Microwave Annealing Effect for Highly Reliable Biosensor: Dual-Gate Ion-Sensitive Field-Effect Transistor Using Amorphous InGaZnO Thin-Film Transistor

We used a microwave annealing process to fabricate a highly reliable biosensor using amorphous-InGaZnO (a-IGZO) thin-film transistors (TFTs), which usually experience threshold voltage instability. Compared with furnace-annealed a-IGZO TFTs, the microwave-annealed devices showed superior threshold voltage stability and performance, including a high field-effect mobility of 9.51 cm²/V·s, a low threshold voltage of 0.99 V, a good subthreshold slope of 135 mV/dec, and an outstanding on/off current ratio of 1.18 × 10⁸. In conclusion, by using the microwave-annealed a-IGZO TFT as the transducer in an extended-gate ion-sensitive field-effect transistor biosensor, we developed a high-performance biosensor with excellent sensing properties in terms of pH sensitivity, reliability, and chemical stability

MOESM4 of Anatomic fat depots and cardiovascular risk: a focus on the leg fat using nationwide surveys (KNHANES 2008–2011)

Additional file 4: Figure S4. Difference in Korean CHD risk according to LF/TF ratio tertiles, subgroup analysis. Proportion of individuals with high CHD risk (>5%) stratified by A hypertension, B diabetes, C metabolic syndrome, and D insulin resistance (HOMA-IR). E Number of cardiovascular risk factors according to LF/TF tertiles. Risk factors are obesity, hypertension, diabetes, hyper LDL-cholesterolemia, and hypertriglyceridemia

MOESM3 of Anatomic fat depots and cardiovascular risk: a focus on the leg fat using nationwide surveys (KNHANES 2008–2011)

Additional file 3: Figure S3. Difference in Framingham CVD risk according to LF/TF ratio tertiles, subgroup analysis. Proportion of individuals with high CVD risk (>20%) stratified by A hypertension, B diabetes, C metabolic syndrome, and D insulin resistance (HOMA-IR). E Number of cardiovascular risk factors according to LF/TF tertiles. Risk factors are obesity, hypertension, diabetes, hyper LDL-cholesterolemia, and hypertriglyceridemia

MOESM1 of Anatomic fat depots and cardiovascular risk: a focus on the leg fat using nationwide surveys (KNHANES 2008–2011)

Additional file 1: Figure S1. Proportion of individuals with CVD risk score tertiles by LF/TF ratio

Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression

<div><p>Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from <i>in vitro</i> functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated <i>VEGFR3</i> mRNA by directly binding to the <i>VEGFR3</i> promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.</p></div

Snail upregulates VEGF receptor 3 (VEGFR3).

<p>(A) Western blot and RT-PCR analyses showing Snail, early growth response protein-1 (Egr-1), VEGF receptor 3 (VEGFR3), and VEGFR2 expression. HRECs were seeded at a density of 2–2.5×10⁴ cells/cm² on FN- (for western blot and RT-PCR) or PLL (for western blot)-coated dishes and cultured for the indicated time points. (B) Western blot analysis showing the effect of Snail knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siSnail on FN-coated dishes, and cultured for the indicated time. (C) Quantitative RT-PCR analysis showⁱng the effect of Snail knockdown on VEGFR3 expression. SiSnail-transfected ECs were reseeded and cultured on FN-coated dishes for 8 h. *, p<0.01. (D) Western blot and quantitative RT-PCR analyses showing the effect of Snail overexpression on VEGFR3. HUVECs were transfected with Snail. On the next day, the medium was changed, and the transfected cells were cultured for 8 h (quantitative RT-PCR; right) or 16 h (western blot; left). *, p<0.01.</p

Proposed model of capillary branching morphogenesis in postnatal mice.

<p>(A) Outline of Snail stabilization by ECM-mediated signaling. Snail is rapidly degraded by the GSK3β-dependent proteosomal system. On exposure of ECs to ECM, they activate Akt, which can suppress GSK3β-dependent system by phosphorylating GSK3β (pGSK3β). This process stabilizes Snail by releasing it from GSK3β system. Thereby, the formation of Snail-Egr-1 complex promotes VEGFR3 expression by binding to the <i>VEGFR3</i> promoter region to facilitate EC morphogenesis, such as EC sprouting, extension, and branching. pSnail, phosphorylated Snail by GSK3β; pAkt, Akt phosphorylation; E, Egr-1; EC, endothelial cell. (B) Capillary branching morphogenesis is controlled by Snail. In P7–P8 mice, venous ECs in the superficial plexus start to extend capillary branching toward the deep retina in response to tissue needs. The sprouting ECs at the border between the GCL and IPL are exposed to ECM, which subsequently contributes to Snail induction and stabilization, followed by enhanced VEGFR3 expression. Snail/VEGFR3-expressing ECs vertically migrate toward deep retina. At P9–P11 mice, vertically migrating ECs reach in the boundary of INL and turn sideways to form the deep capillary plexus in the OPL region. Snail knockdown attenuates the initiation of EC sprouting, which subsequently impairs the formation of the deep capillary plexus.</p

Snail knockdown attenuates retinal vessel sprouting and deep capillary plexus formation.

