Search

NA

Framing through the concept of precarity, we share our arts-based research on the experiences of creating a collaborative performance-making project focused on connecting students from different education levels, to create a film-dance integrated performance to advocate for social justice issues in education. We, as instructors and researchers, embarked on an arts-based research journey creating sketches, poems, videos, and a dance performance to analyze and represent our research findings. Our performance from the performative inquiry shows our understanding of the collaboration project through the same art form we required our students to utilize: film and dance-integrated arts performance.

What should science, technology, engineering, art, and math (STEAM) education look like? In the context of educational policy, the STEAM conversation has intensified and spread across the United St...

Redefining the educational model of science, technology, engineering, and math curriculum (STEM) to also include art to become STEAM (Fournier, 2013; Guyotte, Sochacka, Constantino, Walther, & Kellam, 2014) is a current method to encourage students to actively participate in 21st-century learning (Saxena, 2014). The skills emphasized for 21st-century learning include critical thinking and problem solving; collaboration and communication; and creativity and innovation (Partnership for 21st Century Skills, 2007). Encouraging students to pursue a STEM career has become an important U.S. educational policy that emphasizes teaching these 21st-century skills along with STEM knowledge in the discourse of future economic growth.

Objective— MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. Approach and Results— The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p–transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1 /Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. Conclusions— UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

Aims The aim of this study was to investigate anti-apoptotic gene therapy using ultrasound-mediated plasmid delivery of survivin, an inhibitor of apoptosis protein, to prevent apoptosis and to attenuate left ventricular (LV) systolic dysfunction in a model of heart failure induced by doxorubicin. Methods and results Effect of survivin transduction was investigated in vitro in rat cardiomyoblasts. After survivin transduction, survivin protein was detected in cell culture supernate confirming secretion of extracellular survivin. Under doxorubicin stimulation, survivin-transduced cells had significantly reduced apoptosis; however, incubation with survivin-conditioned media also showed reduced apoptosis that was absent with null-conditioned media. Doxorubicin-induced cardiomyopathy was established in Fischer rats. Subsets of animals underwent ultrasound-mediated survivin gene delivery or empty vector gene delivery at Week 3. Control rats received doxorubicin alone. Animals were studied using PCR, immunohistochemistry, echocardiography, and invasive haemodynamic studies out to Week 6. By Week 6, LV % fractional shortening by echocardiography and systolic function by pressure–volume loops were greater in survivin treated when compared with control- and empty-treated animals. There was reduced apoptosis by TUNEL and caspase activity in survivin-treated animals compared with control and empty treated at Week 4, with reduced interstitial fibrosis at Week 6. Conclusion Survivin gene therapy can attenuate the progression of LV systolic dysfunction in doxorubicin cardiomyopathy. This effect can be attributed to decreased myocyte apoptosis and prevention of maladaptive LV remodelling, by both direct myocyte transfection and potentially by paracrine mechanisms.

Jellyfish salad. The meat is often cut into strips and mixed with cucumber and chili pepper to make a popular dish often served as an appetizer at celebrations in Taiwan. Growing up, I ate it at several wedding banquets. I remember it on a small white plate encircling the petals of a bright purple orchid set in the middle. The dried jellyfish looks like translucent skin, so Chinese menus call it cold jellyfish skin. Live jellyfish look like gelatin, but the meat has a texture somewhere between udon noodles and cloud ear fungus. On its own, jellyfish doesn’t have much flavor, but it absorbs whatever dressing you put on it. Semi-opaque, jellyfish salad creates an in-between space. It takes the edge of hunger but makes you want more. It gives you a taste, a scent, or suggestion, but it does not fill your belly. It comes at the very beginning of the grand feast, the Christine Liao University of North Carolina, Wilmington

