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1.  Precise Gene Modification Mediated by TALEN and Single-Stranded Oligodeoxynucleotides in Human Cells 
PLoS ONE  2014;9(4):e93575.
The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms, speeds up the process of drug screening, and raises the feasibility of using cell replacement therapy in clinics. However, the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly, high efficiency and faithful gene targeting. In this study, we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall, our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application.
doi:10.1371/journal.pone.0093575
PMCID: PMC3972112  PMID: 24691488
2.  Accurate and Economical Detection of ALK Positive Lung Adenocarcinoma with Semiquantitative Immunohistochemical Screening 
PLoS ONE  2014;9(3):e92828.
Right detection of anaplastic lymphoma kinase (ALK) gene rearrangement is pivotal to selection of patients with lung adenocarcinoma for ALK-targeted therapy. We explored the potential of combination of immunohistochemistry (IHC) screening and fluorescence in situ hybridization (FISH) as an affordable practice. We analyzed 410 unselected lung adenocarcinomas by ALK IHC (D5F3 clone) and FISH. Some equivocal cases were further analyzed by RT-PCR. The EGFR mutation was detected by pyrosequencing assay. In total 368 cases which got all IHC, FISH, EGFR mutation results were eligible for analysis. Cases were evaluated as IHC score 3+ (n = 26), score 2+ (n = 9), score 1+ (n = 51), and score 0 (n = 282), respectively. 23 of 26 IHC 3+ and 5 of 9 IHC 2+ cases were FISH positive, whereas 3 of 26 IHC 3+, 4 of 9 IHC 2+ and all 333 IHC 1+/0 cases were FISH negative. If considering FISH as the standard, the sensitivity and specificity of ALK IHC 3+/2+ as ALK positive were 100% and 97.9%, respectively. Three IHC 3+ cases reported as FISH “negative” were actually ALK positive confirmed by ALK RT-PCR or re-detected. Based on the final classify, ALK IHC 3+/2+ was 100% sensitive and 98.8% specific. However, FISH was 90.3% sensitive and 100% specific. IHC 2+ was regarded as equivocal and need to be confirmed by FISH or RT-PCR. In the 368 cases, 8.4% cases had ALK positive, 52.2% cases had EGFR mutation, and only one case had a coexisting. Manually semiquantitative ALK IHC (primary antibody D5F3 coupled with secondary DAKO Envision system) used as the initial screening combined with auxiliary FISH confirmation is a reliable, economical approach to identify ALK positive lung adenocarcinoma. The IHC can find some ALK positive cases which would be missed by FISH only.
doi:10.1371/journal.pone.0092828
PMCID: PMC3965450  PMID: 24667320
3.  The THO Ribonucleoprotein Complex Is Required for Stem Cell Homeostasis in the Adult Mouse Small Intestine 
Molecular and Cellular Biology  2013;33(17):3505-3514.
RNA processing and transport are mediated by cotranscriptionally assembled ribonucleoprotein (RNP) complexes. RNPs have been postulated to help specify coordinated gene expression, but the requirements for specific RNP complexes in mammalian development and tissue homeostasis have not been extensively evaluated. THO is an evolutionarily conserved RNP complex that links transcription with nuclear export. THO is not essential for Saccharomyces cerevisiae viability, but it is essential for early mouse embryonic development. Embryonic lethality has limited the characterization of THO requirements in adult tissues. To overcome this limitation, a mouse model has been generated that allows widespread inducible deletion of Thoc1, which encodes an essential protein subunit of THO. Widespread Thoc1 deletion disrupts homeostasis within the small intestine but does not have detectable effects in other epithelial tissues such as the related mucosa of the large intestine. Thoc1 loss compromises the proliferation and lineage-generating capacity of small intestinal stem cells, disrupting the supply of differentiated cells in this rapidly renewing tissue. These findings demonstrate that the effects of THO deficiency in the adult mouse are tissue and cell type dependent.
doi:10.1128/MCB.00751-13
PMCID: PMC3753850  PMID: 23816884
4.  Genetic and environmental influences on blood pressure variability: a study in twins 
Journal of hypertension  2013;31(4):10.1097/HJH.0b013e32835e2a4a.
