Dense Granule Protein GRA64 Interacts with Host Cell ESCRT Proteins during Toxoplasma gondii Infection.

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In an attempt to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a transmembrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrated that GRA64 appears to interact with components of the host endosomal sorting complexes required for transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence or encystation in mice, as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Δgra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCE Toxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immunocompromised individuals. Part of this parasite's success lies in its ability to infect diverse organisms and host cells and to persist as a latent infection within parasite-constructed structures called tissue cysts. In this study, we characterized a protein that is secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuolar membrane, this protein is exposed to the host cell cytosol and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

Intravesical Oxybutynin for Urgent Bladder Rescue in a Newborn with Posterior Urethral Valves.

Posterior urethral valves are the most common cause of bladder outlet obstruction in male newborns. Initial catheter drainage alleviates the urethral obstruction before definitive management by valve ablation. Newborns with posterior urethral valves often present with hypercontractile bladders that may inhibit upper tract drainage despite bladder catheterization. Anticholinergic agents are commonly used to treat detrusor hyperactivity, with oxybutynin being the most commonly used. We report the first case of a newborn with posterior urethral valves and ureterovesical junction obstruction caused by detrusor hypertrophy who underwent urgent intravesical instillation of oxybutynin at high doses in an attempt to avoid a diversion procedure.

Análisis comparativo de predictores potenciales de prácticas disciplinarias severas con preescolares, antes y después de un entrenamiento para padres / Comparative analysis of predictive factors of severe disciplinary practices with preschoolers, before and after parent training / Análise comparativa de preditores potenciais de práticas disciplinares severas com pré-escolares, antes e depois de um treinamento para pais

El objetivo de este estudio consistió en determinar si un entrenamiento en pautas de crianza dirigido a padres con niños de tres a cinco años de edad modificaría las prácticas disciplinarias severas, así como algunos predictores potenciales de esas prácticas, como son el coraje-agresión y el estrés de los padres, al igual que el estrés en la interacción entre padres e hijos, y los problemas de conducta y el estrés de los niños. Sesenta padres (41 mamás y 19 papás) contestaron los cuestionarios correspondientes. La muestra se dividió al azar en grupo experimental (GE: expuesto a un entrenamiento en crianza) y grupo control (GC: sin entrenamiento). Los padres contestaron los mismos cuestionarios diez semanas después. Un análisis de regresión jerárquica (ARJ) con la muestra total mostró que el estrés en la interacción y las conductas problema fueron las principales variables predictoras de las prácticas disciplinarias (p < .01). En la segunda evaluación, los resultados indicaron la modificación significativa de las prácticas disciplinarias y de todas las variables del estudio en el GE, pero no en el GC; además, nuevos ARJ con el GC y con el GE indicaron la importancia del coraje-agresión parental como variable predictora de las prácticas disciplinarias. Los resultados se discuten en términos de la importancia de la modificación de las prácticas disciplinarias severas y sus efectos negativos en los niños, así como del uso adecuado de técnicas cognitivo-conductuales para modificar positivamente las relaciones negativas entre los padres y el niño.

The objective of this study consisted on determining whether training parents of preschool children on rearing practices would modify harsh disciplinary practices as well as some potential predictors of these practices, such as anger-aggression and stress of parents, children's stress and behavioral problems, as well as parent-child stress. Sixty parents (41 moms and 19 dads) answered the corresponding questionnaires. The sample was randomly divided into an experimental group (EG), exposed to parent training, and a control group without training (CG). Parents answered the same questionnaires 10 weeks later. Hierarchical regression analysis (HRA) showed that parent-child stress and behavioral problems were the main predictor variables of disciplinary practices (p < .01) in the total sample. In the second assessment, results indicated significant changes in disciplinary practices and all the variables for the EG, but not for the CG. In addition, HRA performed on the CG and the EG pointed out the importance of parental anger-aggression as a variable predictive of disciplinary practices. Results are discussed in terms of the importance of modifying harsh disciplinary practices and their negative effects on children, as well as the proper use of cognitive-behavioral techniques to modify negative relationships between parents and children.

O objetivo deste estudo consistiu em determinar se um treinamento em pautas de criação dirigido a pais com crianças de três a cinco anos de idade modificaria as práticas disciplinares severas, bem como alguns preditores potenciais dessas práticas, como por exemplo a coragem-agressão e o estresse dos pais, da mesma forma que o estresse na interação entre pais e filhos, e os problemas de conduta e o estresse das crianças. Sessenta pais (41 mães e 19 pais) responderam os questionários correspondentes. A mostra foi dividida aleatoriamente em grupo experimental (GE: exposto a um treinamento em criação) e o grupo de controle (GC: sem treinamento). Os pais responderam os mesmos questionários dez semanas depois. Uma análise de regressão hierárquica (ARH) com a mostra total assinalou que o estresse na interação e as condutas problema foram as principais variáveis preditoras das práticas disciplinares (p < .01). Na segunda avaliação, os resultados indicaram a modificação significativa das práticas disciplinares e de todas as variáveis do estudo no GE, mas não no GC; além disso, novos ARH com o GC e com o GE indicaram a importância da coragem-agressão parental como variável preditora das práticas disciplinares. Os resultados discutem-se em termos da importância da modificação das práticas disciplinares severas e sus efeitos negativos nas crianças, bem como do uso adequado de técnicas cognitivo-condutuais para modificar positivamente as relações negativas entre os pais e a criança.

Análisis exploratorio de la percepción en niños preescolares sobre la interacción recíproca con sus madres / Exploratory Analysis of Preschoolers' Perception of the Reciprocal Interaction with their Mothers / Análise exploratório da percepçÃo de crianças pré-escolares sobre a interaçÃo recíproca com suas mÃes

Objetivo. Aunque los avances conceptuales y las evidencias empíricas con niños en edad escolar y adolescentes sugieren que las percepciones de los niños son relevantes para comprender las interacciones recíprocas padres - hijos, el estado de conocimiento es prácticamente nulo en niños preescolares. El principal objetivo de este estudio buscó explorar la percepción de niños preescolares sobre las relaciones con sus madres y analizar si los puntajes diferenciales de la percepción de los niños indican diferencias en la competencia parental, así como en otros factores que tienen relación con su crianza. Método. La muestra estuvo conformada por 50 mamás que contestaron los autorreportes y sus niños (n = 50) que contestaron una prueba pictórica de percepción de las relaciones con sus madres. Resultados. Los resultados indicaron que las percepciones de los niños sobre las interacciones adecuadas o inadecuadas fueron congruentes con los puntajes de los cuestionarios, en particular, con la competencia parental, el estrés y el bienestar subjetivo maternos y con la adaptación psicosocial, el estrés y los problemas de comportamiento de los niños, así como con el temperamento de ambos. Conclusión. Se discuten los resultados y sus implicaciones para establecer congruencia entre el avance conceptual y las evidencias empíricas sobre las interacciones recíprocas.

Objective. Although conceptual advances and empirical evidence with school-age children and adolescents suggest that children's perceptions are relevant in understanding the reciprocal reactions between parents and children, the level of knowledge in the case of pre - school children is almost non - existent. The main objective of this study was to explore the perception of preschool children of the relationships with their mothers, and to determine whether scores were related with differences on parenting competence, as well as with other parenting factors. Method. Data were collected from 50 mothers who responded to several questionnaires and their children (n = 50), who answered a pictorial test of perception of the relationships with their mothers. Results. Results indicated that scores from the questionnaires were consistent with perceptions of children regarding appropriate or inappropriate interactions: for mothers on parental competence, stress, and subjective well - being, and for children on psychosocial adaptation, stress, and behavior problems; as well as with scores relating to the temperament of both. Conclusion. The results and their implications are discussed in terms of establishing coherence between conceptual advances and the empirical evidence on reciprocal interactions.

Escopo. Ainda que os avanços conceituais e as evidencias empíricas com crianças em idade escolar e adolescentes sugerem que as percepções das crianças sÃo relevantes para compreender as interações recíprocas pais-filhos, o estado de conhecimento é praticamente nulo com crianças pré - escolares. O principal escopo de este estudo foi o de explorar a percepçÃo de crianças pré - escolares sobre as relações com suas mÃes e analisar sim pontuações diferentes da percepçÃo das crianças indicam diferencias na competência parental, assim como em outros fatores que tem relaçÃo com a educaçÃo. Metodologia. A mostra foi formada com 50 mÃes que contestaram os auto - reportes e suas crianças (n = 50) que contestaram uma prova pictórica de percepçÃo das relações com suas mÃes. Resultados. Os resultados indicaram que as percepções das crianças sobre as interações adequadas ou inadequadas foram congruentes com as pontuações dos questionários; em particular, com a competência parental, o stress e o bem-estar subjetivo maternos e com a adaptaçÃo psicossocial, o stress e os problemas de comportamento das crianças; assim como o temperamento de ambos. ConclusÃo. Foram discutidos os resultados e suas implicações para estabelecer congruência entre o avanço conceitual e as evidencias empíricas sobre interações recíprocas.

Relaciones entre la crianza y factores protectores o de riesgo, antes y después de una intervención para padres / Relationships between parenting and risk or protective factors, before and after a parenting intervention

Los objetivos del estudio fueron: identificar las relaciones entre la crianza y factores que pueden ejercer influencia protectora o de riesgo para la socialización de los niños y analizar los cambios que fomenta una intervención en crianza. La intervención elegida (Paternidad Estrella), fomenta expectativas justas, y disciplina y crianza positivas; los factores incluidos fueron comportamientos pro sociales de los niños, su desarrollo psicosocial y sus comportamientos problema; así como con el estrés y el coraje/agresión de los padres. Participaron padres con un niño de 3 a 5 años de edad, distribuidos en un grupo experimental, con niños con problemas de conducta (n = 30), y uno control (n = 30). Los resultados confirman que las intervenciones tempranas modifican problemas de comportamiento; además, mostraron que las expectativas y las prácticas disciplinarias de los padres están relacionadas con los comportamientos pro sociales, el desarrollo y los comportamientos problema de los niños, y con el estrés y el coraje/agresión de los padres. Los resultados se discuten en términos de los esfuerzos por aclarar qué factores de influencia en la crianza pueden ser modificados exitosamente por programas de intervención, y aportan la identificación de componentes específicos que pueden ser incluidos en otros programas.

The objectives of this study were to identify the relationships between parenting and risk factors and protective factors that exert influence on children's socialization process, and to analyze changes in the relationships as are promoted by a programme for parents of young children. The dimensions fostered by the chosen intervention are fair expectations, and discipline and nurturing practices; factors chosen were children pro social behaviors, psycho-social development, behavior problems, as well as anger-aggression and stress from parents. Parents of young children (3 to 5 yrs. old) with behavior problems were assigned to an experimental group (n = 30) and parents of children without behavior problems were assigned to a control group (n = 30). Results support previous research indicating the importance of early interventions for treatment of behavior problems. Moreover, results allowed the identification of parents' expectations and discipline practices as two components related to children pro social behaviors, psycho-social development, behavior problems, as well as anger-aggression and stress from parents. Results are discussed in terms of present efforts for identifying which factors may be successfully modified, and demonstrate the benefits of specific parenting dimensions which may be adopted by other parenting programs.

Valoración objetiva del estilo de crianza y las expectativas de parejas con niños pequeños / Objective assessment of parenting styles and expectations of couples with young children

Los propósitos de este estudio consistieron en conocer la frecuencia de estilos de crianza en parejas con estilo común versus con estilo diferente, analizar sus diferencias, y conocer susexpectativas. Se eligió un modelo de exigencias-responsividad que se operacionalizó con una medida objetiva de prácticas disciplinarias y de crianza. Se evaluaron 485 parejas con niñospequeños. Los resultados indicaron que en parejas con estilo común el estilo predominante fue el indulgente, pero en parejas con estilo diferente, predominó el estilo autoritario en las madresy el autorizativo en los padres. Las parejas autorizativas, con excepción de las parejasindulgentes, tuvieron más prácticas de crianza y las más altas expectativas. Se resalta la necesidad de más investigación con muestras latinoamericanas.

The purposes of the present study were to determine the frequency of parenting styles in couples with same style versus different style, to analyze differences between these groups, and to explore their parental expectations. A demandingness-responsiveness model was chosen, and it was operationalized by means of an objective measure of discipline and nurturing practices. In the study participated 485 couples with young children. Results revealed moreindulgent parents in couples with the same style. Among parents who didn’t share parenting style, there were more authoritarian mothers and more authoritative fathers. Authoritativecouples, with the exception of indulgent couples, had more nurturing practices and the highest developmental expectations among styles and groups. This study highlights the need of moreLatin-American research.

Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability.

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

Specific oncolytic effect of a new hypoxia-inducible factor-dependent replicative adenovirus on von Hippel-Lindau-defective renal cell carcinomas.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are responsible for a hereditary cancer syndrome characterized by high susceptibility to hemangioblastomas of the retina and central nervous system, pheochromocytomas, and renal cell carcinomas. In agreement with its role as a tumor suppressor, the vast majority of spontaneous clear cell carcinomas of the kidney present loss of heterozygosity at the VHL locus. Recently, it has been shown that VHL works as the substrate recognition component of an E3 ubiquitination complex that targets the hypoxia-inducible factor (HIF) for proteosomal degradation. Under normal oxygen tension, the half-life of HIF transcription factors is extremely short because of its high degradation rate by the proteasome, resulting in undetectable HIF activity in normal cells. However, in VHL-deficient tumor cells, the HIF transcriptional pathway is constitutively activated because of impaired ubiquitination of this transcription factor. To target VHL-deficient tumors, we have exploited this feature to develop a conditionally replicative adenovirus (Ad9xHRE1A), the replication of which is HIF dependent. In this new oncolytic adenovirus, the expression of the E1A gene is controlled by an optimized minimal promoter containing HIF recognition elements. Here, we show that the induction of the E1A gene, as well as the viral replication and cytolytic effect of Ad9xHRE1A, are dependent on HIF activity. As a consequence, this virus efficiently kills VHL-deficient cells both in vitro and in vivo, as well as cells growing under hypoxic conditions. These data suggest that Ad9xHRE1A could be used as a highly specific therapy for VHL-deficient cancers and probably many other tumors that show extensive hypoxic areas or increased HIF activity by genetic alterations other than VHL loss.

The von Hippel Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of the HIF-proline hydroxylase genes in response to low oxygen.

Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIFalpha proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIFalpha is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalyze HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIFalpha escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIFalpha in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIFalpha resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIFalpha.

Inhibiting expression of specific genes in mammalian cells with 5' end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA.

Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278-5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5' end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5' end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5' end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5' end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model.