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Background: Recent increases in patient cost-sharing for health care have lent increasing importance to monitoring cost-related changes in health care use. Despite the widespread use of survey questions to measure changes in health care use and related behaviors, scant data exists on the reliability of such questions. Methods: We administered a cross-sectional survey to a stratified random sample of families in a New England health plan’s high deductible health plan (HDHP) with ≥ $500 in annualized out-of-pocket expenditures. Enrollees were asked about their knowledge of their plan, information seeking, behavior change associated with having a deductible, experience of delay in care due in part to cost, and hypothetical delay in care due in part to cost. Initial respondents were mailed a follow-up survey within two weeks of each family returning the original survey. We computed several agreement statistics to measure the test-retest reliability for select questions. We also conducted continuity adjusted chi-square, and McNemar tests in both the original and follow-up samples to measure the degree to which our results could be reproduced. Analyses were stratified by self-reported income. Results: The test-retest reliability was moderate for the majority of questions (0.41 - 0.60) and the level of test-retest reliability did not differ substantially across each of the broader domains of questions. The observed proportions of respondents with delayed or foregone pediatric, adult, or any family care were similar when comparing the original and follow-up surveys. In the original survey, respondents in the lower-income group were more likely to delay or forego pediatric care, adult care, or any family care. All of the tests comparing income groups in the follow-up survey produced the same result as in the original survey. Conclusions: In this population of HDHP beneficiaries, we found that survey questions concerning plan knowledge, information seeking, and delayed or foregone care were moderately reliable. Our results offer reassurance for researchers using survey information to study the effects cost sharing on health care utilization.

Background: Spatial global clustering tests can be used to evaluate the geographical distribution of health outcomes. The power of several of these tests has been evaluated and compared using simulated data, but their performance using real unadjusted data and data adjusted for individual- and area-level covariates has not been reported previously. We evaluated data on prostate cancer histologic tumor grade and stage of disease at diagnosis for incident cases of prostate cancer reported to the Maryland Cancer Registry during 1992–1997. We analyzed unadjusted data as well as expected counts from models that were adjusted for individual- level covariates (race, age and year of diagnosis) and area-level covariates (census block group median household income and a county-level socioeconomic index). We chose 3 spatial clustering tests that are commonly used to evaluate the geographic distribution of disease: Cuzick-Edwards' k-NN (k-Nearest Neighbors) test, Moran's I and Tango's MEET (Maximized Excess Events Test). Results: For both grade and stage at diagnosis, we found that Cuzick-Edwards' k-NN and Moran's I were very sensitive to the percent of population parameter selected. For stage at diagnosis, all three tests showed that the models with individual- and area-level adjustments reduced clustering the most, but did not reduce it entirely. Conclusion: Based on this specific example, results suggest that these tests provide useful tools for evaluating spatial clustering of disease characteristics, both before and after consideration of covariates.

Background: Berry size is considered as one of the main selection criteria in table grape breeding programs. However, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. Considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. To approach this issue, an RNA-Seq experiment based on Illumina platform was performed (14 libraries), including seedless segregants with contrasting phenotypes for berry weight at fruit setting (FST) and 6-8 mm berries (B68) phenological stages., Results: A group of 526 differentially expressed (DE) genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: 101 genes from the FST stage and 463 from the B68 stage. Also, we integrated differential expression, principal components analysis (PCA), correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. After this, 68 DE genes were selected as candidate genes, and seven candidate genes were validated by real time-PCR, confirming their expression profiles., Conclusions: We have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. Our findings contributed to the understanding of the mechanisms involved in berry weight determination. Also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs.

Background: Grapevine (Vitis vinifera L.) is the most important Mediterranean fruit crop, used to produce both wine and spirits as well as table grape and raisins. Wine and table grape cultivars represent two divergent germplasm pools with different origins and domestication history, as well as differential characteristics for berry size, cluster architecture and berry chemical profile, among others. 'Sultanina' plays a pivotal role in modern table grape breeding providing the main source of seedlessness. This cultivar is also one of the most planted for fresh consumption and raisins production. Given its importance, we sequenced it and implemented a novel strategy for the de novo assembly of its highly heterozygous genome., Results: Our approach produced a draft genome of 466 Mb, recovering 82% of the genes present in the grapevine reference genome; in addition, we identified 240 novel genes. A large number of structural variants and SNPs were identified. Among them, 45 (21 SNPs and 24 INDELs) were experimentally confirmed in 'Sultanina' and six SNPs in other 23 table grape varieties. Transposable elements corresponded to ca. 80% of the repetitive sequences involved in structural variants and more than 2,000 genes were affected in their structure by these variants. Some of these genes are likely involved in embryo development, suggesting that they may contribute to seedlessness, a key trait for table grapes., Conclusions: This work produced the first structural variants and SNPs catalog for grapevine, constituting a novel and very powerful tool for genomic studies in this key fruit crop, particularly useful to support marker assisted breeding in table grapes.

Background: Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine., Results: A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses., Conclusions: Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.

Background: Stenospermocarpy is a mechanism through which certain genotypes of Vitis vinifera L. such as Sultanina produce berries with seeds reduced in size. Stenospermocarpy has not yet been characterized at the molecular level., Results: Genetic and physical maps were integrated with the public genomic sequence of Vitis vinifera L. to improve QTL analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. Major QTLs co-positioning for both traits on chromosome 18 defined a 92-kb confidence interval. Functional information from model species including Vitis suggested that VvAGL11, included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development.Characterization of VvAGL11 at the sequence level in the experimental progeny identified several SNPs and INDELs in both regulatory and coding regions. In association analyses performed over three seasons, these SNPs and INDELs explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. Moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. Transcriptional analysis lent additional support to the putative role of VvAGL11's regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. These results transform VvAGL11 into a functional candidate gene for further analyses based on genetic transformation.For breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site., Conclusion: We propose that VvAGL11 is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. Current knowledge of the function of its orthologous genes, its expression profile in Vitis varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the D-lineage MADS-box gene VvAGL11 corresponds to the Seed Development Inhibitor locus described earlier as a major locus for seedlessness. These results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development.