Methylation profiles of the sense and antisense strands of the <i>GSTP1</i> gene by BSP sequencing of DNA isolated from normal liver and diseased liver tissues.

<p>(A) Diagram of the locations of bisulfite sequencing primers and the CpG sites, indicated by vertical bars, in the promoter and the first exon regions of the <i>GSTP1</i> gene (Genbank accession #M24485, nt. 999–1387). The transcription start site (TSS) is also indicated. The CpG sites are bracketed by the bisulfite sequencing primers for the forward (F) and reverse (R) sense strands (GSTP1_S_F and GSTP1_S_R) and the antisense strands (GSTP1_AS_F and GSTP1_AS_R). (B) Methylation status of each CpG site in both sense (S) and antisense (AS) strands of the promoter and the first exon regions of the <i>GSTP1</i> gene from −28 to +4 on the basis of the sense strand 5′ to 3′ direction relative to TSS in hepatocellular carcinoma (HCC, n = 20) tissue, matched adjacent non-HCC liver tissue (Adj Non-HCC, n = 20), and normal (n = 6), hepatitis (n = 5), and cirrhosis (n = 5) tissues. The filled boxes indicate methylation detected and open boxes indicate no methylation detected. (C) Analysis of the extent of methylation at each CpG site of the sense strand <i>GSTP1</i> gene by BS-PCR sequencing of DNA isolated from normal liver and diseased liver tissues. CpG site locations, BS-PCR sequencing assay, and DNA samples are the same as in panel B. The filled boxes indicate a high level of methylation detected (more than 50%); hatched boxes indicate a low level of methylation detected (50% or less); and open boxes indicate no methylation detected.</p

Scatter plot distribution of serum AFP levels (x-axis) and the amount of methylated 5′-end of the <i>GSTP1</i> DNA (<i>mGTSP1</i>) (y-axis) for 115 HCC samples.

<p>Each circle represents the value for an individual HCC case. A vertical reference line intersects at an AFP value of 20 ng/ml. A horizontal reference line intersects right above the MSP value of 0 as the reference for undetectable (ND), which is less than 10 copies per assay. The number of HCC cases and the percent of the total HCC in each of four areas are indicated.</p

Aberrant methylation of the <i>GSTP1</i> in HCC stratified by clinicopathological characteristics.

1<p>Kruskal-Wallis test.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus.</p

Comparison of the specificity of the 5′-end and the 3-end of the <i>mGSTP1</i> as a biomarker to distinguish HCC samples from tissue samples of other liver diseases, as determined by MSP assays.

<p>(A) Locations of forward (F) and reverse (R) primers and TaqMan probe (P) of the 5′-end MSP (5′-MSP) (including the TaqMan probe) and 3′-end MSP (3′-MSP) SybrGreen assays. The CpG sites (vertical bars) and the transcription start site (TSS) are indicated. Receiver operating characteristic (ROC) curves of the methylated <i>GSTP1</i> gene as a marker to discriminate HCC (n = 120) from non-HCC liver tissues including hepatitis (n = 35) and cirrhosis (n = 35) (B), or hepatitis, cirrhosis, and adjacent non-HCC (C), generated by 5′-end MSP and 3′-end MSP assays, respectively, as indicated. The amount of methylated DNA was the average of two duplicate MSP assays as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035789#s4" target="_blank">Materials and Methods</a>. The area under the curve of each ROC (AUROC) curve and the specificity and sensitivity determined by the cutoff of 10 copies per input of 300 copies of DNA are shown in the inserted table. Note that the CpG sites included in each primer and probe are as follows; 5′-end MSP (F: −27 to −24; P: −23 to −19; R: −11 to −10) and 3′-end MSP (F: −4 to −2; R: +4 to +7).</p

Examples of the variable specificities of methylated <i>GSTP1</i> genes for HCC reported in previous studies.

1<p>The CpG site number is referred to the transcription start site.</p><p>HCC, hepatocellular carcinoma; MSP, methylation-specific PCR.</p

Comparison of the extent of CpG methylation of the GSTP1 sense strand promoter region among various normal and diseased liver tissues¹.

1<p>The extent of methylation at each CpG site was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035789#pone-0035789-g001" target="_blank">Figure 1C</a>: no methylation (no methylation detected); low methylation (50% or less); high methylation (more than 50%).</p>2<p>The CpG sites included in the 5′-end and the 3′-end are described in the text.</p>3<p>The percent of CpG methylation was calculated as the number of methylated CpG sites per category/total CpG sites analyzed×100%.</p>4<p>p value was determined using Student's <i>t</i> test.</p><p>HCC, hepatocellular carcinoma.</p

Summary of clinicopathological characteristics of the tissues analyzed using the MSP assays.

