Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells

Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets

Modeling complex word reading : Examining influences at the level of the word and child on mono-and polymorphemic word reading

Purpose The probability of a child reading a word correctly is influenced by both child skills and properties of the word. The purpose of this study was to investigate child-level skills (set for variability and vocabulary), word-level properties (concreteness), word structure (mono- vs polymorphemic), and interactions between these properties and word structure within a comprehensive item-level model of complex word reading. This study is unique in that it purposely sampled both mono- and polymorphemic polysyllabic words. Method A sample of African American (n = 69) and Hispanic (n = 6) students in grades 2–5 (n = 75) read a set of mono- and polymorphemic polysyllabic words (J = 54). Item-level responses were modeled using cross-classified generalized random-effects models allowing variance to be partitioned between child and word while controlling for other important child factors and word features. Results Set for variability and the interaction between concreteness and word structure (i.e., mono- vs polymorphemic) were significant predictors. Higher probabilities of reading poly- over monomorphemic words were identified at lower levels of concreteness with the opposite at higher levels of concreteness. Conclusions Results indicate important predictors at both the child- and word-level and support the importance of morphological structure for reading abstract polysyllabic words

The effect of facilitative versus inhibitory word training corpora on word reading accuracy growth in children with dyslexia

We modeled word reading growth in typically developing (n = 118) and children with dyslexia (n = 20), Grades 2–5, across multiple exposures to 30 words. We explored the facilitative versus inhibitory effects of exposures to differential mixes of words that support high- versus low-frequency vowel pronunciations. One training corpus contained a ratio of 80%–20% high- to low-frequency pronunciations (e.g., for ea; 80% ea pronounced as /i/ as in bead and 20% ea pronounced /ε/ as in dead), whereas the other consisted of a ratio of 20%–80%. We also modeled accuracy at the final exposure for a subset of 12 shared words across conditions using item-level crossed-random effects models with reading skill (i.e., typically developing vs. dyslexic), condition, word frequency, and vowel pronunciation (i.e., high- vs. low-frequency vowel pronunciation) as predictors in the model. We were particularly interested in the interaction between condition and vowel pronunciation across reading groups. Results suggest typically developing children were influenced by the interaction between condition and vowel pronunciation, suggesting both facilitation and inhibition, whereas children with dyslexia were influenced by condition and vowel pronunciation without an interaction. Results are interpreted within the overfitting model of dyslexia

Mechanisms of Cellular Uptake, Intracellular Transportation, and Degradation of CIGB-300, a Tat-Conjugated Peptide, in Tumor Cell Lines

CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate´s phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.Fil: Benavent Acero, Fernando Rodrigo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perera Negrin, Yasser. Centro de Ingeniería Genética y Biotecnología. Laboratorio de Oncología Molecular; CubaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Perea, Silvio E.. Centro de Ingeniería Genética y Biotecnología. Laboratorio de Oncología Molecular; CubaFil: Gomez, Daniel Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin