Transcription through the eye of a needle: daily and annual cyclic gene expression variation in Douglas-fir needles.

BACKGROUND: Perennial growth in plants is the product of interdependent cycles of daily and annual stimuli that induce cycles of growth and dormancy. In conifers, needles are the key perennial organ that integrates daily and seasonal signals from light, temperature, and water availability. To understand the relationship between seasonal cycles and seasonal gene expression responses in conifers, we examined diurnal and circannual needle mRNA accumulation in Douglas-fir (Pseudotsuga menziesii) needles at diurnal and circannual scales. Using mRNA sequencing, we sampled 6.1 × 109 reads from 19 trees and constructed a de novo pan-transcriptome reference that includes 173,882 tree-derived transcripts. Using this reference, we mapped RNA-Seq reads from 179 samples that capture daily and annual variation. RESULTS: We identified 12,042 diurnally-cyclic transcripts, 9299 of which showed homology to annotated genes from other plant genomes, including angiosperm core clock genes. Annual analysis revealed 21,225 circannual transcripts, 17,335 of which showed homology to annotated genes from other plant genomes. The timing of maximum gene expression is associated with light intensity at diurnal scales and photoperiod at annual scales, with approximately half of transcripts reaching maximum expression +/- 2 h from sunrise and sunset, and +/- 20 days from winter and summer solstices. Comparisons with published studies from other conifers shows congruent behavior in clock genes with Japanese cedar (Cryptomeria), and a significant preservation of gene expression patterns for 2278 putative orthologs from Douglas-fir during the summer growing season, and 760 putative orthologs from spruce (Picea) during the transition from fall to winter. CONCLUSIONS: Our study highlight the extensive diurnal and circannual transcriptome variability demonstrated in conifer needles. At these temporal scales, 29% of expressed transcripts show a significant diurnal cycle, and 58.7% show a significant circannual cycle. Remarkably, thousands of genes reach their annual peak activity during winter dormancy. Our study establishes the fine-scale timing of daily and annual maximum gene expression for diverse needle genes in Douglas-fir, and it highlights the potential for using this information for evaluating hypotheses concerning the daily or seasonal timing of gene activity in temperate-zone conifers, and for identifying cyclic transcriptome components in other conifer species.

Healthier side dishes at restaurants: an analysis of children's perspectives, menu content, and energy impacts.

BACKGROUND: Children consume restaurant-prepared foods at high rates, suggesting that interventions and policies targeting consumption of these foods have the potential to improve diet quality and attenuate excess energy intake. One approach to encouraging healthier dietary intake in restaurants is to offer fruits and vegetables (FV) as side dishes, as opposed to traditional, energy-dense accompaniments like French fries. The aims of the current study were to examine: children's views about healthier side dishes at restaurants; current side dish offerings on children's menus at leading restaurants; and potential energy reductions when substituting FV side dishes in place of French fries. METHODS: To investigate children's attitudes, a survey was administered to a nationally representative sample of U.S. 8- to 18-year-olds (n = 1178). To examine current side dish offerings, children's menus from leading quick service (QSR; n = 10) and full service restaurant chains (FSR; n = 10) were analyzed. Energy reductions that could result from substituting commonly-offered FV side dishes for French fries were estimated using nutrition information corresponding to the children's menu items. RESULTS: Two-thirds of children reported that they would not feel negatively about receiving FV sides instead of French fries with kids' meals. Liking/taste was the most common reason that children gave to explain their attitudes about FV side dishes. Nearly all restaurants offered at least 1 FV side dish option, but at most restaurants (60% of QSR; 70% of FSR), FV sides were never served by default. Substituting FV side dishes for French fries yielded an average estimated energy reduction of at least 170 calories. CONCLUSIONS: Results highlight some healthy trends in the restaurant context, including the majority of children reporting non-negative attitudes about FV side dishes and the consistent availability of FV side dish options at leading QSR and FSR. Yet the minority of restaurants offer these FV sides by default. Promoting creative, appealing FV side dishes can result in healthier, less energy-dense meals for children. Substituting or displacing energy-dense default side dishes with such FV dishes show promise as part of continued, comprehensive efforts to increase the healthfulness of meals consumed by children in restaurant settings.

Children's perceptions of weight, obesity, nutrition, physical activity and related health and socio-behavioural factors.

