61 research outputs found

    KRAB-Induced Heterochromatin Effectively Silences <i>PLOD2</i> Gene Expression in Somatic Cells and is Resilient to TGFβ1 Activation

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    Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression

    Inhalation characteristics and their effects on in vitro drug delivery from dry powder inhalers .2. Effect of peak flow rate (PIFR) and inspiration time on the in vitro drug release from three different types of commercial dry powder inhalers

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    Three commercial dry powder inhalers with completely different dosing and powder disintegration principles were evaluated in an in vitro deposition study. A four-stage cascade impactor was used for the range of flow rates between 20 and 60 1/min. Turbuhaler, Diskhaler and Spinhaler showed increasing amounts of drug discharged from the dose system with increasing peak inspiratory flow rate (PIFR). Only for the Spinhaler, was discharge influenced by total inspiration time as well. All three inhalers also showed improved powder disintegration with increasing PIFR. Highest fine particle yield was obtained from the Turbuhaler, reaching a maximum of 35-40% of the nominal dose at flow rates of 50-60 1/min. In comparison, less than 10% of the nominal dose from the Spinhaler and on average 23% from the Diskhaler were released as fine drug particles at 60 1/min. From the work of inspiration involved, it has been concluded that a short and fast inspiration through the Turbuhaler gives an optimal result from fine particle output and from efficiency point of view
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