1<p>4 of the 6 normal livers are “normal” liver tissues with concomitant cholangiocarcinoma.</p>2<p>Across all subjects (n = 196), age was analyzed by the Student <i>t</i> test and gender by Fisher's exact test.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; SD, standard deviation.</p

Summary of clinicopathological characteristics of the tissues analyzed by BSP sequencing.

1<p>4/6 of the 6 normal livers are “normal” liver tissues with concomitant cholangiocarcinoma.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; SD, standard deviation.</p

A Cluster Randomized Controlled Trial of the Archena Infancia Saludable Project on 24-h Movement Behaviors and Adherence to the Mediterranean Diet among Schoolchildren: A Protocol Study

Objective: The aim of this paper is to describe the protocol of pilot cluster randomized controlled trial (RCT) that will evaluate the effects of a lifestyle-based intervention. The Archena Infancia Saludable project will have several objectives. The primary objective of this project is to determine the 6-month effects of a lifestyle-based intervention on adherence to 24-h movement behaviors and Mediterranean diet (MedDiet) in schoolchildren. The secondary objective of this project is to test the intervention effects of this lifestyle-based intervention on a relevant set of health-related outcomes (i.e., anthropometric measurements, blood pressure, perceived physical fitness, sleep habits, and academic performance). The tertiary objective is to investigate this intervention’s “halo” effect on parents’/guardians’ 24-h movement behaviors and adherence to the MedDiet. Methods: The Archena Infancia Saludable trial will be a cluster RCT submitted to the Clinical Trials Registry. The protocol will be developed according to SPIRIT guidelines for RCTs and CONSORT statement extension for cluster RCTs. A total of 153 eligible parents/guardians with schoolchildren aged 6–13 years will be randomized into an intervention group or a control group. This project focuses on two fundamental pillars: 24-h movement behaviors and MedDiet. It will mainly focus on the relationship between parents/guardians and their children. Behavior change strategies for dietary and 24-h movement behaviors in schoolchildren will be based on healthy lifestyle education for parents/guardians through infographics, video recipes, brief video clips, and videos. Conclusions: Most of the current knowledge on 24-h movement behaviors and adherence to the MedDiet is based on cross-sectional or longitudinal cohort studies, warranting a need to design and conduct RCTs to obtain more robust evidence on the effect of a healthy lifestyle program to increase 24-h movement behaviors and to improve adherence to the MedDiet in schoolchildren

A Cluster Randomized Controlled Trial of the Archena Infancia Saludable Project on Adherence to 24-h Movement Guidelines and Mediterranean Diet among Schoolchildren: A Protocol Study

Objective: The aim of this paper is to describe the protocol of pilot cluster randomized controlled trial (RCT) that will evaluate the effects of a lifestyle-based intervention. The Archena Infancia Saludable project will have several objectives. The primary objective of this project is to determine the 6-month effects of a lifestyle-based intervention on adherence to 24-h movement behaviors and Mediterranean diet (MedDiet) in schoolchildren. The secondary objective of this project is to test the intervention effects of this lifestyle-based intervention on a relevant set of health-related outcomes (i.e., anthropometric measurements, blood pressure, perceived physical fitness, sleep habits, and academic performance). The tertiary objective is to investigate this intervention’s “halo” effect on parents’/guardians’ 24-h movement behaviors and adherence to the MedDiet. Methods: The Archena Infancia Saludable trial will be a cluster RCT submitted to the Clinical Trials Registry. The protocol will be developed according to SPIRIT guidelines for RCTs and CONSORT statement extension for cluster RCTs. A total of 153 eligible parents/guardians with schoolchildren aged 6–13 years will be randomized into an intervention group or a control group. This project focuses on two fundamental pillars: 24-h movement behaviors and MedDiet. It will mainly focus on the relationship between parents/guardians and their children. Behavior change strategies for dietary and 24-h movement behaviors in schoolchildren will be based on healthy lifestyle education for parents/guardians through infographics, video recipes, brief video clips, and videos. Conclusions: Most of the current knowledge on 24-h movement behaviors and adherence to the MedDiet is based on cross-sectional or longitudinal cohort studies, warranting a need to design and conduct RCTs to obtain more robust evidence on the effect of a healthy lifestyle program to increase 24-h movement behaviors and to improve adherence to the MedDiet in schoolchildren