Objectives
Blood pressure variability (BPV) and its reduction in response to antihypertensive treatment are predictors of clinical outcomes; however, little is known about its heritability. In this study, we examined the relative influence of genetic and environmental sources of variance of BPV and the extent to which it may depend on race or sex in young twins.
Methods
Twins were enrolled from two studies. One study included 703 white twins (308 pairs and 87 singletons) aged 18–34 years, whereas another study included 242 white twins (108 pairs and 26 singletons) and 188 black twins (79 pairs and 30 singletons) aged 12–30 years. BPV was calculated from 24-h ambulatory blood pressure recording.
Results
Twin modeling showed similar results in the separate analysis in both twin studies and in the meta-analysis. Familial aggregation was identified for SBP variability (SBPV) and DBP variability (DBPV) with genetic factors and common environmental factors together accounting for 18–40% and 23–31% of the total variance of SBPV and DBPV, respectively. Unique environmental factors were the largest contributor explaining up to 82–77% of the total variance of SBPV and DBPV. No sex or race difference in BPV variance components was observed. The results remained the same after adjustment for 24-h blood pressure levels.
Conclusions
The variance in BPV is predominantly determined by unique environment in youth and young adults, although familial aggregation due to additive genetic and/or common environment influences was also identified explaining about 25% of the variance in BPV.
doi:10.1097/HJH.0b013e32835e2a4a
PMCID: PMC3848497  PMID: 23470779
blacks; blood pressure variability; heritability; meta-analysis; twin study
5.  PROSTASIN: A POSSIBLE CANDIDATE GENE FOR HUMAN HYPERTENSION 
American journal of hypertension  2008;21(9):10.1038/ajh.2008.224.
Background
Prostasin, a serine protease, is suggested to be a novel mechanism regulating the epithelial sodium channel expressed in the distal nephron. This study aimed to evaluate whether the human prostasin gene is a novel candidate gene underlying blood pressure (BP) elevation.
Methods
In a sample of healthy African American (AA) and European American (EA) twin subjects aged 17.6±3.3 years (n=920, 45% AAs), race-specific tagging single nucleotide polymorphisms (tSNPs) were identified to tag all the available SNPs ± 2Kb up- and downstream of the prostasin gene from HapMap at r2 of 0.8 – 1.0. Selection yielded four tSNPs in AAs and one in EAs, with one tSNP (rs12597511: C to T) present in both AAs and EAs.
Results
For rs12597511, CT and TT genotypes exhibited higher systolic BP than CC genotype (115.9±1.1 mmHg vs. 113.7±0.6 mmHg, p=0.025 [AAs]; and 110.7±0.5 mmHg vs. 109.6±0.6 mmHg, p=0.115 [EAs]). CT and TT genotypes compared to CC genotype showed a significant increase in diastolic BP in both racial groups (62.5±0.7 mmHg vs. 60.4±0.4 mmHg, p=0.003 [AAs]; and 58.2±0.3 mmHg vs. 56.7±0.4 mmHg, p=0.007 [EAs]). Furthermore, there was an increase in radial pulse wave velocity (PWV) in subjects with CT and TT genotype as compared to those with CC genotype (6.5±0.1 vs. 6.1±0.1 m/s, p<0.0001) [EAs]; and 6.7±0.1 vs. 6.6±0.1 m/s, p=0.354 [AAs]). Analyses combining AAs and EAs consistently demonstrated a statistical significance of rs1259751 on all the phenotypes including systolic/diastolic BP, and PWV.
