Supplemental Material - Parental Stress Scale: Validation study with a Portuguese population of parents of children from 3 to 10 years old

<p>Supplemental Material for Parental Stress Scale: Validation study with a Portuguese population of parents of children from 3 to 10 years old by Susana Algarvio, Isabel Leal and João Maroco in Journal of Child Health Care</p

McrBC based methylation analysis.

<p>Leaves of Pokkali and IR29 rice varieties in control and salt stress conditions (24h of 200 mM NaCl) were used. (A) Transposable elements (TEs) and repetitive sequences (B) Genes involved in rice responses to salt stress and constitutively expressed genes. The position of primers for all selected TE elements and stress related genes is indicated.</p

Global DNA methylation levels in salt tolerant and sensitive rice varieties.

<p>Genomic DNA from leaves and roots of ‘IR29’, ‘Nipponbare’, ‘Pokkali’ and ‘FL478’ in control and salt stress conditions (1h and 24h of 200 mM NaCl) were used to determine the relative global DNA methylation using a commercial ELISA-based kit. The methylation values represented in the plots correspond to percent methylation of the samples relative to a methylated control DNA supplied with the kit. (A) In leaves, there were statistically significant differences between control and 24h of salt stress in the Nipponbare, Pokkali and FL478 varieties (F(2,24) = 6.68: p = 0.005; F(2,24) = 6.39: p = 0.006; F(2,24) = 4.993: p = 0.015, respectively). (B) For the methylation % in the roots of Nipponbare at 24h of salt stress imposition, there was a significant effect of the treatment on the roots percentage methylation (F(2,24) = 3.601; p = 0.043), although the Tukey’s HSD test only revealed marginally significant differences between the control and 24h treatments (p = 0.06). In ‘FL478’ the methylation % was higher in 24h of salt stress than in control (F(2,24) = 10.946; p<0.001). Statistical significance was assumed for p<0.05. The “p” value was calculated according to the Tukey HSD. N.S: Not Statistically Significant.</p

Phenotypic evaluation of epigenetic rice mutants under salt stress.

<p>The assays “1” and “2” (A and B, respectively) refer to the application of salt stress at distinct developmental stages for different time periods. The length and biomass values correspond to a % of change in salt relative to control. For details, see description in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124060#sec002" target="_blank">material and methods</a>. (C) Shoot and root length of WT, <i>osdrm2</i> and <i>oshac704</i> in salt stress relative to control conditions. (D) Percentage of water content in shoots and roots of WT, <i>osdrm2</i> and <i>oshac704</i> in salt stress relative to control conditions. (E) Biomass of shoots and roots of WT, <i>osdrm2</i> and <i>oshac704</i> in salt stress relative to control conditions.</p

DNA demethylases (<i>DNG701</i> and <i>DNG710</i>) and DNA methyltransferase (<i>OsDRM2</i>) expression studies.

<p>Quantitative real-time PCR reactions were performed with cDNA prepared from total RNA extracted from leaves and roots of 14-days-old seedlings of ‘Pokkali’ (A-C) and ‘IR29’ (D-F) subjected to salinity for 1h or 24h (200 mM NaCl). The data were normalized to the internal control Ubiquitin-conjugating Enzyme E2 (<i>OsUBC2)</i>. The mean expression value of control was normalized to 1 and the other means values represent fold change in expression. Error bars represent standard deviation.</p

Global DNA methylation levels in rice mutants.

<p>Leaves of the <i>osdrm2</i> and <i>oshac704</i> rice mutants and WT (Dongjin) in control or salt stress conditions (24h of 200 mM NaCl) were used. The methylation values represented in the plots correspond to percent methylation of the samples relative to a methylated control DNA supplied with the kit. There were statistically significant differences between varieties (F(2, 18) = 6.628; p = 0.007) as well as treatment (F(1,8) = 14.307; p = 0.001). The control samples always had higher % of methylation than the salt stress ones. The <i>oshac704</i> mutant had the lowest methylation percentage (p<0.05).</p

3D imaging of DNA methylation in single interphase nuclei.

<p>Immunofluorescence with a specific antibody against 5-methylcytosine was performed in rice root sections of ‘Pokkali’ (A-F) and ‘IR29’ (G-L) 14-days-old seedlings in control conditions or after 24h of exposure to salinity stress (200 mM NaCl). Bar = 10 μM. (M) Rice root-tip section obtained with a vibratome, and stained with DAPI. (N) Fluorescence intensity was calculated using Image J. The values obtained are shown as arbitrary units (AU).</p

Factor structure of the original model CASQ.

<p>Factor structure of the original model (CFA: λ = .13–.81; χ²/df = 18.002, CFI = .761, GFI = .846 RMSEA = .122, AIC = 1,020.112, BCC = 1,020.666, BIC = 1,141.043, σ² = 0,53) and the refined model fitted to the Brazilian sample of patients with cancer (CFA: λ = .34–.70; χ²/df = 8.532, CFI = .936, GFI = .954, RMSEA = .081, AIC = 312.505, BCC = 312.973, BIC = 433.436, σ² = 0,64).</p

Sociodemographic and Clinical Characterization of the study Participants.

<p>Sociodemographic and Clinical Characterization of the study Participants.</p

Summary Measures and Content Validity Ratio (CVR) for the Items of the Portuguese Version of the Appetite and Symptoms Questionnaire for Cancer Patients (CASQ).

<p>Summary Measures and Content Validity Ratio (CVR) for the Items of the Portuguese Version of the Appetite and Symptoms Questionnaire for Cancer Patients (CASQ).</p