OBJECTIVE: Approximately one-third of children in the USA are either overweight or obese. Understanding the perceptions of children is an important factor in reversing this trend. DESIGN: An online survey was conducted with children to capture their perceptions of weight, overweight, nutrition, physical activity and related socio-behavioural factors. SETTING: Within the USA. SUBJECTS: US children (n 1224) aged 8-18 years. RESULTS: Twenty-seven per cent of children reported being overweight; 47·1% of children overestimated the rate of overweight/obesity among US children. A higher percentage of self-classified overweight children (81·9%) worried about weight than did self-classified under/normal weight children (31·1%). Most children (91·1%) felt that it was important to not be overweight, for both health-related and social-related reasons. The majority of children believed that if someone their age is overweight they will likely be overweight in adulthood (93·1%); get an illness such as diabetes or heart disease in adulthood (90·2%); not be able to play sports well (84·5%); and be teased or made fun of in school (87·8%). Children focused more on food/drink than physical activity as reasons for overweight at their age. Self-classified overweight children were more likely to have spoken with someone about their weight over the last year than self-classified under/normal weight children. CONCLUSIONS: Children demonstrated good understanding of issues regarding weight, overweight, nutrition, physical activity and related socio-behavioural factors. Their perceptions are important and can be helpful in crafting solutions that will resonate with children.

Healthy kids out of school: using mixed methods to develop principles for promoting healthy eating and physical activity in out-of-school settings in the United States.

INTRODUCTION: Widespread practices supporting availability of healthful foods, beverages, and physical activity in out-of-school-time (OST) settings would further obesity prevention efforts. The objective of this article was to describe principles to guide policy development in support of healthy eating and physical activity practices in out-of-school settings to promote obesity prevention. METHODS: The Institute of Medicine's L.E.A.D. framework (Locate Evidence, Evaluate it, Assemble it, and Inform Decisions) was used to identify practices relevant to children's healthful eating in most OST settings: 1) locate and evaluate information from a national survey of children's perceptions of healthful-food access; published research, reports, policies and guidelines; and roundtables with OST organizations' administrators; 2) assemble information to prioritize actionable practices; and 3) inform programmatic direction. RESULTS: Three evidence-informed guiding principles for short-duration OST resulted: 1) drink right: choose water instead of sugar-sweetened beverages; 2) move more: boost movement and physical activity in all programs; and 3) snack smart: fuel up on fruits and vegetables. CONCLUSION: Healthy Kids Out of School was launched to support the dissemination and implementation of these guiding principles in short-duration OST settings, complementing efforts in other OST settings to shift norms around eating and physical activity.

A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation.

BACKGROUND: Douglas-fir (Pseudotsuga menziesii), one of the most economically and ecologically important tree species in the world, also has one of the largest tree breeding programs. Although the coastal and interior varieties of Douglas-fir (vars. menziesii and glauca) are native to North America, the coastal variety is also widely planted for timber production in Europe, New Zealand, Australia, and Chile. Our main goal was to develop a SNP resource large enough to facilitate genomic selection in Douglas-fir breeding programs. To accomplish this, we developed a 454-based reference transcriptome for coastal Douglas-fir, annotated and evaluated the quality of the reference, identified putative SNPs, and then validated a sample of those SNPs using the Illumina Infinium genotyping platform. RESULTS: We assembled a reference transcriptome consisting of 25,002 isogroups (unique gene models) and 102,623 singletons from 2.76 million 454 and Sanger cDNA sequences from coastal Douglas-fir. We identified 278,979 unique SNPs by mapping the 454 and Sanger sequences to the reference, and by mapping four datasets of Illumina cDNA sequences from multiple seed sources, genotypes, and tissues. The Illumina datasets represented coastal Douglas-fir (64.00 and 13.41 million reads), interior Douglas-fir (80.45 million reads), and a Yakima population similar to interior Douglas-fir (8.99 million reads). We assayed 8067 SNPs on 260 trees using an Illumina Infinium SNP genotyping array. Of these SNPs, 5847 (72.5%) were called successfully and were polymorphic. CONCLUSIONS: Based on our validation efficiency, our SNP database may contain as many as ~200,000 true SNPs, and as many as ~69,000 SNPs that could be genotyped at ~20,000 gene loci using an Infinium II array-more SNPs than are needed to use genomic selection in tree breeding programs. Ultimately, these genomic resources will enhance Douglas-fir breeding and allow us to better understand landscape-scale patterns of genetic variation and potential responses to climate change.

