blends ; Crystallization ; Relaxation behavior ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: In this paper we focus on miscible blends of two engineering polymers: poly(butylene terephthalate) (PBT) and a polyarylate (PAr). The issue of transesterification in these blends will be addressed, followed by a discussion of the crystallization kinetics of PBT, poly(ethylene terephthalate) and several PBT/PAr blends. The ability to estimate polymer-polymer interaction parameters in blends from melting point depression will also be discussed. The amorphous phase behavior of the PBT/PAr blends has been explored primarily using dielectric spectroscopy. For blends in which PBT has crystallized, we observe two relaxations associated with Tg-like motion, and this behavior is interpreted in light of our recent work on order-disorder interphases in crystalline blends.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Bognor Regis [u.a.] : Wiley-Blackwell
    ISSN: 0887-6266
    Keywords: polymer solution ; light scattering ; poly(isobornyl methacrylate) ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Physics
    Notes: Thermodynamic and hydrodynamic properties of dilute solutions of poly (isobornyl methacrylate) (PIMA) in tetrahydrofuran (THF) were characterized by using viscosity, static, and dynamic light scattering measurements. PIMA samples with different molecular weight were obtained by fractional precipitation of PIMA solution. Chain dimension parameters (Rg and RH), together with second virial coefficient A2 and intrinsic viscosity [η], were used to calculate various solution parameters characterizing polymer chains in polymer solutions. The experimental results are compared with calculation, indicating that PIMA behaves as a flexible coil in THF. © 1994 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 0021-8995
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Physics
    Notes: A series of hydroxyl-terminated maleopimaric acid esters (HTMAEs) and rigid polyurethane (PU) foams based on these HTMAEs were synthesized using chemically modified natural gum rosin and its derivative maleopimaric acid as raw materials. Thermal stability of these polyols and their corresponding rigid PU foams was studied by a thermogravimetric method and a dimensional stability measurement. It was shown that the thermal stability of the final foams was strongly dependent on the structure of their corresponding polyols. The thermogravimetric analysis curves of these rosin-based rigid PU foams displayed two distinct regions of weight loss. It has been shown that at the initial stage of weight loss the process was dominated by polyol component degradation; the second stage was governed by isocyanate component degradation. © 1995 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0021-8995
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Physics
    Notes: A series of rosin-based rigid polyurethane foams of different composition were synthesized directly from chemically modified gum rosin. The effect of the composition of these rosin-based rigid polyurethane foams on their thermal stability and compression strength was measured. It was shown that the onset temperature of weight loss and the dimensional stability at high temperature increased with increase of the molar ratio of NCO/OH. The TGA data further confirmed that the second stage weight loss in the two-stage weight loss process of these polyurethane foams was governed by thermal degradation of the isocyanate component. Although density had no significant influence on the TGA curves of the rosin-based rigid polyurethane foams, it had great influence on the dimensional stability at high temperature and compression strength of the foams. It has been shown that the inclusion of rosin in rigid polyurethane foams increases the strength and thermal stability compared with that of polyether-based ones. © 1996 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 0269-3879
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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