Schematic diagram of rHSA-mediated co-delivery technology.

<p>Recombinant HSA-delivery complexes were conceived as a co-delivery technology in that 1) therapeutic peptides can be fused to the C-terminal of HSA for both extracellular and intracellular targeting and 2) FA-Drugs can form stable complexes with rHSA fusion proteins to promote synergistic therapeutic efficacy. </p

rHSA-P53i and rHSA-PMI bind to MDM2 and MDMX.

<p>To detect the interaction between MDM2/MDMX and rHSA fusion proteins, 4 µg each of biotinylated rHSA (lane 1), rHSA-P53i (lane 2), or rHSA-PMI (lane 3) were added to 200 µg of SJSA-1 whole cell lysate. MDM2 or MDMX antibody was added to the lysate followed by pulling down MDM2/MDMX and rHSA complexes using Protein A/G (1:1) resins. Samples were then analyzed by SDS-PAGE and Western blotting using MDM2, MDMX, and Streptavidin-HRP (Strep-HRP) antibodies. </p

rHSA-p53 and rHSA-PMI are efficiently taken up into SJSA-1 cells.

<p>FITC-labeled rHSA (5 μM), FITC-rHSA-P53i (5 μM), and FITC-rHSA-PMI (5 μM) were added to SJSA-1 cells as described in <i>Methods</i>. Visualization at 60X magnification revealed efficient uptake of FITC-rHSA (A), FITC-rHSA-P53i (B) and FITC-rHSA-PMI (C) occurred following 24-hour incubation. FITC staining of vesicular cargo suggests significantly greater uptake of rHSA-P53i and rHSA-PMI, compared to rHSA.</p

Methods to develop the bone's tomographic image.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g003" target="_blank">Fig. 3a</a> Reconstructed tomogram by applying Eq. (4) after using SCS. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g003" target="_blank">Fig. 3b</a> Tomographic boundary extracted by Eq. (5). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g003" target="_blank">Fig. 3c</a> Circle set up by the average radius of the tomographic image, where the center is on the COM of the tomographic image. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g003" target="_blank">Fig. 3d</a> Developed tomographic boundaries by Eqs. (6) and (7) based upon the circumference taken as sea level. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g003" target="_blank">Fig. 3e</a> Flattened tomographic boundaries by Eq. (8).</p

rHSA-P53i and rHSA-PMI promote cytotoxicity in SJSA-1 cells via caspase activation.

<p>rHSA fusion proteins, as well as nutlin (to serve as a p53-MDM2 antagonist control) were added at the indicated concentrations and allowed to incubate for 24 hrs. A). Cytotoxicities were measured by CyQuant Assay and normalized according to 10 μM rHSA-treated cells. B). Caspase activation was quantitated using the Homogeneous Caspase Assay as described in <i>Methods</i> and normalized according to untreated cells. Results are displayed as percent cell death (A) or fold change (B) relative to 10 μM rHSA-treated wells. Data are representative of 3 independent experiments performed in triplicate. Error bars indicate ± SD.</p

rHSA fusion proteins are able to form stable complexes with FA-FITC.

<p><b>A</b>). rHSA-PMI (lane 1-3), rHSA-P53i (lane 4-6) and rHSA (lane 7-9) were incubated at the indicated molar ratios (rHSA:FA-FITC) with FA-FITC (lane 1, 4, and 7 (1:1); lane 2, 5, and 8 (1:2); lane 3, 6, and 9 (1:4); lane 10, FA-FITC only). The upper band in the gel corresponds to the HSA/FA-FITC complex, while the lower band indicates unbound FA-FITC. Incorporation of FA-FITC into rHSA was achieved up to a 1:4 rHSA:FA-FITC molar ratio. <b>B</b>). rHSA/FA-FITC complexes were pre-formed at a 1:4 molar ratio (HSA:FA-FITC; 30 pmol:120 pmol) as described in <i>Methods</i>. Unlabeled FA was then added at the indicated concentrations to mimic the competition of free FA present under physiological conditions. The minimal dissociation of FA-FITC from pre-formed rHSA/FA-FITC complexes at the 8 times excess concentration of unlabeled FA (lane 8) indicates FA-FITC and rHSA complex was highly stable in the presence of free FA. <b>C</b>). Pre-formed biotin-rHSA and FA-FITC complexes (biotin-rHSA:FA-FITC; 1:2) were incubated with PBS (Lane 1 and 3) and 10% serum (lane 2 and 4) for 1 and 24 hours. Lanes 1 and 2 represent total FA-FITC incorporation into rHSA without and with 10% FBS, respectively, prior to the addition of streptavidin resins. Lane 3 (with PBS only) and lane 4 (with 10% FBS) correspond to the supernatants of samples after incubation with streptavidin resins and pulling down rHSA/FA-FITC complexes. The absence of rHSA/FA-FITC complexes in lane 3 indicates that all biotin-rHSA/FA-FITC complexes (in PBS) were efficiently pulled down. Any FA-FITC present in lane 4 would imply the displacement of rHSA-bound FA-FITC by serum components. The presence of only a weak band in lane 4 indicates the majority of FA-FITC remained bound to rHSA (pulled down by streptavidin resins). Quantitation of the amount of FA-FITC in lane 4 (Image J, <i>NIH</i>) revealed approximately 15% and 17% of FA-FITC was removed from biotin-rHSA in the presence of serum following 1 and 24 hour incubations, respectively. </p

Relationship between muscle tension and load.

<p>The foot arch is here simplified as a triangle truss, of which the truss width (foot length) is standardized as 1, the truss height (foot arch height) is the foot arch index, and the magnitude of the concentration force is 1. Its direction is changed from <i>45</i> degrees to <i>135</i> degrees, and its point application is at the top of the truss.</p

Analysis of bone strength to the fourth metatarsal (left foot) of eight wrestlers and volleyball players, respectively.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6a</a> Comparison of bone density, volume and area of the fourth metatarsal (left foot) of eight wrestlers and volleyball players. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6b</a> Relationship between the fourth metatarsal (left foot) bone tissue density and the tissue distribution radius from eight wrestlers and volleyball players. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6c</a> Differences of the map of the fourth metatarsal (left foot) average value from eight wrestlers and volleyball players. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6d</a> P value distribution of the fourth metatarsal (left foot) map from eight wrestlers and volleyball players. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6a</a>, the bone's density is defined as , where , stands for the gray value of the <i>i</i>-th volume element, stands for the gray value of water. Volume and area are results from <i>Mimics</i> software. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6b</a>, for detailed method, see Reference 30. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6c</a>, when , or , is colored, where and stand for the altitude values for the positions of of the fourth metatarsal from volleyball players and wrestlers, respectively, where blue shows and red (). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032926#pone-0032926-g006" target="_blank">Fig. 6d</a>, the calculation is done by <i>SPSS</i>, where blue shows p<0.05 and red p<0.01.</p

Basic information of subjects' feet.

<p>*p<0.05,</p><p>**p<0.01.</p

A Practical and Efficient Approach to the Preparation of Bioactive Natural Product Wogonin

A scalable and practical route to wogonin, a bioactive natural product with multiple pharmacological activities which is currently under phase I/II clinical studies, is described. Wogonin was obtained via a four-step process starting from commercially available chrysin and including the Elbs oxidation, benzylation, methylation, and the final debenzylation. The whole procedure gives the target product in a 38% overall yield with >99% purity. Key steps in this process including oxidation and methylation are discussed in detail. The optimized process has been successfully demonstrated on the kilogram scale to support ongoing clinical development of wogonin