A genome-wide view of Caenorhabditis elegans base-substitution mutation processes.

Knowledge of mutation processes is central to understanding virtually all evolutionary phenomena and the underlying nature of genetic disorders and cancers. However, the limitations of standard molecular mutation detection methods have historically precluded a genome-wide understanding of mutation rates and spectra in the nuclear genomes of multicellular organisms. We applied two high-throughput DNA sequencing technologies to identify and characterize hundreds of spontaneously arising base-substitution mutations in 10 Caenorhabditis elegans mutation-accumulation (MA)-line nuclear genomes. C. elegans mutation rate estimates were similar to previous calculations based on smaller numbers of mutations. Mutations were distributed uniformly within and among chromosomes and were not associated with recombination rate variation in the MA lines, suggesting that intragenomic variation in genetic hitchhiking and/or background selection are primarily responsible for the chromosomal distribution patterns of polymorphic nucleotides in C. elegans natural populations. A strong mutational bias from G/C to A/T nucleotides was detected in the MA lines, implicating oxidative DNA damage as a major endogenous mutagenic force in C. elegans. The observed mutational bias also suggests that the C. elegans nuclear genome cannot be at equilibrium because of mutation alone. Transversions dominate the spectrum of spontaneous mutations observed here, whereas transitions dominate patterns of allegedly neutral polymorphism in natural populations of C. elegans and many other animal species; this observation challenges the assumption that natural patterns of molecular variation in noncoding regions of the nuclear genome accurately reflect underlying mutation processes.

TileQC: a system for tile-based quality control of Solexa data.

BACKGROUND: Next-generation DNA sequencing technologies such as Illumina's Solexa platform and Roche's 454 approach provide new avenues for investigating genome-scale questions. However, they also present novel analytical challenges that must be met for their effective application to biological questions. RESULTS: Here we report the availability of tileQC, a tile-based quality control system for Solexa data written in the R language. TileQC provides a means of recognizing bias and error in Solexa output by graphically representing data generated by flow cell tiles. The data represented in the images is then made available in the R environment for further analysis and automation of error detection. CONCLUSION: TileQC offers a highly adaptable and powerful tool for the quality control of Solexa-based DNA sequence data.

Estimating impacts of a breakfast in the classroom program on school outcomes.

IMPORTANCE: Short-term impacts of breakfast consumption on diet quality and cognitive functioning have been reported, but more evidence is needed to draw causal inferences about long-term impacts of school breakfast on indicators of school engagement and academic achievement. OBJECTIVE: To estimate the impact of a Breakfast in the Classroom (BIC) program on School Breakfast Program participation, school attendance, and academic achievement. DESIGN, SETTING, AND PARTICIPANTS: This quasi-experimental study included a sample of 446 public elementary schools from a large, urban US school district that served predominantly low-income, racial/ethnic minority students. INTERVENTIONS: A total of 257 schools (57.6%) implemented a BIC program during the 2012-2013 academic year, whereas 189 (42.4%) did not. MAIN OUTCOMES AND MEASURES: School- and grade-level data from 2012-2013 and grade-level achievement data from the prior year were collected from school district records across the elementary schools. Hypotheses that a BIC program would improve school breakfast participation at the school level, school attendance at the grade level (kindergarten through sixth grade), and academic achievement at the grade level (second through sixth grades) were tested using propensity score weights to adjust for demographic differences between the BIC and non-BIC schools. RESULTS: The BIC program was linked with increased breakfast participation during the academic year (F10,414=136.90, P<.001), with mean participation rates of 73.7% in the BIC group vs 42.9% in the non-BIC group. The BIC program was also linked with greater overall school attendance rates (95.5% vs 95.3% in the non-BIC group; F1,2772=8.40, P = .004). When performing attendance analyses in the subset of grade levels for which achievement data were available, results were mostly consistent, although there was a group × time interaction (F10,1891=1.94, P=.04) such that differences between least squares means in the BIC vs non-BIC groups did not reach statistical significance at every month. There were no group differences in standardized test performance in math (57.9% in the BIC group vs 57.4% in the non-BIC group; F1,1890=0.41, P=.52) or reading (44.9% in the BIC group vs 44.7% in the non-BIC group; F1,1890=0.15, P=.70). CONCLUSIONS AND RELEVANCE: Findings add to the evidence that BIC can increase school breakfast participation substantially and suggest that it has the potential to improve overall school attendance rates. Additional research is needed to explore the generalizability of these findings and the potential impacts on achievement for longer periods and on additional outcomes, such as weight status.