Conclusion
Genetic variation of the prostasin gene may be implicated in the development of hypertension in youths.
doi:10.1038/ajh.2008.224
PMCID: PMC3812941  PMID: 18583984
Prostasin; ENaC; Polymorphisms; Blood Pressure; Arterial stiffness
6.  Neural Encoding of Acupuncture Needling Sensations: Evidence from a fMRI Study 
Deqi response, a psychophysical response characterized by a spectrum of different needling sensations, is essential for Chinese acupuncture clinical efficacy. Previous neuroimaging research works have investigated the neural correlates of an overall deqi response by summating the scores of different needling sensations. However, the roles of individual sensations in brain activity and how they interact with each other remain to be clarified. In this study, we applied fMRI to investigate the neural correlates of individual components of deqi during acupuncture on the right LV3 (Taichong) acupoint. We selected a subset of deqi responses, namely, pressure, heaviness, fullness, numbness, and tingling. Using the individual components of deqi of different subjects as covariates in the analysis of percentage change of bold signal, pressure was found to be a striking sensation, contributing to most of negative activation of a limbic-paralimbic-neocortical network (LPNN). The similar or opposite neural activity in the heavily overlapping regions is found to be responding to different needling sensations, including bilateral LPNN, right orbitofrontal cortex, and bilateral posterior parietal cortex. These findings provide the neuroimaging evidence of how the individual needle sensations interact in the brain, showing that the modulatory effects of different needling sensations contribute to acupuncture modulations of LPNN network.
doi:10.1155/2013/483105
PMCID: PMC3766991  PMID: 24062782
7.  Genetic influences on heart rate variability at rest and during stress 
Psychophysiology  2009;46(3):458-465.
We tested whether the heritability of heart rate variability (HRV) under stress is different from rest and its dependency on ethnicity or gender. HRV indexed by root mean square of successive differences (RMSSD) and high-frequency (HF) power was measured at rest and during 3 stressors in 427 European and 308 African American twins. No ethnic or gender differences were found for any measures. There was a nonsignificant increase in heritability of RMSSD (from 0.48 to 0.58) and HF (from 0.50 to 0.58) under stress. Up to 81% and 60% of the heritabilities of RMSSD and HF under stress could be attributed to genes influencing rest levels. The heritabilities due to genes expressed under stress were 0.11 for RMSSD and 0.23 for HF. The findings suggest that, independent of ethnicity and gender, HRV regulation at rest and under stress is largely influenced by the same genes with a small but significant contribution of stress-specific genetic effects.
doi:10.1111/j.1469-8986.2009.00793.x
PMCID: PMC3713464  PMID: 19226307
Heart rate variability; Stress; Ethnicity; Twin study
8.  Genetic influence on blood pressure measured in the office, under laboratory stress and during real life 
To determine to what extent the genetic influences on blood pressure (BP) measured in the office, under psychologically stressful conditions in the laboratory and during real life are different from each other. Office BP, BP during a video game challenge and a social stressor interview, and 24-h ambulatory BP were measured in 238 European American and 186 African American twins. BP values across the two tasks were averaged to represent stress levels. Genetic model fitting showed no ethnic or gender differences for any of the measures. The model fitting resulted in heritability estimates of 63, 75 and 71% for office, stress and 24-h systolic BP (SBP) and 59, 67 and 69% for diastolic BP (DBP), respectively. Up to 81% of the heritability of office SBP and 71% of office DBP were attributed to genes that also influenced stress BP. However, only 45% of the heritability of 24-h SBP and 49% of 24-h DBP were attributed to genes that also influence office BP. Similarly, about 39% of the heritability of 24-h SBP and 42% of 24-h DBP were attributed to genes that also influence stress BP. Substantial overlap exists between genes that influence BP measured in the office, under laboratory stress and during real life. However, significant genetic components specific to each BP measurement also exist. These findings suggest that partly different genes or sets of genes contribute to BP regulation in different conditions.
