Summary of band-specific increases versus decreases in EEG/MEG functional connectivity in ASD (compared to NT controls).

<p>The “N” column list the total size of the sample (i.e., sum of participants in all groups). Frequency is varying along the x-axis, from 1 Hz to 120 Hz.</p

PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flowchart describing the paper selection process.

<p>PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flowchart describing the paper selection process.</p

Research questions needing clear answers to provide a solid foundation for linking connection length versus EEG/MEG functional connectivity in autism.

<p>Research questions needing clear answers to provide a solid foundation for linking connection length versus EEG/MEG functional connectivity in autism.</p

Increasing vascular permeability enhances the accumulation of doxorubicin into the tumor.

<p>Doxorubicin was injected intravenously subsequent to administration of PBS or VEGF and its uptake at the tumor site in real time was estimated using its natural fluorescence. <b>A–B.</b> Representative images of doxorubicin uptake over time, tumors (green) and doxorubicin uptake (red) are shown. <b>C.</b> Heat map of doxorubicin uptake after 3 hours in control and VEGF-treated tumors. <b>D.</b> Graph showing relative uptake of doxorubicin in the tumor in the presence or absence of systemic VEGF treatment. <b>E.</b> Graph showing relative uptake of doxorubicin in the normal tissues distal to the tumor in the presence or absence of systemic VEGF treatment. N = 4 per treatment; data were analyzed by 2 way ANOVA.</p

Assessment of regional permeability and vascular integrity in human tumors.

<p><b>A.</b> Representative fluorescence micrographs from two human HEp3 tumors displaying peri-tumoral (i) and tumor core (ii) vascular leak are shown with the 2000 kDa FITC-dextran (green) and 158 kDa TRITC-dextran (red). The normalized images were generated by subtracting the 0 hour image from the 3 hour image, and represent the net vascular leak. Tumor induced vascular leak is localized primarily to the tumor and especially to the central, necrotic core of the tumor. <b>B.</b> Areas utilized for regional vascular leak analyses are delineated. The solid circle represents an area of non-tumor tissue; the dashed circle denotes tumor and the dotted line indicates the avascular necrotic core. <b>C.</b> Quantitation of leak of large (green) and small (red) dextrans is shown for non-tumor tissue, the entire tumor and the core of the tumor. The relative leak of both dextrans was normalized to time zero; n = 6 for each analysis. Two-way ANOVA, (p<0.05) followed by Bonferroni post-tests, (p<0.05) was used to assess significant leak of the TRITC-dextran of either tumor versus non-tumour tissue, and necrotic core versus non-tumour tissue at each timepoint. Timepoints that demonstrated significance are indicated by an asterisk.</p

Discovery of Novel Integrin Ligands from Combinatorial Libraries Using a Multiplex “Beads on a Bead” Approach

The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex “beads on a bead” strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. Novel non-RGD peptides that target α_vβ₃ integrin were discovered that do not affect cancer or endothelial cell biology. The peptides identified here represent novel integrin-targeted agents that can be used to develop targeted nanomedicines without the risk of increased tumor invasion and metastasis

The Miles assay measures vascular permeability changes in the CAM.

<p><b>A.</b> Bright field images of CAM vasculature following injection of Evan's blue dye subsequent to the systemic administration of PBS (left panel) or VEGF (right panel). Arrows indicate areas of visible vascular leak. <b>B.</b> When VEGF or PEP is injected intravenously distal to the site of analysis, a significant level of vascular permeability is observed in the CAM (left). Topically administered VEGF but not PEP induces a significant level of vascular permeability (right). <b>C.</b> Vascular permeability changes in the CAM were evaluated in the presence of human tumor xenografts. Increased vascular permeability was observed at the tumor site, particularly in HEp3 tumors. Systemically administered PEP (0.1 nM) further increases vascular permeability. Data are presented as Mean +/− SEM, n>15 for each group. * indicates statistical significance, p<0.05, ** p<0.01.</p

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

<div><p>Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 <i>versus</i> Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.</p></div

mTORC2 inactivation inhibits focal adhesion kinase activity.

<p>HUVECs were transfected with siRict¹ or siAkt1, then stimulated with 20 ng/mL VEGF for 10 minutes as indicated. <b>A</b>) A representative Western blot of EC phospho-focal adhesion kinase (P-FAK), total FAK, total Akt1, total rictor and actin. <b>B)</b> Quantitation of P-FAK (n = 4 independent experiments, *<i>P</i><0.05 by ANOVA). <b>C)</b> A representative Western blot of EC phospho-eNOS, and phospho-Src, illustrates that mTORC2 disruption, but not Akt1 inactivation, blocks VEGF-stimulated Src activation (n = 3 independent experiments). Knockdown of either rictor or Akt1 similarly blunts eNOS phosphorylation. <b>D)</b> Quantitation of P-Src (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). <b>E)</b> The effect of sustained mTORC1 or mTORC1/2 inhibition on EC FAK activation. HUVECs were treated with PP242 or rapamycin and stimulated with VEGF overnight. A representative Western blot of EC P-FAK, and P-S6K (n = 3 independent experiments). <b>F)</b> Quantitation of P-FAK (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p

mTORC1/2 dual inhibition blocks VEGF-mediated angiogenesis.

<p>HUVEC-coated Cytodex beads were embedded in fibrin gels as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135245#pone.0135245.g002" target="_blank">Fig 2</a>, then the EC were stimulated with 50 ng/ml VEGF, and were treated with PP242 or carrier as indicated. <b>A)</b> Representative images of EC sprouts after 18 hours incubation. <b>B)</b> Quantitation of the number of sprouts per bead. <b>C)</b> Quantitation of the length of the sprouts (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA, scale bar = 95 um). <b>D)</b> Collagen gel onplants containing VEGF (100 ng/onplant) and PP242 or carrier, were placed on chicken embryo CAM as described in Methods. Quantitation of neovascularization after 64 hours of exposure to VEGF supplemented with 1, 5, 10 or 50 uM PP242 (n > 48 onplants or 16 chicken embryos per group, <i>*P</i><0.05 by ANOVA).</p