Right atrial systolic flow patterns (streamlines) viewed from the right sagittal (left hand column) and anteroposterior (right hand column) projections during late ventricular systole/atrial filling phase.

<p>SVC flow (red) and IVC flow (green).</p

Streamline visualization (velocity encoded) of ‘typical clockwise’ right atrial vortex during late ventricular systole/atrial filling phase in a control subject viewed from the right sagittal aspect, demonstrating positioning of the planes and contours in the caval veins and associated flow waveform across the cardiac cycle (*peak systolic flow, _†peak diastolic flow, note negative SVC flow reflects directionality; flow magnitude only is reported in the results).

<p>Streamline visualization (velocity encoded) of ‘typical clockwise’ right atrial vortex during late ventricular systole/atrial filling phase in a control subject viewed from the right sagittal aspect, demonstrating positioning of the planes and contours in the caval veins and associated flow waveform across the cardiac cycle (*peak systolic flow, _†peak diastolic flow, note negative SVC flow reflects directionality; flow magnitude only is reported in the results).</p

Type of stroke, clinical management and findings of routine investigations in subjects.

<p>Type of stroke, clinical management and findings of routine investigations in subjects.</p

Summary Data for Bland-Altman Plots to Characterize Inter and Intra Observer and Scan-Scan Reproducibility of Atrial 4D Flow Measurements.

<p>Summary Data for Bland-Altman Plots to Characterize Inter and Intra Observer and Scan-Scan Reproducibility of Atrial 4D Flow Measurements.</p

Distance between vena cava in the right left plane (judged by comparing points at which centerlines of respective caval vein flows intersected with right atrial junction, S1 Fig) in CS-PFO group and controls.

<p>Distance between vena cava in the right left plane (judged by comparing points at which centerlines of respective caval vein flows intersected with right atrial junction, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173046#pone.0173046.s001" target="_blank">S1 Fig</a>) in CS-PFO group and controls.</p

Right Atrial Size and Flow Characteristics in CS-PFO group and matched controls.

<p>Right Atrial Size and Flow Characteristics in CS-PFO group and matched controls.</p

Left atrial, anatomy, size and flow characteristics in CS-PFO group and matched controls.

<p>Left atrial, anatomy, size and flow characteristics in CS-PFO group and matched controls.</p

Peripheral leukocytosis after primary percutaneus coronary intervention (PPCI).

<p>Absolute number of total leukocytes, neutrophil granulocytes and monocytes (<b>A</b>), as well as lymphocytes and the main T-cell subsets (<b>B</b>) in peripheral blood. Concentration of leukocyte populations are displayed for patients 24 h following PPCI in STEMI (n = 31), 3 months post PPCI (n = 24), and in age-matched healthy Controls (n = 18). 1-way ANOVA with Tukeys' post-hoc test was performed. *p<0.05, ***p<0.001, ns not significant.</p

Molecular characterization of the <i>Cited2 ^T166N</i>, <i>Cited2 ^MRG1</i> and <i>Cited2 ^HUM</i> alleles.

<p>(A) Western blot of total protein lysates from mouse embryonic fibroblasts (MEFs), probed with anti-CITED2 antibody. CITED2 and CITED2-MRG1 are indicated, as is a non-specific band (N.S.) that migrates at 25 kDa. (B) RT-PCR showing RNA products expressed by embryos of various genotypes. PCR primers were designed to differentiate between the endogenous mouse <i>Cited2</i> transcript and the <i>Cited2 ^MRG1</i> transcript by their size difference. Wild type mouse <i>Cited2</i>, containing the SRJ domain produces the larger 725 bp band. (C) Southern blots of <i>Cited2 ^T166N</i> allele. Top, Southern blot of <i>EcoRI</i> digested genomic DNA probed with a 5′-probe. Middle, Southern blot of <i>BglII</i> digested genomic DNA, probed with a 3′-probe. Probe positions are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046256#pone-0046256-g003" target="_blank">Fig. 3</a>. Bottom, Southern blot of <i>SpeI</i> digested genomic DNA hybridized with an internal (Neomycin) probe, to confirm single copy integration. (D) Southern blots of <i>Cited2 ^MRG1</i> and <i>Cited2 ^HUM</i> alleles. Top, Southern blot of <i>EcoRI/SacII</i> digested genomic DNA, probed with a 5′-probe. Middle, Southern blot of <i>BglII</i> digested genomic DNA, probed with a 3′-probe. The position of the probes is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046256#pone-0046256-g003" target="_blank">Fig. 3</a>. Bottom, Southern blot of <i>EcoRI</i> digested genomic DNA hybridized with an internal (Puromycin) probe to confirm single copy integration.</p

Functional Significance of SRJ Domain Mutations in <em>CITED2</em>

<div><p>CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of <em>Cited2</em> in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 <em>in vitro</em> at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance <em>in vivo</em>, we generated a T166N mutation of mouse <em>Cited2</em>. We also used PhiC31 integrase-mediated cassette exchange to generate a <em>Cited2</em> knock-in allele replacing the mouse <em>Cited2</em> coding sequence with human <em>CITED2</em> and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using <em>in vivo</em> models and that predictions based on structural conservation and <em>in vitro</em> functional assays, or even <em>in vivo</em> global loss of function models, may be insufficient.</p> </div