Effect of a school-based asthma clinic on asthma outcomes.

Background: The St. Louis Children's Hospital Healthy Kids Express Asthma (HKEA) program was developed to improve asthma control in children who attend schools with the highest asthma prevalence in the metropolitan area. The HKEA program differs from other programs because unscheduled visits occur at school without parents present. Objective: To assess the effectiveness of the HKEA program via a retrospective quality assurance study. Methods: A chart review was performed to evaluate the change in health-care utilization, absenteeism, staff and student education, inhaler technique checks, and parent satisfaction surveys before and after participation in the program. The Wilcoxon signed rank test, two-way analysis of variance, and descriptive statistics were used to analyze the data. Results: The HKEA program recruited 1076 participants ages 5-15 years during 3 school years, from 2008 to 2011. The participants showed a reduction in emergency department visits (36.9% to 14.2%) and hospitalizations (7.1% to 5.0%) from the year before beginning the program to the third year of the program. Absenteeism was significantly improved, from 59.1% to 27.1%. Staff and student knowledge of asthma improved significantly after completing asthma education programs. More than 90% of participants completed three technique checks of their inhaler and spacer technique and showed significant improvement in their tech check (an inhaler/aero chamber technique check) scores. Parent satisfaction with the HKEA program was rated excellent or very good by 96.9% of the parents. Conclusion: The HKEA program is a novel school-based asthma clinic that is well accepted by parents, and results in less health-care utilization and school absences as well as improved asthma knowledge in participants and the school staff.

Asthma control and obesity in urban African American children.

OBJECTIVE: To examine the relationship between body mass index (BMI), gender, age, controller medication use, household smoke exposure, season, and allergic rhinitis status with asthma control in a group of lower income, African American children. We hypothesized that non-obese children would have better asthma control. METHODS: Baseline data from a longitudinal study of children in a school-based asthma program in a Midwest urban area were analyzed. 360 children, ages 4-15 years, who were enrolled in either the 2012-2013 or 2013-2014 program were included. Asthma control was classified using criteria from the 2007 National Asthma Education and Prevention Program. Multiple ordinal regression was performed. RESULTS: The median age was 9 years, 61% had well-controlled asthma, and 29% were obese. The model included all main effects plus two interaction terms and was significant (χ2(7) = 22.17, p =.002). Females who were normal weight (OR, 2.78; 95% CI, 1.38-5.60, p =.004) or overweight (OR, 3.12; 95% CI, 1.26-7.72, p =.014) had better asthma control than obese females. For males, there were no differences by BMI category but males without allergic rhinitis had significantly better asthma control (OR, 2.23; 95% CI, 1.25-3.97, p =.006) than those with allergic rhinitis. CONCLUSIONS: Non-obese girls and non-allergic males had better asthma control. Promotion of healthy activity and nutrition as well as management of allergic rhinitis should be part of the asthma plan in school-based programs in low income urban areas. Innovative approaches to address asthma care in low income populations are essential.

Conserved properties of Drosophila and human spermatozoal mRNA repertoires.

It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilization, however, their function remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are unlikely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilization and can be detected before, and at least until, the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular, a highly significant enrichment for transcripts encoding ribosomal proteins (RPs). The substantial functional coherence of spermatozoal transcripts in humans and the fly opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules in sperm and in the oocyte following fertilization.

The impact of quantitative optimization of hybridization conditions on gene expression analysis.

BACKGROUND: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. RESULTS: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. CONCLUSIONS: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.

Neighbourhood continuity is not required for correct testis gene expression in Drosophila.

It is now widely accepted that gene organisation in eukaryotic genomes is non-random and it is proposed that such organisation may be important for gene expression and genome evolution. In particular, the results of several large-scale gene expression analyses in a range of organisms from yeast to human indicate that sets of genes with similar tissue-specific or temporal expression profiles are clustered within the genome in gene expression neighbourhoods. While the existence of neighbourhoods is clearly established, the underlying reason for this facet of genome organisation is currently unclear and there is little experimental evidence that addresses the genomic requisites for neighbourhood organisation. We report the targeted disruption of three well-defined male-specific gene expression neighbourhoods in the Drosophila genome by the synthesis of precisely mapped chromosomal inversions. We compare gene expression in individuals carrying inverted chromosomes with their non-inverted but otherwise identical progenitors using whole-transcriptome microarray analysis, validating these data with specific quantitative real-time PCR assays. For each neighbourhood we generate and examine multiple inversions. We find no significant differences in the expression of genes that define each of the neighbourhoods. We further show that the inversions spatially separate both halves of a neighbourhood in the nucleus. Thus, models explaining neighbourhood organisation in terms of local sequence interactions, enhancer crosstalk, or short-range chromatin effects are unlikely to account for this facet of genome organisation. Our study challenges the notion that, at least in the case of the testis, expression neighbourhoods are a feature of eukaryotic genome organisation necessary for correct gene expression.

Asthma prevalence in low-income urban elementary school students in St. Louis, 1992 and 2004.