doi:10.1038/hr.2010.218
PMCID: PMC3713474  PMID: 21068740
African American; ambulatory blood pressure; heritability; stress; twin
9.  A 15-year longitudinal study on ambulatory blood pressure tracking from childhood to early adulthood 
This study evaluates the tracking stability of office blood pressure (BP), ambulatory BP (ABP), BP variability (BPV) and nocturnal BP drops (dipping) from childhood to early adulthood, and their dependence on ethnicity, gender and family history (FH) of essential hypertension (EH). Generalized estimating equations (GEEs) were used to estimate tracking coefficients for 295 European Americans and 252 African Americans, with a maximum of 12 measurements over a 15-year period. Office BP and ABP had moderate-to-relatively high tracking coefficients (r= 0.30–0.59; P≤0.001). Twenty-four hour readings tracked better than office readings for diastolic BP (DBP; 0.57 vs. 0.46, P=1.72×10−6) and pulse pressure (PP) (0.59 vs. 0.51, P=2.70×10−4), and equally well for systolic BP (SBP; 0.55 vs. 0.54, P=0.805). Daytime readings tracked better than their night-time counterparts for SBP (0.50 vs. 0.37, P=7.62×10−13), DBP (0.49 vs. 0.30, P=7.98×10−32) and PP (0.55 vs. 0.50, P=0.0061). All BPV (r=0.08–0.28; P≤0.001) and dipping measures (r=0.07–0.12; odds ratio, 1.60–1.73; P≤0.001) had low tracking coefficients. Males had significantly higher tracking stability for office SBP, DBP and ambulatory PP than females (P<0.01). Subjects with a positive FH of EH had significantly higher tracking stability for daytime and night-time DBP and dipping indexed by continuous variables than those with a negative FH (P<0.001). No significant ethnic differences were observed. The high tracking stability of 24-h ABP highlights the importance of using ambulatory BP monitoring in both research and clinical settings.
doi:10.1038/hr.2009.32
PMCID: PMC3713484  PMID: 19325561
ambulatory blood pressure; blood pressure variability; dipping; longitudinal study; tracking
10.  Genetic influences on daytime and night-time blood pressure: similarities and differences 
Journal of hypertension  2009;27(12):2358-2364.
Objective
To examine whether the genetic influences on blood pressure (BP) during night-time are different from those during daytime and the extent to which they depend on ethnicity or sex.
Methods
Ambulatory BP was measured in 240 European–American and 190 African–American twins (mean ± SD age, 17.2 ± 3.4). Individuals with night-time BP falls more than 10% of the daytime values were defined as dippers. A bivariate analysis of the daytime and the night-time BP levels, as well as a liability-threshold model of dippers vs. nondippers were used.
Results
Bivariate model fitting showed no ethnic or sex differences for any of the measures, with heritabilities of 0.70 and 0.68 for SBP and 0.70 and 0.64 for DBP at daytime and at night-time. The genetic influences on daytime and night-time were not significantly different for SBP or DBP. The bivariate analysis also indicated that about 56 and 33% of the heritabilities of night-time SBP and DBP could be attributed to genes that also influenced daytime levels. The specific heritabilities due to genetic effects only influencing night-time values were 0.30 for SBP and 0.43 for DBP. The heritabilities of systolic and diastolic dipping were 0.59 and 0.81, respectively.
Conclusion
Independent of ethnicity and sex, an overlap exists between genes that influence daytime and night-time BP, as well as a significant genetic component that is specific to the night-time BP. These findings suggest that different genes or sets of genes contribute to BP regulation at daytime and night-time.
doi:10.1097/HJH.0b013e328330e84d
PMCID: PMC3713486  PMID: 19657283
ambulatory blood pressure monitoring; blacks; dipping; heritability; twin study
11.  A longitudinal study of blood pressure variability in African–American and European American youth 
Journal of hypertension  2010;28(4):715-722.
Objectives
High blood pressure variability is increasingly used as a predictor of target-organ damage and cardiovascular events. However, little is known about blood pressure variability changes with age and its possible sociodemographic, anthropometric, and genetic moderators.
Methods
Twenty-four-hour ambulatory blood pressure was measured up to 12 times over a 15-year period in 344 European Americans and 297 African–Americans with an average age of 14 years at the initial visit. Blood pressure variability was indexed by the weighted 24-h standard deviation of ambulatory blood pressure recordings.