Changes in children's meal orders following healthy menu modifications at a regional U.S. restaurant chain.

OBJECTIVE: To examine changes in children's meal orders, price, and revenue following the implementation of a healthier children's menu in a full-service restaurant chain. METHODS: In April 2012, the healthier menu was implemented, featuring more meals meeting nutrition standards, healthy side dishes by default, and removal of French fries and soda (which could be substituted). Orders (n = 352,192) were analyzed before (September 2011 to March 2012; PRE) and after (September 2012 to March 2013; POST) implementation. RESULTS: Children's meal prices increased by $0.79 for breakfasts and $0.19 for non-breakfast meals from PRE to POST. Revenue continued to increase post-implementation. Orders of healthy meals, strawberry and vegetable sides, milk, and juice increased, and orders of French fries and soda decreased (P < 0.0001). Orders at POST were more likely to include healthy sides (P < 0.0001) and substitutions (P < 0.0001) and less likely to include a la carte sides (P < 0.0001) and desserts (P < 0.01), versus PRE. Total calories ordered by children accepting all defaults decreased (684.2 vs. 621.2; P < 0.0001) and did not change for those not accepting defaults (935.0 vs. 942.9; P = 0.57). CONCLUSIONS: Healthy children's menu modifications were accompanied by healthier ordering patterns, without removing choice or reducing revenue, suggesting that they can improve child nutrition while restaurants remain competitive.

Parent perspectives on nutrition and physical activity during out-of-school time.

OBJECTIVE: Out-of-school time (OST) programs serve a large, diverse population of children, including those at increased obesity risk. In this study, parents' perspectives about nutrition and physical activity (PA) during OST were assessed. DESIGN: Survey. SETTING: Online. PARTICIPANTS: Six hundred parents with a school-aged child participating in programs from selected OST organizations. MAIN OUTCOME MEASURES: Parent perspectives about the importance and availability of different foods, beverages, and PA opportunities during OST, and OST program and parent involvement in promoting healthful environments. ANALYSIS: Frequencies were used to describe parents' perspectives. Wilcoxon tests and logistic regression analyses were conducted to test for significant differences. RESULTS: Most parents reported that it was important that their children have water (96.2%), fresh fruits and vegetables (79.0%), and PA (97.2%) during OST; fewer parents reported that these were regularly available. About two thirds of parents agreed that OST programs should promote healthy environments for children, whereas one third agreed that parents alone should be responsible for children's nutrition and PA. CONCLUSIONS AND IMPLICATIONS: Results suggest that many parents would support efforts to improve OST nutrition and PA. Yet, there is a need to further understand the perceptions and motivations of different subgroups to enact successful obesity prevention efforts during OST.

Variation in base-substitution mutation in experimental and natural lineages of Caenorhabditis nematodes.

Variation among lineages in the mutation process has the potential to impact diverse biological processes ranging from susceptibilities to genetic disease to the mode and tempo of molecular evolution. The combination of high-throughput DNA sequencing (HTS) with mutation-accumulation (MA) experiments has provided a powerful approach to genome-wide mutation analysis, though insights into mutational variation have been limited by the vast evolutionary distances among the few species analyzed. We performed a HTS analysis of MA lines derived from four Caenorhabditis nematode natural genotypes: C. elegans N2 and PB306 and C. briggsae HK104 and PB800. Total mutation rates did not differ among the four sets of MA lines. A mutational bias toward G:C→A:T transitions and G:C→T:A transversions was observed in all four sets of MA lines. Chromosome-specific rates were mostly stable, though there was some evidence for a slightly elevated X chromosome mutation rate in PB306. Rates were homogeneous among functional coding sequence types and across autosomal cores, arms, and tips. Mutation spectra were similar among the four MA line sets but differed significantly when compared with patterns of natural base-substitution polymorphism for 13/14 comparisons performed. Our findings show that base-substitution mutation processes in these closely related animal lineages are mostly stable but differ from natural polymorphism patterns in these two species.