Left ventricular hypertrophy (LVH) is commonly found in chronic dialysis (CD) recipients, and is associated with impaired microvascular cardiac perfusion and heart failure. In response to LVH and cardiac ischemia, early outgrowth pro-angiogenic cellS(EPCs) mobilize from the bone marrow to facilitate angiogenesis and endothelial repair. In the general population, EPC number and function correlate inversely with cardiovascular risk. In end-stage renal disease (ESRD), EPC number and function are generally reduced.To test whether left ventricular abnormalities retain their potent ability to promote EPC reparative responses in the setting of ESRD.Cross-sectional study.St. Michael's Hospital, Toronto, Ontario, Canada.47 prevalent chronic dialysis recipients.(1) circulating CD34(+) and CD133(+) EPC number, (2) cultured EPC migratory ability, in vitro differentiation potential, and apoptosis rate, and (3) cardiac magnetic resonance-measured LV mass, volume and ejection fraction.Bivariate correlation analysis was performed with Spearman's rho test.Of the 47 patients (mean age: 54 ± 13 years), the mean delivered urea reduction was 74 ± 10 %. Mean LV mass was 123 ± 38 g. Circulating CD34(+) and CD133(+) EPCs represented 0.14 % (IQR: 0.05 - 0.29 %) and 0.05 % (IQR: 0.01 - 0.10 %) of peripheral blood mononuclear cells. There were no significant correlations between any EPC parameter and measures of LV mass or ejection fraction.Lack of a non-ESRD control population, and the inability to measure all parameters of EPC function due to limitations in blood sampling. Our inability to measure cardiac VEGF expression prevented an assessment of changes in cardiac EPC mobilization signals.These data suggest that in ESRD, the reparative EPC response to cardiac hypertrophy may be blunted. Further investigation of the effects of uremia on EPC physiology and its relationship to cardiac injury are required.L’hypertrophie ventriculaire gauche (HVG), qui est associée à la perfusion cardiaque microvasculaire alterée et à l’insuffisance cardiaque, n’est pas rare chez les patients qui reçoivent une dialyse chronique. En réponse à l’HVG et à l’ischémie cardiaque, les cellules proangiogéniques à croissance précoce (CPCP) se mobilisent au sein de la moelle osseuse afin de faciliter l’angiogenèse et la réparation endothéliale. Dans l’ensemble de la population, le nombre et l’activité des CPCP sont inversement proportionnels au risque cardiovasculaire. Dans le cas d’insuffisance rénale chronique terminale (IRT), le nombre et l’activité des CPCP sont généralement réduits.Vérifier si les anomalies du ventricule gauche demeurent aussi efficaces à promouvoir les actions réparatrices des CPCP dans le contexte de l’IRT.St. Michael’s Hospital, à Toronto, en Ontario, au Canada.47 cas prévalents de patients qui reçoivent une dialyse chronique.(1) le nombre de CPCP CD34L’analyse de la corrélation simple a été effectuée au moyen du coefficient de corrélation des rangs de Spearman.On a observé une réduction de 74 ± 10 % en moyenne parmi les 47 participants (moyenne d’âge : 54 ± 13 ans). La masse ventriculaire gauche était de 123 ± 38 g en moyenne. Les CPCP CD34L’absence d’une population témoin non atteinte d’IRT, de même que l’inhabilité de mesurer tous les paramètres de l’activité des CPCP, en raison des limites de l’échantillon sanguin. Notre incapacité à mesurer l’expression du facteur de croissance endothéliale vasculaire nous a empêchés d’effectuer l’analyse des modifications dans les signaux cardiaques de mobilisation des CPCP.Ces données suggèrent que dans le cas d’une IRT, la réponse réparatrice des CPCP à l’hypertrophie cardiaque peut être atténuée. Des observations plus poussées sur les effets de l’urémie sur la physiologie des CPCP et sur le lien avec les lésions cardiaques seraient nécessaires.

Teneurins are a family of highly conserved pair-rule proteins involved in morphogenesis and development of the central nervous system. Their function in adult tissues and in disease is largely unknown. Recent evidence suggests a role for dysregulated expression of Teneurins in human tumors, but systematic investigations are missing. Here, we investigated Teneurin-2 and Teneurin-4 expression in various cancer cell lines and in ovarian tumor tissues. Teneurin-2 and Teneurin-4 were expressed in most of the breast cancer cell lines tested. Teneurin-4 was also detected in ovarian cancer cell lines, and throughout ovarian tumors and normal ovary tissue. Ovarian tumors with low Teneurin-4 expression showed less differentiated phenotypes and these patients had shorter mean overall survival. Similarly, Teneurin-2 expression correlated with overall survival as well, especially in patients with serous tumors. In the various cell lines, 5-Aza-cytidine-induced changes in DNA methylation did not alter expression of Teneurin-2 and Teneurin-4, despite the existence of predicted CpG islands in both genes. Interestingly, however, we found evidence for the control of Teneurin-2 expression by the oncogenic growth factor FGF8. Furthermore, we identified multiple transcript splicing variants for Teneurin-2 and Teneurin-4, indicating complex gene expression patterns in malignant cells. Finally, downregulation of Teneurin-4 expression using siRNA caused a cell-type dependent increase in proliferation and resistance to cisplatin. Altogether, our data suggest that low Teneurin-4 expression provides a growth advantage to cancer cells and marks an undifferentiated state characterized by increased drug resistance and clinical aggressiveness. We conclude that Teneurin-2 and Teneurin-4 expression levels could be of prognostic value in ovarian cancer.

