34 research outputs found

    Modular function of long noncoding RNA, COLDAIR, in the vernalization response

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    <div><p>The long noncoding RNA COLDAIR is necessary for the repression of a floral repressor <i>FLOWERING LOCUS C</i> (<i>FLC</i>) during vernalization in <i>Arabidopsis thaliana</i>. The repression of <i>FLC</i> is mediated by increased enrichment of Polycomb Repressive Complex 2 (PRC2) and subsequent trimethylation of Histone H3 Lysine 27 (H3K27me3) at <i>FLC</i> chromatin. In this study we found that the association of COLDAIR with chromatin occurs only at the <i>FLC</i> locus and that the central region of the COLDAIR transcript is critical for this interaction. A modular motif in COLDAIR is responsible for the association with PRC2 <i>in vitro</i>, and the mutations within the motif that reduced the association of COLDAIR with PRC2 resulted in vernalization insensitivity. The vernalization insensitivity caused by mutant COLDAIR was rescued by the ectopic expression of the wild-type COLDAIR. Our study reveals the molecular framework in which COLDAIR lncRNA mediates the PRC2-mediated repression of <i>FLC</i> during vernalization.</p></div

    Ectopic expression of COLDAIR restores vernalization response in COLDAIR_Mut lines.

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    <p><b>(A)</b> Flowering times of the wild-type plants (<i>FRI_Col</i>), COLDAIR_Mut plants, and and COLDAIR_Mut plants complemented with the 35S::COLDAIR at NV and 40V (<i>n</i> = 16). <b>(B)</b> Representative flowering behaviors of wild-type (<i>FRI_Col</i>) plants, <i>flc-2FRI</i>, COLDAIR_WT, COLDAIR_Mut, and COLDAIR_Mut complemented with 35S::COLDAIR without (NV) and with (40V) vernalization. <b>(C)</b> Levels of <i>FLC</i> mRNA during the course of vernalization in COLDAIR_WT, COLDAIR_Mut, and COLDAIR_Mut complemented with 35S::COLDAIR. <b>(D)</b> Schematic model of interactions of COLDAIR with the PRC2 complex over <i>FLC</i> during vernalization. A structured region of the COLDAIR transcript, which originates from the first intron of <i>FLC</i>, interacts with CLF-containing PRC2 complex during vernalization. Whether the interaction of COLDAIR-PRC2 complex with DNA is direct or other DNA-binding proteins (DBP) are involved remains to be determined.</p

    Stable repression of <i>FLC</i> by vernalization is impaired in transgenic lines that express COLDAIR_Mut.

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    <p><b>(A)</b> Relative COLDAIR transcript amounts during the course of vernalization in wild-type (<i>FRI</i>_Col) plants, transgenic lines expressing COLDAIR_WT, and transgenic lines expressing COLDAIR_Mut. (<b>B)</b> Relative fold changes in COLDAIR RNA retrieved by RNA immunoprecipitation using anti-CLF antibody followed by qRT-PCR from wild type (<i>FRI</i>_Col) and COLDAIR_Mut plants. <b>(C)</b> Levels of <i>FLC</i> mRNA during the course of vernalization in the wild-type (<i>FRI</i>_Col) plants and transgenic lines expressing COLDAIR_WT and COLDAIR_Mut. Data plotted are means ± SD; <i>n</i> = 3; * <i>p</i><0.1.</p

    Mapping of CLF-interacting region of COLDAIR.

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    <p><b>(A)</b> Schematic representation of full length and truncated transcripts of COLDAIR used in RNA binding assay. <b>(B~D)</b> RNA-binding assays using biotinylated RNAs and nuclear extracts of transgenic plants that express GFP-CLF. Anti-GFP antibody (ab290, Abcam) was used in western blot analyses. tRNA was used as a random RNA control (<b>D</b>).</p

    COLDAIR_Mut transgenic plants exhibit late flowering after vernalization.

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    <p><b>(A)</b> Predicted secondary structures and sequences of wild-type COLDAIR and COLDAIR_Mut (<a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi" target="_blank">http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi</a>). Mutated nucleotides are indicated in red. <b>(B)</b> Flowering time of wild-type and primary transgenic plants. Top) Flowering times of wild-type (<i>FRI_Col</i>) plants after 40 days vernalization. Middle) Flowering times of the primary transgenic lines carrying wild-type genomic <i>FLC</i> transgene (COLDAIR_WT) in <i>flc-2</i> after 40 days vernalization. Bottom) Flowering times of the primary transgenic lines carrying mutated genomic <i>FLC</i> transgene (COLDAIR_Mut) in <i>flc-2</i> after 40 days vernalization. <i>X</i>-axis: rosette leaf numbers at flowering. <b>(C)</b> Photographs of representative plants showing the flowering behaviors of <i>flc-2 FRI</i> transformed with the wild-type <i>FLC</i> transgene (left) and <i>flc-2 FRI</i> that express COLDAIR_Mut (right) after 40 days of vernalization. (<b>D)</b> Flowering time as number of rosette leaves on wild-type (<i>FRI_Col</i>), COLDAIR_WT, and COLDAIR_Mut plants that were non-vernalized (NV) and vernalized (40V). Arrows indicate the flowering time with more than 100 leaves.</p

    Vernalization-mediated histone modifications over <i>FLC</i> are impaired in COLDAIR_Mut line.

