Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1–MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.KEY WORDS: Myotonic dystrophy, Splicing, Luciferase, In vivo screening, Minigene     

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.     

Effectiveness of Music Education for the Improvement of Reading Skills and Academic Achievement in Young Poor Readers: A Pragmatic Cluster-Randomized,Controlled Clinical Trial

Introduction

Difficulties in word-level reading skills are prevalent in Brazilian schools and may deter children from gaining the knowledge obtained through reading and academic achievement. Music education has emerged as a potential method to improve reading skills because due to a common neurobiological substratum.

Objective

To evaluate the effectiveness of music education for the improvement of reading skills and academic achievement among children (eight to 10 years of age) with reading difficulties.

Method

235 children with reading difficulties in 10 schools participated in a five-month, randomized clinical trial in cluster (RCT) in an impoverished zone within the city of São Paulo to test the effects of music education intervention while assessing reading skills and academic achievement during the school year. Five schools were chosen randomly to incorporate music classes (n = 114), and five served as controls (n = 121). Two different methods of analysis were used to evaluate the effectiveness of the intervention: The standard method was intention-to-treat (ITT), and the other was the Complier Average Causal Effect (CACE) estimation method, which took compliance status into account.

Results

The ITT analyses were not very promising; only one marginal effect existed for the rate of correct real words read per minute. Indeed, considering ITT, improvements were observed in the secondary outcomes (slope of Portuguese = 0.21 [p<0.001] and slope of math = 0.25 [p<0.001]). As for CACE estimation (i.e., complier children versus non-complier children), more promising effects were observed in terms of the rate of correct words read per minute [β = 13.98, p<0.001] and phonological awareness [β = 19.72, p<0.001] as well as secondary outcomes (academic achievement in Portuguese [β = 0.77, p<0.0001] and math [β = 0.49, p<0.001] throughout the school year).

Conclusion

The results may be seen as promising, but they are not, in themselves, enough to make music lessons as public policy.     

Increased Numbers of Circulating CD8 Effector Memory T Cells before Transplantation Enhance the Risk of Acute Rejection in Lung Transplant Recipients

The effector and regulatory T cell subpopulations involved in the development of acute rejection episodes in lung transplantation remain to be elucidated. Twenty-seven lung transplant candidates were prospectively monitored before transplantation and within the first year post-transplantation. Regulatory, Th17, memory and naïve T cells were measured in peripheral blood of lung transplant recipients by flow cytometry. No association of acute rejection with number of peripheral regulatory T cells and Th17 cells was found. However, effector memory subsets in acute rejection patients were increased during the first two months post-transplant. Interestingly, patients waiting for lung transplant with levels of CD8⁺ effector memory T cells over 185 cells/mm³ had a significant increased risk of rejection [OR: 5.62 (95% CI: 1.08-29.37), p=0.04]. In multivariate analysis adjusted for age and gender the odds ratio for rejection was: OR: 5.89 (95% CI: 1.08-32.24), p=0.04. These data suggest a correlation between acute rejection and effector memory T cells in lung transplant recipients. The measurement of peripheral blood CD8⁺ effector memory T cells prior to lung transplant may define patients at high risk of acute lung rejection.     

Comparative Phylogeography of Direct-Developing Frogs (Anura: Craugastoridae: Pristimantis) in the Southern Andes of Colombia

The Andes of South America hosts perhaps the highest amphibian species diversity in the world, and a sizable component of that diversity is comprised of direct-developing frogs of the genus Pristimantis (Anura: Craugastoridae). In order to better understand the initial stages of species formation in these frogs, this study quantified local-scale spatial genetic structuring in three species of Pristimantis. DNA sequences of two mitochondrial gene fragments (16S and COI) were obtained from P. brevifrons, P. palmeri and P. jubatus at different locations in the Cordillera Occidental. We found high levels of genetic diversity in the three species, with highly structured populations (as measured by F _ST) in P. brevifrons and P. palmeri while P. jubatus showed panmixia. Large effective population sizes, inferred from the high levels of genetic diversity, were found in the three species and two highly divergent lineages were detected within P. jubatus and P. palmeri. Estimated divergence times among populations within P. brevifrons and P. palmeri coincide with the Pleistocene, perhaps due to similar responses to climatic cycling or recent geological history. Such insights have important implications for linking alpha and beta diversity, suggesting regional scale patterns may be associated with local scale processes in promoting differentiation among populations in the Andes.     