Background: Ultrasound-targeted microbubble destruction (UTMD) is a non-invasive gene transfection technique using carrier microbubbles and targeted high power ultrasound, primarily used to deliver plasmid DNA (pDNA). Minicircle-DNA (mcDNA) is a novel gene vector, which has recently been shown to have an improved and persistent transfection, compared to conventional pDNA. Hypothesis: We hypothesized that UTMD of mcDNA in a hind-limb model would exhibit a more potent and prolonged gene expression compared to conventional pDNA. Methods: In vitro , HUVECs, fibroblasts (3T3) and neonatal cardiomyocytes were transfected with molar equivalents of GFP-minicircle and GFP-plasmid. GFP expression was measured by RT-PCR and fluorescent microscopy for 28 days. We then performed a comparison of bubble binding capacities of both vectors to cationic lipid microbubbles. In vivo , for UTMD to the left proximal hind-limb adductor muscle, 500μg and 214μg of GFP-plasmid and GFP-minicircle respectively were charge-coupled with 1x10 9 cationic microbubbles and delivered via UMGD into Sprague-Dawley rats (n=30). The animals were followed for 28 days with GFP measured by RT-PCR and immunohistochemistry. Results: In vitro results showed greater GFP expression by mcDNA across all cell lines, with 7-10 fold transfection efficacy compared to pDNA. mcDNA demonstrated greater binding capacity to cationic microbubbles compared to pDNA. For plasmid and minicircle DNA, binding saturations were reached at ~6000 copies per microbubble, and ~20000 copies per microbubble respectively, suggesting a higher minicircle bubble binding efficiency. In vivo results showed higher GFP levels as early as 6 hours post minicircle UTMD, demonstrating minicircle to be a faster acting therapeutic agent over conventional plasmid. A significantly greater (p Conclusions: In summary, UTMD using mcDNA results in more rapid and sustained transfection compared to conventional pDNA, and may be a more effective vector for translational studies of UTMD.

Background: Diabetes mellitus is a chronic metabolic disease associated with various vascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue including delivery of pro-angiogenic growth factors have shown disappointing results. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We assessed the hypothesis that temporally separated delivery of multiple pro-angiogenic growth factors will improve the efficacy of the angiogenic response in the setting of both diabetes and non-diabetes in vitro. Methods: 96-well tissue culture plates were coated with 60 μL growth factor reduced Matrigel. After gelation, non-diabetic human dermal microvascular endothelial cells (HMVECs) and type-II diabetic cells (D-HMVECs) were plated at 1x104 cells/well with 100 μL basal media EBM-2 with 0.1% bovine serum, and incubated at 37°C. Combinations of growth factors including recombinant human VEGF (50 ng/mL), angiopoietin (Ang)-1 and -2 (250 ng/mL) were administrated at start of the assay and/or at 6 hours into the assay. Images were taken at 3, 6, 9, 12, 18 and 24 hours and used for tube formation quantification by Inverted Microscope at 10x magnification. Results: In untreated control, D-HVECs showed reduced tube formation in compared to HMVECs (n=12; p Conclusion: Diabetic endothelial cells showed impaired angiogenesis compared to non-diabetic endothelial cells. Combinations of temporally separated pro-angiogenic growth factors were able to optimize the angiogenic effects and yield the most significant efficacy in the setting of both diabetes and non-diabetes. Future studies will focus on in vivo temporally separated combinations of multiple growth factors.

Secondary prevention can improve outcomes in high risk patients. This study investigated the magnitude of cardiovascular risk reduction associated with consumption of a modified portfolio diet in parallel with medical management.30 patients with type II diabetes, 6 weeks post bypass surgery received dietary counseling on a Modified Portfolio Diet (MPD) (low fat, 8 g/1000 kcal viscous fibres, 17 g/1000 kcal soy protein and 22 g/1000 kcal almonds). Lipid profiles, endothelial function and markers of glycemic control, oxidative stress and inflammation were measured at baseline and following two and four weeks of intervention. Seven patients with no diet therapy served as time controls.Consumption of the MPD resulted in a 19% relative reduction in LDL (1.9 ± 0.8 vs 1.6 ± 0.6 mmol/L, p0.001) with no change in HDL cholesterol. Homocysteine levels dropped significantly (10.1 ± 2.7 vs 7.9 ± 4 μmol/L, p = 0.006) over the study period. Flow mediated dilatation increased significantly in treated patients (3.8 ± 3.8% to 6.5 ± 3.6%, p = 0.004) while remaining constant in controls (p = 0.6). Endothelial progenitor cells numbers (CD34+, CD 133+ and UEA-1+) increased significantly following MPD consumption (p0.02) with no difference in migratory capacity. In contrast, time controls showed no significant changes.Dietary intervention in medically managed, high risk patients resulted in important reductions in risk factors. Clinical Trials registry number NCT00462436.

Moving Mountains is a collaborative venture by eight art educators who explore the notion of distributed leadership to transcend boundaries of proximity, ideation, and artistic production. Their distributed leadership, enacted through both human and non-human performers, involved sharing knowledge and skills to create a cyberformance and machinima. They completed these projects from conceptualization to artistic production without a designated leader and without hierarchical constraints. In this chapter, the authors view distributed leadership in Moving Mountains collaborations through actor-network theory, crowdsourcing, and transformative potential. Moving Mountains collaborators continue to create video art, written work, curriculum, and virtual world performances, through distributed leadership, in order to challenge oppression and transgress borders.

This article looks at the connections between avatars and art and proposes avatars as an art medium and the assembling or creating of avatars as an art-making process. The term assembling is used to indicate that avatars are assemblages in that they are combinations of representations (images), and the process of constructing an avatar involves putting together body parts (and clothes).Framed by Deleuze and Guattari’s idea of assemblage, avatar assembling constitutes the in-between space wherein new connections between art and identity play form and the possibility of a new art genre likewise takes shape. Three ways to conceive the connections between avatars and art are discussed. The first focuses on avatars as the subject of art, the second on avatars as performance art, and the third on connecting avatar reassembling with the process of art-making.

Summary Background and objectives Angiogenesis is a key response to tissue ischemia that may be impaired by uremia. Although early-outgrowth endothelial progenitor-like cells promote angiogenesis in the setting of normal renal function, cells from uremic patients are dysfunctional. When compared with conventional hemodialysis, it was hypothesized that nocturnal hemodialysis would improve the in vivo angiogenic activity of these cells in a well described model of ischemic vascular disease. Design, setting, participants, and measurements Early-outgrowth endothelial progenitor-like cells were cultured from healthy controls (n = 5) and age- and gender-matched conventional hemodialysis (12 h/wk, n = 10) and nocturnal hemodialysis (30 to 50 h/wk, n = 9) patients. Cells (5 × 105) or saline were injected into the ischemic hindlimb of athymic nude rats 1 day after left common iliac artery ligation. Results Although conventional dialysis cell injection had no effect versus saline, nocturnal hemodialysis and healthy control cell injection significantly improved ischemic hindlimb perfusion and capillary density. Nocturnal hemodialysis cell injection was also associated with significant increases in endogenous angiopoietin 1 expression in the ischemic hindlimb compared with saline and conventional dialysis cell injection. Conclusions In contrast to a conventional dialytic regimen, nocturnal hemodialysis is associated with a significantly improved ability of early-outgrowth endothelial progenitor-like cells to promote angiogenesis and thus restore perfusion in a model of ischemic vascular disease.

Background Most forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs). Methodology/Principal Findings In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-ß. The antifibrotic effects of CMCs and SCs were then evaluated in the 5/6 nephrectomy model of chronic cardio-renal disease. While intravascular infusion of 106 SCs had no effect, 106 CMCs reduced renal fibrosis compared to saline in the glomeruli (glomerulosclerosis index: 0.8±0.1 v 1.9±0.2 arbitrary units) and the tubulointersitium (% area type IV collagen: 1.2±0.3 v 8.4±2.0, p

Aims Imaging methods to track the fate of progenitor cells after their delivery would be useful in assessing the efficacy of cell-based therapies. We hypothesized that contrast-enhanced ultrasound (CEU) using microbubbles targeted to a genetically engineered cell-surface marker on endothelial progenitor cells (EPCs) would allow the targeted imaging of vascular engraftment. Methods and results Rodent bone marrow-derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or Firefly luciferase served as controls. Control microbubbles (MBC) and microbubbles targeted to H-2Kk expressed on EPCs (MBH-2Kk) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented Matrigel plugs in rats. In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. Although numbers of adhered MBC were also low, there was greater and more diffuse attachment of MBH-2Kk to plated H-2Kk-transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented Matrigel plug for MBH-2Kk, whereas contrast enhancement was low for MBC. Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MBH-2Kk when compared with MBC. Conclusion Microbubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro . These targeted microbubbles bind to engrafted EPCs in vivo within Matrigel plugs and can be detected by their enhancement on CEU imaging.

The perceptual grouping of a four-tone cycle was studied as a function of differences in fundamental frequencies and the frequencies of spectral peaks. Each tone had a single formant and at least 13 harmonics. In Experiment 1 the formant was created by filtering a flat spectrum and in Experiment 2 by adding harmonics. Fundamental frequency was found to be capable of controlling grouping even when the spectra spanned exactly the same frequency range. Formant peak separation became more effective as the sharpness (amplitude of the peak relative to a spectral pedestal) increased. The effect of each type of acoustic difference depended on the task. Listeners could group the tones by either sort of difference but were also capable of resisting the disruptive effect of the other one. This was taken as evidence for the presence of a schema-based process of perceptual grouping and the relative weakness of primitive segregation.

BACKGROUND: Diabetes mellitus increases the risk of developing cardiovascular diseases and is associated with macro and microvascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue have been investigated, including delivery of pro-angiogenic growth factors and the genes that encode them, but results have been disappointing. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We sought to examine the revascularization effect of multiple pro-angiogenic genes on chronic hindlimb ischemia in both non-diabetic and diabetic mouse models. METHODS: Spontaneously type II diabetic male mice (db/db; with C57/Bl6 genetic Background) and their control (db/m) between 8and 10-week-old were studied. Unilateral hindlimb ischemia was induced in all animals by ligation and stripping of the left femoral artery. For gene delivery, plasmids containing pro-angiogenic genes were administrated intramuscularly directly into the ischemic distal hindlimb muscle, below the ligation site at 4 weeks (for early delivery) and at 6 weeks (for late delivery) after ligation procedure. Gene combinations included vascular endothelial growth factor (VEGF) and angiopoietin 1, 2 (Ang-1, 2). Relative blood flow to the foot was measured under standardized conditions by laser Doppler perfusion imaging (LDPI). Perfusion measurements were performed on postoperative days 1, 3, 7, week 2, 3, 4, 5, 6, 7 and 8. The perfusion ratio (occludedto-non-occluded leg) was calculated for each animal. Animals from all groups were sacrificed at various times after gene delivery for tissue sampling (PCR, westerns, immunohistochemistry). RESULTS: Diabetic mice showed reduced ability to induce angiogenesis following ischemia compared to non-diabetic mice. In response to VEGF gene therapy, non-diabetic mice responded with greater neovascularization compared to diabetic mice. Ang-1 mono-therapy promoted an angiogenic response in diabetic mice, but not in non-diabetic mice. Ang2 mono-therapy did not promote angiogenesis in either model. Temporally separated delivery of VEGF (early) and Ang-1 (late) promoted more neovascularization than any single gene therapy. Delivery of VEGF and Ang-2 (early) and Ang-1 (late) showed the greatest angiogenic response in both diabetic and non-diabetic mice. CONCLUSION: Diabetic mice show a reduced angiogenic response to VEGF and increased angiogenic response to Ang1 in compared to non-diabetic mice. Temporally delivery of VEGF, Ang-2 and Ang-1 gene combination resulted a greater perfusion recovery and a greater angiogenic response in ischemic hindlimb skeletal muscle, compared to single gene or multiple gene combinations without temporal separation in both diabetic and non-diabetic settings.

Ultrasound-targeted microbubble destruction (UTMD) can be used to deliver silencing gene therapy to tumors. We hypothesized that UTMD would be effective in suppressing angiogenesis within tumors, and that modulation of the ultrasound pulsing intervals (PI) during UTMD would affect the magnitude of target knockdown. We performed UTMD of vascular endothelial growth factor receptor-2 (VEGFR2) short hairpin (sh)RNA plasmid in an heterotopic mammary adenocarcinoma model in rats, evaluating PIs of 2, 5, 10, and 20 seconds. We demonstrated that UTMD with a PI of 10 seconds resulted in the greatest knockdown of VEGFR2 by PCR, immunostaining, western blotting, smaller tumor volumes and perfused areas, and lower tumor microvascular blood volume (MBV) and flow by contrast-enhanced ultrasound (CEU) compared with UTMD-treated tumors at 2, 5, and 20 seconds, control tumors, tumors treated with intravenous shRNA plasmid and scrambled plasmid. CEU perfusion assessment using the therapeutic probe demonstrated that tumors were fully replenished with microbubbles within 10 seconds, but incompletely replenished at PI-2 and PI-5 seconds. In conclusion, for anti-VEGFR2 cancer gene therapy by UTMD, PI of 10 seconds results in higher target knockdown and a greater anti-angiogenic effect. Complete replenishment of tumor vasculature with silencing gene-bearing microbubbles in between destructive pulses of UTMD is required to maximize the efficacy of anti-angiogenic cancer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e94; doi:10.1038/mtna.2013.20; published online 21 May 2013.

BACKGROUND: Efficient and targeted delivery strategies for microRNA (miR) are currently under investigation. Ultrasoundmediated gene delivery (UMGD) is a method for delivery of plasmid DNA and siRNA in numerous pre-clinical models; however, UMGD has not been used with microRNA (miR). We aimed to develop a method of conjugating microRNA to cationic microbubbles, and to test it for the purpose of ultrasound-mediated microRNA delivery in vivo. METHODS: Cationic microbubbles were incubated with increasing concentrations (0, 1, 2.5, 5 ig/ml) of miR-126. After incubation, conjugated microbubbles were assessed for clumping, size and amount of bound microRNA. Time course of circulating miR-126 concentration was then assessed using qRTPCR, without and with microbubble conjugation at various time points after intravenous injection in rats. Ultrasound-mediated gene delivery (UMGD) of miR-126 (1x109 cationic microbubbles 2.5 ig of miR-126) (n 6) or scrambled miR (miR-SCR) (n 4) was performed in a subset of animals. Control animals (n 4) receiving no treatment. RESULTS: Binding of miR-126 to microbubbles (1x109) saturated at approximately 120,000 miR-126 molecules per microbubble with the addition of 2.5ig miR (n 5). Upon visual inspection and size analysis, there was no microbubble clumping before or after miR-126 conjugation. Upon intravenous administration, greater concentrations of miR-126 were found in the plasma after injection of miR-126 conjugated microbubbles as compared to miR-126 alone, microbubbles alone or sham. PCR results for miR-126 levels after UMGD into the distal hindlimb showed an approximate 15-fold increase over control at 3h, with persistent expression up to day3 and normalizing by day7 (n 4 per time point). MiR-126 targets (SPRED1, PIK3R2) were knocked down at 3h (SPRED1; 72 10% and PIK3R2 73 13%, p 0.05 v. Control), day1 (SPRED1; 48 5% and PIK3R2 44 11%, p 0.01 v. Control) and day3 (SPRED1; 69 13% and PIK3R2 80 20%, p 0.05 v. Control) with normalization by day 7. MiR-SCR delivery had no effect on SPRED1 or PIK3R2 levels as compared to controls and there was no detectable transfection seen in remote organs (liver, lung, kidney and spleen). CONCLUSION: MiR-126 had avid binding to cationic microbubbles, and miR-conjugated microbubbles led to prolonged circulation times of miR-126. In vivo transfection data shows robust transfection of miR-126 in hindlimb skeletal muscle after UMGD, with significant downregulation of targets, SPRED1 and PIK3R2. UMGD of miR results in targeted transfection and is a promising in vivo miR delivery technique. Canadian Cardiovascular Society (CCS) CCS246 Oral HERITABLE ARRHYTHMIAS AND SUDDEN DEATH Monday, October 29, 2012

were treated with a MMP inhibitor, doxycycline, post-surgery. Doxycycline prevented the reduction in plaque size in the AVF group (p 0.05). Plaque MMP-9 expression was increased in AVF vs. control and sham (10.8% 1.4, 3.9% 0.7, 4.6% 1.1, respectively; p 0.0001). MMP-3 expression was increased in AVF vs. control (9.5% 1.5, 5.0% 0.8, respectively; p 0.05). MMP-2 expression was decreased in AVF vs. sham (5.7% 0.5, 11.3% 1.6, respectively; p 0.05). In addition, we overexpressed the tissue inhibitor of metalloproteinase (TIMP-1) in mice before surgery. The increased Timp-1 prevented the reduction in plaque size in the AVF group (p 0.05). All groups had comparable body weight, blood pressure, lipid levels and hematological parameters. There was also no difference in smooth muscle cell and collagen content between groups. To further understand the role of MMPs, we devised an endothelial cell (EC)-macrophage co-culture system where the ECs were exposed to high (15 dynes/cm) or low (0.5 dynes/cm) shear stress. Macrophage migration exposed to EC effluent was performed using a scratch assay. There was a 2.6 fold increase in migration in the high flow condition compared to the low (p 0.05). When the cells were exposed to the MMP inhibitor drug, GM6001, migration was decreased 2 fold (p 0.05). CONCLUSION: Our findings suggest that MMPs play a role in shear stress inducedplaque regression. Shear stress acting on ECs may influence the cells within the plaque by increasing MMP expression allowing for better macrophage mobility and egress which are characteristics of regressing plaques.

The fusion of two amplitude-modulated (AM) tones presented simultaneously was studied. Subjects were presented with an AM tone (A) followed by a copy of itself (B) which was accompanied by another AM tone (C). In different experiments, the subjects were asked either to rate how clearly they could hear Tone B in the BC mixture or whether Tone B was present or not. The stronger the fusion of Tones B and C, the harder it was to "hear out" Tone B. It was found that fusion was strongest when Tones B and C were modulated at the same rate; segregation curves were obtained for the degree of mismatch of modulation frequency.

Objectives The aim of this study was to compare temporally separated vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 delivery with concomitant delivery or single VEGF delivery, for therapeutic angiogenesis in chronic ischemia. Background Single gene delivery of VEGF results in immature neovessels that ultimately regress. Endogenously, VEGF acts early to initiate angiogenesis, whereas Ang-1 acts later to induce vessel maturation. Timing VEGF and Ang-1 gene delivery to mimic endogenous angiogenesis might be more effective for sustained neovascularization. Methods Unilateral hindlimb ischemia was induced in 170 rats. Ultrasound-mediated gene delivery was performed with cationic microbubbles and plasmid deoxyribonucleic acid. Groups included VEGF at 2 weeks, VEGF/Ang-1 at 2 weeks, VEGF at 2 weeks with Ang-1 at 4 weeks, and untreated control subjects. At 2, 4, and 8 weeks after ligation, blood flow and flow reserve (FR) were assessed by contrast-enhanced ultrasound. Vascular density, organization, and supporting cell coverage were assessed by fluorescent microangiography and immunohistochemistry. Results In untreated control subjects, blood flow, FR, and vessel density remained reduced. The VEGF delivery improved flow and vessel density at 4 weeks; however, FR remained low, supporting cell coverage was poor, and flow and vessel density regressed by 8 weeks. The VEGF/Ang-1 co-delivery marginally increased flow and vessel density; however, FR and supporting cell coverage improved. After temporally separated VEGF and Ang-1 delivery, blood flow, vessel density, and FR increased and were sustained, with improved pericyte coverage at 8 weeks. Conclusions In conclusion, temporally separated VEGF and Ang-1 gene therapy results in sustained and functional neovascularization.