Results
Both systolic and diastolic blood pressure variability increased with age and ambulatory blood pressure mean values. Men had higher levels of blood pressure variability (P<0.001) and showed steeper linear increase rates with age than women. African–Americans showed higher values of blood pressure variability (P<0.05) than European Americans. Body mass index and waist circumference were also associated with higher blood pressure variability levels (P< 0.001). Individuals with higher father’s education level showed lower blood pressure variability. In the full model which included all the above factors, ethnic difference in systolic blood pressure variability was no longer significant.
Conclusion
The results of the present study suggest that men and African–Americans have higher blood pressure variability than women and European Americans. Apart from these ethnicity and sex effects, blood pressure variability increases with increases in age (especially in men), ambulatory blood pressure mean values and adiposity as well as decreased socioeconomic status
doi:10.1097/HJH.0b013e328336ed5b
PMCID: PMC3713487  PMID: 20075746
blood pressure variability; ethnicity; longitudinal study; sex; youth
12.  Deqi Sensations of Transcutaneous Electrical Nerve Stimulation on Auricular Points 
Deqi sensation, a psychophysical response characterized by a spectrum of different needling sensations, is essential for Chinese acupuncture clinical efficacy. Previous research works have investigated the component of Deqi response upon acupuncture on acupoints on the trunk and limbs. However, the characteristics of Deqi sensations of transcutaneous electrical nerve stimulation (TENS) on auricular points are seldom reported. In this study, we investigated the individual components of Deqi during TENS on auricular concha area and the superior scapha using quantitative measurements in the healthy subjects and depression patients. The most striking characteristics of Deqi sensations upon TENS on auricular points were tingling, numbness, and fullness. The frequencies of pressure, warmness, heaviness, and soreness were relatively lower. The dull pain and coolness are rare. The characteristics of Deqi were similar for the TENS on concha and on the superior scapha.
doi:10.1155/2013/371543
PMCID: PMC3722844  PMID: 23935663
13.  TGFβ1 and HGF protein secretion by esophageal squamous epithelial cells and stromal fibroblasts in oesophageal carcinogenesis 
Oncology Letters  2013;6(2):401-406.
Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) affect tumorigenesis by creating an environment primed for growth and invasion through the secretion of factors, including hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1). In the present study, the levels of α-smooth muscle actin (α-SMA), TGFβ1 and HGF were determined immunohistochemically in oesophageal precancerous lesions (low- and high-grade intraepithelial neoplasia; LGIEN and HGIEN, respectively), carcinoma in situ (CIS) and squamous cell carcinoma (SCC). Immunoreactivity was observed in the cytoplasm of oesophageal epithelial cells and stromal fibroblasts. Expression levels of α-SMA, TGFβ1 and HGF increased significantly in the following order: normal, LGIEN, HGIEN, CIS and SCC. In addition, linear correlations between the expression of α-SMA, TGFβ1 and HGF and different lesions were observed. Microvessel density (MVD) was measured in all specimens and increased gradually in the normal, LGIEN, HGIEN, CIS and SCC specimens, successively. A linear correlation between MVD and pathological grade was also observed and the MVD in α-SMA-, HGF- and TGFβ1-positive groups was higher when compared with that of their negative counterparts. The results of the present study indicated that the frequent overexpression of TGFβ1 and HGF proteins, secreted by oesophageal epithelium and stromal fibroblasts, promoted the progression of oesophageal precancerous lesions via the proliferation of epithelial cells and angiogenesis, through the upregulation of vascular endothelial growth factor (VEGF) expression.
doi:10.3892/ol.2013.1409
PMCID: PMC3789106  PMID: 24137336
esophageal squamous cell carcinoma; fibroblast; transforming growth factor-β1; α-smooth muscle actin; hepatocyte growth factor
14.  Heritability of Arterial Stiffness in Black- and White American Youth and Young Adults 
American journal of hypertension  2007;20(10):1065-1072.
Background
To examine the heritability of arterial stiffness measured as pulse wave velocity (PWV) and its dependence on ethnicity, gender and blood pressure (BP).
Methods
As part of the Georgia Cardiovascular Twin Study, we measured aortoradial (radial) and aorto-dorsalis-pedis (foot) PWV in 702 twins (41% black, 49% male) aged 12 – 30 years (mean: 17.7 ± 3.3) including monozygotic and dizygotic pairs of same- as well as opposite-gender. Ethnicity and gender effects on genetic and environmental contributions to PWV were estimated by genetic model fitting.
