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  • 1
    Language: English
    In: The Journal of biological chemistry, 06 February 2015, Vol.290(6), pp.3654-65
    Description: Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the β1- and β3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of β-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.
    Keywords: Atp1a1 ; Fibroblast Growth Factor (Fgf) ; Heparan Sulfate ; Membrane Recruitment ; Membrane Translocation ; Na+/K+-Atpase ; Phosphoinositide ; Plasma Membrane ; Tec Kinase ; Unconventional Secretion ; Secretory Pathway ; Fibroblast Growth Factor 2 -- Metabolism ; Sodium-Potassium-Exchanging Atpase -- Metabolism
    E-ISSN: 1083-351X
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  • 2
    Language: English
    In: Bioorganic & Medicinal Chemistry Letters, 01 October 2014, Vol.24(19), pp.4694-4698
    Description: Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5′- and 3′-modifications include 5′-trimethoxystilbene with 3′-uracil(anthraquinone)-cap, 5′-chloic acid derivative with 3′-uracyl(anthraquinone)-cap and 5′-cholic acid derivative with three 3′-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5′-cholic acid derivative with three 3′-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.
    Keywords: Phosphorothioate ; Chemical Modification ; Terminal Modifier ; Oligonucleotide ; Antisense ; Medicine ; Chemistry ; Anatomy & Physiology
    ISSN: 0960-894X
    E-ISSN: 1464-3405
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  • 3
    Language: English
    In: Biotechnology Journal, Sept, 2015, Vol.10(9), p.1467(11)
    Keywords: Antineoplastic Agents -- Analysis ; Antineoplastic Agents -- Health Aspects ; Rna Interference -- Analysis ; Rna Interference -- Health Aspects ; Melanoma -- Genetic Aspects ; Melanoma -- Analysis ; Melanoma -- Health Aspects ; Computer Vision -- Analysis ; Computer Vision -- Health Aspects ; Phenotypes -- Analysis ; Phenotypes -- Health Aspects ; Genes -- Analysis ; Genes -- Health Aspects ; Gene Expression -- Analysis ; Gene Expression -- Health Aspects
    ISSN: 1860-6768
    Source: Cengage Learning, Inc.
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  • 4
    In: Chemical Communications, 2013, Vol.49(67), pp.7397-7399
    Description: We applied 14-mer 2-OMe RNAs as inhibitors of selected micro RNAs. To improve their properties, we introduced a trimethoxystilbene residue at the 5-terminus and three 2-fluoro-2-deoxynucleotides at the 3-terminus to obtain potent inhibitors, whose mismatch discrimination is substantially better than that of typically applied 〉18-mers.
    Keywords: Micrornas -- Antagonists & Inhibitors ; Oligonucleotides -- Chemistry ; Stilbenes -- Chemistry;
    ISSN: 1359-7345
    E-ISSN: 1364-548X
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  • 5
    Language: English
    In: The Journal of cell biology, 18 February 2013, Vol.200(4), pp.505-22
    Description: Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference-based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110-Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.
    Keywords: Axoneme -- Metabolism ; Cilia -- Metabolism ; Protein-Serine-Threonine Kinases -- Physiology
    ISSN: 00219525
    E-ISSN: 1540-8140
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  • 6
    In: Nature, 2010, Vol.464(7289), p.721
    Description: Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
    Keywords: Sciences (General) ; Physics;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 7
    Language: English
    In: PLoS ONE, 01 January 2012, Vol.7(12), p.e52555
    Description: miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: Histochemistry and Cell Biology, 2014, Vol.141(6), pp.597-603
    Description: We have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution d STORM microscope (direct stochastic optical reconstruction microscopy). The coordinate transfer was based on fixed cells as reference points without the use of additional fiducial markers. This procedure is suitable to combine any kind of fluorescence microscopy technique, in order to gain further insights on the observed specimen at multiple temporal or special scales.
    Keywords: High-throughput and high-content screening ; Single-molecule localization ; STORM
    ISSN: 0948-6143
    E-ISSN: 1432-119X
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  • 9
    Language: English
    In: BMC Bioinformatics, Dec 20, 2011, Vol.12, p.485
    Description: Background High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology. Results We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. Conclusions Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.
    Keywords: Genes -- Identification And Classification ; Genetic Testing -- Methods ; Genetic Testing -- Research ; Rna Interference -- Physiological Aspects ; Rna Interference -- Usage
    ISSN: 1471-2105
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: BMC Bioinformatics, Dec 20, 2011, Vol.12, p.485
    Description: Background High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology. Results We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. Conclusions Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.
    Keywords: Genes -- Identification And Classification ; Genetic Testing -- Methods ; Genetic Testing -- Research ; Rna Interference -- Physiological Aspects ; Rna Interference -- Usage
    ISSN: 1471-2105
    Source: Cengage Learning, Inc.
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