Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus

Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs

Evaluación en la incertidumbre. Caso de estudio: docentes de la facultad de administración de empresa

Con el fin de mejorar la evaluación del docentese realizó una investigación para evaluar de las competencias de 161 docentes a tiempo completo de la Facultad de Administración de Empresa (FADE)de la Escuela Superior Politécnica de Chimborazo en Ecuador (ESPOCH). La determinación del grado de competencia de los docentes evaluados, se hizo a partir la determinación de las distancias relativas utilizando herramientas de la lógica difusa. Por tal motivo, el objetivo de este trabajo es determinar el grado de competencias de los docentes evaluados de forma que se puedan tomar  las estrategias más eficaces para el mejoramiento de las competencias.De igual forma los resultados de esta investigación podrán infieren que es necesario variar el modelo de evaluación del docente particularizado para la ESPOCH.Este trabajo fue realizado en 2014 como parte del trabajo final de pregrado de seis alumnos y liderado por los autores de este artículo

Fusogenic-Oligoarginine Peptide-Mediated Delivery of siRNAs Targeting the CIP2A Oncogene into Oral Cancer Cells

<div><p>Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.</p></div

Optimization of 599 peptide binding to siRNAs.

<p>An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).</p

Optimization of 599 peptide-mediated delivery of siRNAs into oral cancer cells.

<p>(<b>A</b>) Fluorescence microscopy analysis of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) alone or in complex with increasing amounts of 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs). Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm. (<b>B</b>) CAL 27 cells incubated for 2.5 hours with D-siCIP2A alone or in complex with increasing amounts of 599 peptide (ranging from 1 to 100-fold molar excess of siRNAs). For comparison, the cells were also transfected using the commercial transfection reagent, INTERFERin™ (IFN). The amount of siRNA delivered into cells in pmol per mg of protein is reported with each treatment normalized to D-siCIP2A alone. Data are mean ± SEM of four separate experiments, where ***P<0.001, **P<0.01 compared to D-siCIP2A alone treated cells (ANOVA, Dunnett’s Multiple Comparison Test). (<b>C</b>) Assessment of long-term toxicity (as measured by a cell proliferation assay) of CAL 27 cells 48 hours post-treatment with either a non-targeting siRNA (siNT) alone, increasing concentrations of 599 peptide alone, or increasing amounts of 599 peptide (ranging from 1 to 100-fold molar excess of siRNAs) in complex with siNT. For comparison, the cells were also transfected with the commercial transfection reagent, IFN. Untreated cells were defined as 100% viable. Data are mean ± SEM performed in triplicate (n = 3), where ***P<0.001, **P<0.01 compared to untreated cells (ANOVA, Dunnett’s Multiple Comparison Test).</p

Particle characterization of the 599/siCIP2A complex.

<p>(<b>A</b>) Size distribution and zeta potential of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. (<b>B</b>) Darkfield-based optical microscopy image of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. Scale bar: 10,000 nm.</p

The 599 peptide mediates delivery of siRNAs into cells via endocytosis.

<p>(<b>A</b>) Fluorescence microscopy analysis and overlay of a phase contrast image of live CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) complexed with the 599 peptide at a 50∶1 peptide-to-siRNA molar ratio. Scale bar: 50 µm. (<b>B</b>) Indirect immunofluorescence microscopy analysis of CAL 27 oral cancer cells incubated for 2 hours with D-siCIP2A (red) complexed with the 599 peptide at a 50∶1 peptide-to-siRNA molar ratio. Endosomes (green) were stained using a rabbit monoclonal anti-EEA1 antibody. Nuclei (blue) were counterstained with DAPI. The co-localization of D-siCIP2A with endosomes (yellow) is observed in the merged panel and is emphasized by arrows. Scale bar: 25 µm.</p

The 599 peptide-mediated silencing of CIP2A decreases oral cancer cell invasiveness and anchorage-independent growth.

<p>(<b>A</b>) Quantitation of the percentage of invading SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test). (<b>B</b>) Anchorage-independent growth of SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siCIP2A compared with control siNT. Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test).</p

Characterization of 599 peptide-mediated RNAi responses in oral cancer cells.

<p>(<b>A</b>) Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either 100 nM of control non-targeting siRNA (siNT) or siRNA targeting CIP2A (siCIP2A) at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples. (<b>B</b>) Western blot analysis of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide alone (5 µM) or complexed at a 50∶1 peptide-to-siRNA molar ratio to either siNT (100 nM) or decreasing concentrations of siCIP2A. The CIP2A protein levels in untreated cells were also analyzed for comparison. GAPDH protein levels were monitored to ensure equal loading of samples. (<b>C</b>) Western blot analyses of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio, in the presence (+) or absence (-) of serum, compared to untreated cells or cells treated with commercial transfection reagents Lipofectamine® RNAiMAX (RNAiMAX), Lipofectamine® 2000 (LF2000), INTERFERin™ (IFN), and HiPerFect®. GAPDH protein levels were monitored to ensure equal loading of samples.</p

The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.

<p>(<b>A</b>) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (<b>B</b>) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples.</p