Results
Diastolic BP was the most important hemodynamic predictor. Best fitting models showed no ethnicity or gender differences in estimates of genetic and environmental influence and indicated substantial heritabilities of 0.43 (95% CI: 0.30–0.54) and 0.53 (95% CI: 0.42–0.62) for radial and foot PWV, respectively. Over a quarter of these heritabilities (0.19 for radial PWV, 0.14 for foot PWV) were due to genes in common with DBP as based on multivariate models.
Conclusions
Individual differences in arterial stiffness of youth and young adults are substantially heritable and more than 25% of this heritability is explained by genes that also influence DBP. Heritability estimates do not show any differences between blacks and whites or males and females.
doi:10.1016/j.amjhyper.2007.05.013
PMCID: PMC3672691  PMID: 17903689
Arterial Stiffness; Heritability; Twins; ethnicity
15.  A genome-wide methylation study on obesity 
Epigenetics  2013;8(5):522-533.
Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14–20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.
doi:10.4161/epi.24506
PMCID: PMC3741222  PMID: 23644594
African-Americans; epigenome-wide association study (EWAS); genome-wide association study (GWAS); methylation variation; obesity
16.  Role for Dpy-30 in ES Cell-Fate Specification by Regulation of H3K4 Methylation within Bivalent Domains 
Cell  2011;144(4):513-525.
Summary
Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions.
doi:10.1016/j.cell.2011.01.020
PMCID: PMC3572774  PMID: 21335234
17.  A Genome-Wide Methylation Study on Essential Hypertension in Young African American Males 
PLoS ONE  2013;8(1):e53938.
Objective
There is emerging evidence from animal studies suggesting a key role for methylation in the pathogenesis of essential hypertension. However, to date, very few studies have investigated the role of methylation in the development of human hypertension, and none has taken a genome-wide approach. Based on the recent studies that highlight the involvement of inflammation in the development of hypertension, we hypothesize that changes in DNA methylation of leukocytes are involved in the pathogenesis of hypertension.
Method & Results
We conducted a genome-wide methylation analysis on 8 hypertensive cases and 8 normotensive age-matched controls aged 14–23 years and performed validation of the most significant CpG sites in 2 genes in an independent sample of 36 hypertensive cases and 60 normotensive controls aged 14–30 years. Validation of the CpG sites in the SULF1 gene was further conducted in a second replication sample of 36 hypertensive cases and 34 controls aged 15.8–40 years. A CpG site in the SULF1 gene showed higher methylation levels in cases than in healthy controls in the genome-wide step (p = 6.2×10−5), which was confirmed in the validation step (p = 0.011) for subjects ≤30 years old but was not significant for subjects of all ages combined (p = 0.095).
Conclusion
The identification of a difference in a blood leukocyte DNA methylation site between hypertensive cases and normotensive controls suggests that changes in DNA methylation may play an important role in the pathogenesis of hypertension. The age dependency of the effect further suggests complexity of epigenetic regulation in this age-related disease.
doi:10.1371/journal.pone.0053938
PMCID: PMC3542324  PMID: 23325143
18.  XIAP expression is associated with pancreatic carcinoma outcome 
Molecular and Clinical Oncology  2013;1(2):305-308.
Pancreatic carcinoma is an aggressive tumor that is resistant to treatment regimens. Although the X-linked inhibitor of apoptosis protein (XIAP) overexpression in pancreatic carcinoma cells has been previously reported, the aim of this study was to investigate the association of XIAP expression with pancreatic carcinoma outcome. Specimens were obtained from 54 patients who underwent resection for pancreatic cancer. Kaplan-Meier survival analysis was used to estimate post-operative survival. The results of this analysis revealed a significantly shorter overall survival time in patients with a high compared to those with a low XIAP expression (P=0.041). Results of the multivariate analysis revealed that XIAP expression was identified as an independent predictor for pancreatic carcinoma outcome [relative risk, 1.771; 95% confidence interval (CI), 1.099–2.852; P=0.019]. Moreover, XIAP levels were associated with pancreatic carcinoma characteristics, including tumor invasion status and histological grade. XIAP overexpression shortens the survival of pancreatic cancer patients probably by modifying their resistance to apoptosis and the proliferation capacity of pancreatic carcinoma cells. Thus, XIAP expression analysis can help in the identification of patient subgroups at high risk of a poor disease outcome.