OBJECTIVE: We reexamined asthma prevalence in urban public elementary school children after 12 years, during which time poverty had worsened. STUDY DESIGN: We surveyed 152 children in 1992 and 331 in 2004 attending fourth- and fifth-grade classrooms in a low-income area of St. Louis, Missouri. Prevalences of phenotypes (current asthma, previous diagnosis without current asthma, and frequent wheezing without diagnosis) were based on standard published questions. We assessed age, sex, percentage below poverty level, and asthma experience (household member with asthma; friend, relative, or neighbor with asthma; or ever having seen someone have an attack). RESULTS: Prevalences were similar in 1992 and 2004 for current asthma (18% and 20%) and frequent wheezing without diagnosis (24% and 26%), despite higher 2004 percentage below poverty level (40% vs 18%). Prevalences of phenotypes were not associated with demographics or percentage below poverty level but were associated with asthma experience. In multivariate analysis, current asthma was associated with household member with asthma and ever having seen someone have an attack, and previous diagnosis was associated with household member with asthma. CONCLUSIONS: For these fourth- and fifth-grade urban public school children, self-reported asthma prevalence was similar after 12 years despite worsening poverty.

Stability and dynamics of polycomb target sites in Drosophila development.

Polycomb-group (PcG) and Trithorax-group proteins together form a maintenance machinery that is responsible for stable heritable states of gene activity. While the best-studied target genes are the Hox genes of the Antennapedia and Bithorax complexes, a large number of key developmental genes are also Polycomb (Pc) targets, indicating a widespread role for this maintenance machinery in cell fate determination. We have studied the linkage between the binding of PcG proteins and the developmental regulation of gene expression using whole-genome mapping to identify sites bound by the PcG proteins, Pc and Pleiohomeotic (Pho), in the Drosophila embryo and in a more restricted tissue, the imaginal discs of the third thoracic segment. Our data provide support for the idea that Pho is a general component of the maintenance machinery, since the majority of Pc targets are also associated with Pho binding. We find, in general, considerable developmental stability of Pc and Pho binding at target genes and observe that Pc/Pho binding can be associated with both expressed and inactive genes. In particular, at the Hox complexes, both active and inactive genes have significant Pc and Pho binding. However, in comparison to inactive genes, the active Hox genes show reduced and altered binding profiles. During development, Pc target genes are not simply constantly associated with Pc/Pho binding, and we identify sets of genes with clear differential binding between embryo and imaginal disc. Using existing datasets, we show that for specific fate-determining genes of the haemocyte lineage, the active state is characterised by lack of Pc binding. Overall, our analysis suggests a dynamic relationship between Pc/Pho binding and gene transcription. Pc/Pho binding does not preclude transcription, but levels of Pc/Pho binding change during development, and loss of Pc/Pho binding can be associated with both stable gene activity and inactivity.

Variable sexually dimorphic gene expression in laboratory strains of Drosophila melanogaster.

BACKGROUND: Wild-type laboratory strains of model organisms are typically kept in isolation for many years, with the action of genetic drift and selection on mutational variation causing lineages to diverge with time. Natural populations from which such strains are established, show that gender-specific interactions in particular drive many aspects of sequence level and transcriptional level variation. Here, our goal was to identify genes that display transcriptional variation between laboratory strains of Drosophila melanogaster, and to explore evidence of gender-biased interactions underlying that variability. RESULTS: Transcriptional variation among the laboratory genotypes studied occurs more frequently in males than in females. Qualitative differences are also apparent to suggest that genes within particular functional classes disproportionately display variation in gene expression. Our analysis indicates that genes with reproductive functions are most often divergent between genotypes in both sexes, however a large proportion of female variation can also be attributed to genes without expression in the ovaries. CONCLUSION: The present study clearly shows that transcriptional variation between common laboratory strains of Drosophila can differ dramatically due to sexual dimorphism. Much of this variation reflects sex-specific challenges associated with divergent physiological trade-offs, morphology and regulatory pathways operating within males and females.

Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila.

BACKGROUND: Insulator elements are proposed to play a key role in the organization of the regulatory architecture of the genome. In Drosophila, one of the best studied is the gypsy retrotransposon insulator, which is bound by the Suppressor of Hairy-wing (Su [Hw]) transcriptional regulator. Immunolocalization studies suggest that there are several hundred Su(Hw) sites in the genome, but few of these endogenous Su(Hw) binding sites have been identified. RESULTS: We used chromatin immunopurification with genomic microarray analysis to identify in vivo Su(Hw) binding sites across the 3 megabase Adh region. We find 60 sites, and these enabled the construction of a robust new Su(Hw) binding site consensus. In contrast to the gypsy insulator, which contains tightly clustered Su(Hw) binding sites, endogenous sites generally occur as isolated sites. These endogenous sites have three key features. In contrast to most analyses of DNA-binding protein specificity, we find that strong matches to the binding consensus are good predictors of binding site occupancy. Examination of occupancy in different tissues and developmental stages reveals that most Su(Hw) sites, if not all, are constitutively occupied, and these isolated Su(Hw) sites are generally highly conserved. Analysis of transcript levels in su(Hw) mutants indicate widespread and general changes in gene expression. Importantly, the vast majority of genes with altered expression are not associated with clustering of Su(Hw) binding sites, emphasizing the functional relevance of isolated sites. CONCLUSION: Taken together, our in vivo binding and gene expression data support a role for the Su(Hw) protein in maintaining a constant genomic architecture.