<p>(A) Illustration of the siRNA or shRNA injection strategy in mice. Mice were consecutively and intraperitoneally injected from P6 to P7 or from P7 to P10 and then sacrificed at P8-P9 (P8/P9) or P11, respectively. (B) Quantitative RT-PCR demonstrating Snail knockdown at P11 in siSnail-injected mice. (C) Confocal images of iB4 staining in the superficial plexus and deep plexus. SiSnail or siCon injection was performed, as described in <b>A</b>. Whole flat-mount retinas were stained with iB4 at P11. Confocal images were taken in the superficial plexus and then taken in the deep plexus below the superficial plexus by moving the z axis of the confocal microscopic field. The formation of the deep plexus was decreased by Snail knockdown. (D) Representative confocal images of iB4 staining at P11 in siCon- and siSnail-injected mice. SiSnail or siCon injection performed, as described in <b>A</b>. Broken lines indicate the position of veins in the superficial plexus. Arrows indicate sprouting vertical vessels from veins in the superficial plexus. Bar, 100 μm. (E) Quantification of vertical vessels and branching points in the deep plexus at P11. *, p<0.05. (F) Confocal images were collected in 1-μm z-stacks in the xz axis at P11 in siCon- and siSnail-injected mice. S.P., the superficial plexus; D.P., the deep plexus.</p

Vertically sprouting vessels have strong VEGFR3, but weak VEGFR2, expression in the developing retinal vasculature.

<p>(A) Cross-sectional confocal images showing the differential expression pattern of VEGFR2 and VEGFR3 in P11 mice. The immunoreactivity of VEGFR3 was strongly detected in the vertical vessels (IPL and INL; triangles) and deep plexus (OPL, triangles). In contrast, strong immunoreactivity of VEGFR2 was detected in the superficial plexus (GCL, arrows) and neurons (arrow heads). Nuclei were DAPI positive (blue). ONL, outer nuclear layer. Bar, 100 μm. (<b>B</b> and <b>C</b>) Confocal images of VEGFR3 staining in the superficial plexus at P8. Eyeballs from P8 mice were applied to whole flat-mount staining of iB4 and VEGFR3. The region in the box (B) is magnified in <b>C</b> (upper). The region of the vertical vessel was taken below the superficial plexus. (C, lower) The immunoreactivity of VEGFR3 was detected in sprouting vessels from the vein (arrows). Broken lines correspond to the position of vein that appeared in the superficial plexus. A, artery; V, vein. Nuclei were DAPI positive (blue). Bar, 100 μm.</p

Snail knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.

<p>(A) Whole flat-mount images showing the colocalization of Snail and VEGFR3. The immunoreactivity of Snail (green) was observed in the sprouting vessel from the vein in P8 retinal vessels. VEGFR3 immunoreactivity (magenta) was also found in the vein. V, vein. (B) Confocal images of iB4 combined with VEGFR3 staining in shCon or shSnail lentivirus-infected retinas at P8. Mice were consecutively injected intraperitoneally with the shCon or shSnail lentivirus at P6 and P7, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. The shSnail lentivirus was the same virus that was described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g001" target="_blank">Fig 1F</a> (shSnail#2). Images of vertical vessels from superficial plexus were taken. Arrows indicate sprouting vessels from veins. Broken line indicates the position of veins in the superficial plexus. Bar, 100 μm. (C) Confocal images of iB4 combined with VEGFR3 staining in the region of the deep capillary plexus in siCon- or siSnail-injected mice at P11. SiRNA injections were performed, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. Cell nuclei were stained with DAPI (blue). The immunoreactivity of iB4 and VEGFR3 was weaker in siSnail-injected mice than in siCon mice. Bar, 100 μm. (D) Quantification of total vessel area and VEGFR3-positive regions in the deep plexus at P11. Over six fields were analyzed. *, p<0.01. (E) Fibrin bead assay. HRECs were transfected with mock and Snail in a combination with VEGFR3 siRNA (siVEGFR3). Representative spheroids are shown for each condition (left). Sprouting numbers per bead, sprouting lengths from each bead, and branch numbers were calculated to quantify endothelial sprouting (right). *, p<0.01.</p