Detecting areas of endemism with a taxonomically diverse data set: plants,mammals, reptiles,amphibians, birds,and insects from Argentina

The idea of an area of endemism implies that different groups of plants and animals should have largely coincident distributions. This paper analyses an area of 1152 000 km², between parallels 21 and 32°S and meridians 70 and 53°W to examine whether a large and taxonomically diverse data set actually displays areas supported by different groups. The data set includes the distribution of 805 species of plants (45 families), mammals (25 families), reptiles (six families), amphibians (five families), birds (18 families), and insects (30 families), and is analysed with the optimality criterion (based on the notion of endemism) implemented in the program NDM/VNDM. Almost 50% of the areas obtained are supported by three or more major groups; areas supported by fewer major groups generally contain species from different genera, families, or orders. © The Willi Hennig Society 2011.     

Persistence and effect of Metarhizium anisopliae in the fungal community of sugarcane soil

The persistence of Metarhizium anisopliae (Metschnikoff) Sorokin URM5951 in soil of sugarcane crop and the effect of introducing it in the structure of the fungal community were checked, using the colony-forming units and the PCR–DGGE techniques. A suspension of conidia was sprayed on the surface of the soil obtained from the field. The experiment was carried out in vases, each one containing 3?kg of soil, in which a sugarcane seedling was introduced and maintained in greenhouse. Soil samples were collected at 15, 30, 60 and 90?days after the fungus was applied. Soil fungal DNA was PCR amplified using the primer pair FR1GC and FF390, giving fragments of the 18S rDNA. A high level of similarity was observed between the samples inoculated with M. anisopliae and the control samples in every sampling time studied. Isolate URM5951 showed persistence up to 60?days and did not interfere in the soil fungal community in the conditions tested, with these being desirable characteristics in the process of selecting entomopathogenic fungi.     

Health-Related Quality of Life of Latin-American Immigrants and Spanish-Born Attended in Spanish Primary Health Care: Socio-Demographic and Psychosocial Factors

Background

This study compares the health-related quality of life of Spanish-born and Latin American-born individuals settled in Spain. Socio-demographic and psychosocial factors associated with health-related quality of life are analyzed.

Methods

A cross-sectional Primary Health Care multi center-based study of Latin American-born (n = 691) and Spanish-born (n = 903) outpatients from 15 Primary Health Care Centers (Madrid, Spain). The Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) was used to assess health-related quality of life. Socio-demographic, psychosocial, and specific migration data were also collected.

Results

Compared to Spanish-born participants, Latin American-born participants reported higher health-related quality of life in the physical functioning and vitality dimensions. Across the entire sample, Latin American-born participants, younger participants, men and those with high social support reported significantly higher levels of physical health. Men with higher social support and a higher income reported significantly higher mental health. When stratified by gender, data show that for men physical health was only positively associated with younger age. For women, in addition to age, social support and marital status were significantly related. Both men and women with higher social support and income had significantly better mental health. Finally, for immigrants, the physical and mental health components of health-related quality of life were not found to be significantly associated with any of the pre-migration factors or conditions of migration. Only the variable “exposure to political violence” was significantly associated with the mental health component (p = 0.014).

Conclusions

The key factors to understanding HRQoL among Latin American-born immigrants settled in Spain are age, sex and social support. Therefore, strategies to maintain optimal health outcomes in these immigrant communities should include public policies on social inclusion in the host society and focus on improving social support networks in order to foster and maintain the health and HRQoL of this group.