SimArray: a user-friendly and user-configurable microarray design tool.

BACKGROUND: Microarrays were first developed to assess gene expression but are now also used to map protein-binding sites and to assess allelic variation between individuals. Regardless of the intended application, efficient production and appropriate array design are key determinants of experimental success. Inefficient production can make larger-scale studies prohibitively expensive, whereas poor array design makes normalisation and data analysis problematic. RESULTS: We have developed a user-friendly tool, SimArray, which generates a randomised spot layout, computes a maximum meta-grid area, and estimates the print time, in response to user-specified design decisions. Selected parameters include: the number of probes to be printed; the microtitre plate format; the printing pin configuration, and the achievable spot density. SimArray is compatible with all current robotic spotters that employ 96-, 384- or 1536-well microtitre plates, and can be configured to reflect most production environments. Print time and maximum meta-grid area estimates facilitate evaluation of each array design for its suitability. Randomisation of the spot layout facilitates correction of systematic biases by normalisation. CONCLUSION: SimArray is intended to help both established researchers and those new to the microarray field to develop microarray designs with randomised spot layouts that are compatible with their specific production environment. SimArray is an open-source program and is available from http://www.flychip.org.uk/SimArray/.

Robotic spotting of cDNA and oligonucleotide microarrays.

DNA microarrays are a uniquely efficient method for simultaneously assessing the expression levels of thousands of genes. Owing to their flexibility and value, mechanically spotted microarrays remain the most popular platform. Here, we review recent technological advances with a focus on spotted arrays. Robotic spotting still poses numerous technical challenges. To reduce artefacts, many laboratories have recently investigated ways of improving the spotting process. We compare alternative options and discuss implications for next-generation systems. Together with modern approaches to data analysis, such developments bring greatly improved reliability to individual microarray experiments. Advancing towards the ultimate goal of delivering calibrated, truly quantitative gene-expression measurements on a genomic scale, microarray technology remains at the forefront of post-genomic systems biology.

Evolving treatment in a decade of pediatric burn care.

BACKGROUND: Over the last decade, an ambulatory burn care (ABC) and procedural sedation (PS) program was instituted at St Louis Children's Hospital (SLCH). This study assessed the effect of these interventions on resource utilization. METHODS: The authors reviewed the hospital experience comparing 1993 with 2002 data regarding gender, age, burn depth, patient admissions, inpatient days, and ABC visits. Outcome measures included length of stay (LOS), incidence of infection, and hospital charges. RESULTS: Gender, age, and burn depth were similar; 192 patients were admitted in 1993. In 2002, there were 167 admissions and 118 patients treated solely on an ABC basis resulting in a total of 285 burn patients treated (+48%). Hospital days decreased from 2,041 (1993) to 963 (2002 [-53%]). LOS declined from 10.4 +/- 8.3 days (1993) to 5.8 +/- 14.2 days (2002 [-44%; P <.05]). PS was used sporadically in 1993, and increased to 71% in patients in 2002. There were no ABC visits in 1993 and 501 visits in 2002. The incidence of infection was 5.2% in 1993 versus 3.0% in 2002 (P <.05) Average charge per patient fell 45% from 13,286 dollars (1993) to 7,372 dollars (2002), adjusted to 1993 dollars using medical care price index. CONCLUSIONS: Over a 10-year period, the program achieved a significant reduction in resource utilization while increasing the number of patients treated and maintaining a low incidence of infection. This was due in large part to a shift to ABC and the use of PS.

Frequency and voltage dependence of the dielectrophoretic trapping of short lengths of DNA and dCTP in a nanopipette.

The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.

Evidence for differential and redundant function of the Sox genes Dichaete and SoxN during CNS development in Drosophila.

Group B Sox-domain proteins encompass a class of conserved DNA-binding proteins expressed from the earliest stages of metazoan CNS development. In all higher organisms studied to date, related Group B Sox proteins are co-expressed in the developing CNS; in vertebrates there are three (Sox1, Sox2 and Sox3) and in Drosophila there are two (SoxNeuro and Dichaete). It has been suggested there may be a degree of functional redundancy in Sox function during CNS development. We describe the CNS phenotype of a null mutation in the Drosophila SoxNeuro gene and provide the first direct evidence for both redundant and differential Sox function during CNS development in Drosophila. In the lateral neuroectoderm, where SoxNeuro is uniquely expressed, SoxNeuro mutants show a loss or reduction of achaete expression as well as a loss of many correctly specified lateral neuroblasts. By contrast, in the medial neuroectoderm, where the expression of SoxNeuro and Dichaete overlaps, the phenotypes of both single mutants are mild. In accordance with an at least partially redundant function in that region, SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. However, the finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo.