Continues variables measured and their relevant statistics.

<p>Continues variables measured and their relevant statistics.</p

Parameter estimates for the prediction of antiplatelet therapy delivery by significant variables from the occlusion formation study.

<p>Parameter estimates for the prediction of antiplatelet therapy delivery by significant variables from the occlusion formation study.</p

Intra-subject and inter-subject assay variation.

<p>Intra-subject and inter-subject assay variation.</p

Schematic images of the microfluidic chip.

<p>The complete device is shown in (A) with detailed view of the stenosis region (B). From a single inlet, four branching channels subject the blood flow to a range of shear rates from physiological to pathological conditions. Dimensions of all four stenoses within the device are identical, with varying shear rates imposed by resistive tubing at the outlet ports.</p

Significance of shear and dose for ASA as judged by MANOVA with Tukey's Posttest.

<p>Significance of shear and dose for ASA as judged by MANOVA with Tukey's Posttest.</p

Hazard ratio estimates for three therapies compared to each other.

<p>Hazard ratio estimates for three therapies compared to each other.</p

Formation and measurement of thrombus in a channel run within the microfluidic device measured using both microscopy and flow rate at 10000⁻¹ initial shear rate.

<p>Microscopy images (A) show aggregation initiation at the entry of the stenosis, where brighter areas of the images correspond to more platelet mass. Time stamps for images in seconds are shown at the bottom of each image, and correspond with the time axis shown below in (B). As platelets aggregate, the flow rate (B) decreases until it reaches occlusion time. The unstable thrombus detaches, indicated by the sudden increase in flow rate.</p

A Simplified and Versatile System for the Simultaneous Expression of Multiple siRNAs in Mammalian Cells Using Gibson DNA Assembly

<div><p>RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an <i>in vitro</i> recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A <i>in vitro</i> and <i>in vivo</i>. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.</p></div

Functional validation of siRNAs targeting human β-catenin.

<p>(<b>A</b>) Efficient knockdown of endogenous β-catenin in HEK-293 and SW480 cells. Total RNA was isolated from subconfluent 293-siBC4, 293-siControl, SW480-siBC4, and SW480-siControl cells, and subjected to qPCR analysis using primers specific for human β-catenin. All samples were normalized with GAPDH. Each reaction was done in triplicate. Relative β-catenin expression was calculated by dividing β-catenin expression levels with respective GAPDH levels in 293 (<b><i>a</i></b>) and SW480 (<b><i>b</i></b>) cells. The % of remaining β-catenin expression was calculated by dividing the relative β-catenin expression in siBC4 with that of the respective siControl's (<b><i>c</i></b>). “**”, <i>p<0.001</i>. (<b>B</b>) β-Catenin/Tcf transcription activity is significantly reduced in siBC4 cells. Subconfluent 293-siBC4 and 293-siControl cells were co-transfected with TOP-Luc reporter and pCMV-Wnt3A plasmids using Lipofectamine reagent (<b><i>a</i></b>), while SW480-siBC4 and SW480-siControl cells were transfected with TOP-Luc reporter plasmid using Lipofectamine reagent (<b><i>b</i></b>). At 24 h and 48 h after transfection, the cells were lysed and subjected to firefly luciferase assay using the Luciferase Reporter Assay System (Promega). Each assay condition was done in triplicate. “**”, <i>p<0.001</i>. (<b>C</b>) siBCs can effectively block Wnt3a-induced β-catenin accumulation. Subconfluent SW480-siBC4 and SW480-siControl cells fixed and subjected to immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counter stained with DAPI. Control IgG and minus primary antibody were used as negative controls (data not shown).</p

Schematic depiction of the one-step system pSOK for expressing multiple siRNAs.

<p>(<b>A</b>) Schematic representation of a tandem siRNA targeting configuration (4 sites listed as an example). The pSOK vector was constructed based on the previously reported pSOS vector, which contains opposing U6 and H1 promoters to drive siRNA duplex expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064.s001" target="_blank"><b>Figure S1A</b></a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064-Luo1" target="_blank">[15]</a>. The linker sites of the pSOS vector were modified and a SwaI site was created for linearizing the vector for Gibson Assembly (<b><i>a</i></b>). The primers were designed according to the guidelines outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064.s002" target="_blank"><b>Figure S2A</b></a>. Using the pB2B as a template vector (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064.s001" target="_blank"><b>Figure S1B</b></a>), the back-to-back U6-H1 promoter fragments with different siRNA target sites were generated. The first fragment overlaps with the 3′-end of the U6 promoter while the last fragment overlaps with the 3′-end of the H1 promoter (<b><i>b</i></b>). The ends of the middle fragments overlaps the specific siRNA target sequences (<b><i>b</i></b>). After the Gibson Assembly reaction, a single vector expressing 4 siRNA target sites is constructed (<b><i>c</i></b>). It is noteworthy that the siRNA sites may target the same or different genes. (<b>B</b>) The targeting sequences and locations of the designed siRNA sites on human (<b><i>a</i></b>) and mouse (<b><i>b</i></b>) β-catenin open reading frame (ORF). All of these sites have been validated in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064-Luo1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113064#pone.0113064-Tang1" target="_blank">[40]</a>. Note that one of the mouse siRNAs also targets human β-catenin coding sequence.</p