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    <p><b>(A)</b> Schematic representation of <i>FLC</i> gene with regions of primer binding (P1, P2, P3, and P4) used indicated; the P3 binding site is within the COLDAIR coding area. <b>(B)</b> Occupancy of CLF at <i>FLC</i> chromatin in COLDAIR_WT and COLDAIR_Mut lines during the course of vernalization. <b>(C)</b> H3K27me3 at <i>FLC</i> chromatin in COLDAIR_WT and COLDAIR_Mut lines during the course of vernalization. <b>(D)</b> H3K4me3 at <i>FLC</i> chromatin in COLDAIR_WT and COLDAIR_Mut lines during the course of vernalization. Data plotted are means ± SD of qPCR; <i>n</i> = 4; * <i>p</i><0.05.</p

    Detection of COLDAIR-associated chromatin by ChIRP.

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    <p><b>(A)</b> (top) Schematic representation of <i>FLC</i> gene with regions of primer binding (P1, P2, P3, and P4) used in ChIRP detection indicated; the P3 binding site is within the COLDAIR coding area. (bottom) Plot of amount of DNA precipitated. Anti-COLDAIR probes retrieved a significant amount of DNA only in the P3 area (black), whereas anti-LacY probes showed no significant enrichment (grey). Means ± SD are shown (<i>n</i> = 3). **, <i>p</i><0.01. <b>(B)</b> Detection of ChIRP result by qPCR after RNase treatment. Anti-COLDAIR probes showed significant enrichment in P3 area (black), which was reduced after RNase A and RNase H treatment of the chromatin prior to precipitation (white). Anti-LacY probes showed no enrichment (grey). Means ± SD are shown (<i>n</i> = 3). **, <i>p</i><0.01, *, <i>p</i><0.05. <b>(C)</b> Browser tracks of normalized ChIRP-Seq results with anti-COLDAIR probes (upper panel) and anti-LacY probes (lower panel).</p

    Correction to “Dual-Surfactant-Capped Ag Nanoparticles as a Highly Selective and Sensitive Colorimetric Sensor for Citrate Detection”

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    Correction to “Dual-Surfactant-Capped Ag Nanoparticles as a Highly Selective and Sensitive Colorimetric Sensor for Citrate Detection

    Highly Stretchable and Mechanically Stable Transparent Electrode Based on Composite of Silver Nanowires and Polyurethane–Urea

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    Transparent electrodes based on conventional indium–tin oxide (ITO) can hardly meet the requirements of future generations of stretchable electronic devices, including artificial skins, stretchable displays, sensors, and actuators, because they cannot retain high conductivity under substantial stretching and bending deformation. Here we suggest a new approach for fabricating highly stretchable and transparent electrodes with good stability in environments where they would be stretched repeatedly. We designed polyurethane–urea (PUU), a urethane-based polymer, to enhance the adhesion between Ag nanowires (AgNWs) and poly­(dimethylsiloxane) (PDMS). The adhesion could be further improved when irradiated by intense pulsed light (IPL). After delicate optimization of the layered AgNW/PUU/PDMS structure, we fabricated a stretchable transparent electrode that could withstand 100 cycles of 50% stretching–releasing, with exceptionally high stability and reversibility. This newly developed electrode is therefore expected to be directly applicable to a wide range of high-performance, low-cost, stretchable electronic devices

    In Vitro Evaluation of Dendrimer–Polymer Hybrid Nanoparticles on Their Controlled Cellular Targeting Kinetics

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    Although polymeric nanoparticles (NPs) and dendrimers represent some of the most promising cancer-targeting nanocarriers, each of them has drawbacks such as limited tissue diffusivity/tumor penetration and rapid in vivo elimination, respectively. To address these issues, we have designed a multiscale hybrid NP system (nanohybrid) that combines folate (FA)-targeted poly­(amidoamine) dendrimers and poly­(ethylene glycol)-<i>b</i>-poly­(d,l-lactide) NPs. The nanohybrids (∼100 nm NPs encapsulating ∼5 nm targeted dendrimers) were extensively characterized through a series of in vitro experiments that validate the design rationale of the system, in an aim to simulate their in vivo behaviors. Cellular uptake studies using FA receptor (FR)-overexpressing KB cells (KB FR<sup>+</sup>) revealed that the nanohybrids maintained high FR selectivity resembling the selectivity of free dendrimers, while displaying temporally controlled cellular interactions due to the presence of the polymeric NP shells. The cellular interactions of the nanohybrids were clathrin-dependent (characteristic of polymer NPs) at early incubation time points (4 h), which were partially converted to caveolae-mediated internalization (characteristic of FA-targeted dendrimers) at longer incubation hours (24 h). Simulated penetration assays using multicellular tumor spheroids of KB FR<sup>+</sup> cells also revealed that the targeted dendrimers penetrated deep into the spheroids upon their release from the nanohybrids, whereas the NP shell did not. Additionally, methotrexate-containing systems showed the selective, controlled cytotoxicity kinetics of the nanohybrids. These results all demonstrate that our nanohybrids successfully integrate the unique characteristics of dendrimers (effective targeting and penetration) and polymeric NPs (controlled release and suitable size for long circulation) in a kinetically controlled manner
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