doi:10.3892/mco.2013.58
PMCID: PMC3915639  PMID: 24649165
X-linked inhibitor of apoptosis protein; outcome; pancreatic carcinoma; apoptosis
19.  Characterization of De Qi with Electroacupuncture at Acupoints with Different Properties 
Abstract
Objectives
This study sought to explore the characteristics of de qi using electroacupuncture at acupoints with different properties in the meridian category, histological type, and nerve innervations.
Methods
Electroacupuncture was performed on 21 healthy volunteers at paired acupoints of ST36-GB34, CV4-CV12, ST36-ST28, PC6-PC7, and ST36-CV4. Upon acupuncture de qi, the intensities and the prevalence of individual sensations, sensation transmission, and the amplitude of electrical current were recorded. Chi-square, Fischer's exact test, Wilcoxon test, and two-sample paired t test were used to compare the components of de qi within each paired group.
Results
Overall intensities and prevalence of individual sensations are fullness, numbness, soreness, tingling, heaviness, pressure, dull pain, warmness, and coolness in decreasing order. No significant difference was found in the prevalence of needling sensations between the two paired points (p>0.05). However, significant intensity differences (p<0.05) were showed in soreness, fullness, and heaviness between ST36-ST28, in fullness and numbness between ST36-CV4, and in fullness between CV4-CV12. Tingling sensation was stronger than heaviness and pressure at acupoints PC6-PC7 and ST36 (as paired with CV4). Sharp pain occurred in 10/216 tests (4.63%). Sensation transmission occurred highly (77.78%) on participants, and sensations mostly propagate over one joint but no further than two joints. Overall electrical current was 2.35±0.07 mA (mean±standard error). No significant difference was found between paired acupoints for the distance of sensation transmission and the amplitude of electrical current.
Conclusions
Fullness, numbness, and soreness were the most common and obvious sensations associated with electroacupuncture. The difference of sensation intensities may be associated with different nerve innervations.
doi:10.1089/acm.2010.0652
PMCID: PMC3221509  PMID: 21999154
20.  GENOME-WIDE ARRAY-BASED COPY NUMBER PROFILING IN HUMAN PLACENTAS FROM UNEXPLAINED STILLBIRTHS 
Prenatal diagnosis  2011;31(10):932-944.
Introduction
Accumulating evidence suggests that genomic structural variations, particularly copy number variations (CNV), are a common occurrence in humans that may bear phenotypic consequences for living individuals possessing the variant. While precise estimates vary, large-scale karyotypic abnormalities are present in 6–12% of stillbirths (SB). However, due to inherent limitations of conventional cytogenetics, the contribution of genomic aberrations to stillbirth is likely underrepresented. High-resolution copy number variant (CNV) analysis by genomic array-based profiling may overcome such limitations.
Methods
Prospectively acquired SB cases >22 weeks underwent classification of "unexplained" stillbirth by Wigglesworth and Aberdeen criteria after extensive testing and rigorous multidisciplinary audit. Genome-wide analysis was conducted using high resolution Illumina SNP arrays (HumanCNV370 Duo) on placental and fetal samples. Potential alternate detection methods were completed by one or more of three independent means (quantitative PCR, Illumina1M or Agilent105K CGH arrays).
Results
In our cohort of 54 stillbirths, 29 met strict unexplained criteria. Among these, we identified 24 putative novel CNVs. Subsequent interrogation detected18 of 24 CNVs (75%) in placental samples, 8 of which were also confirmed in available fetal samples; none were present in maternal blood.
Conclusion
We describe the potential of whole-genome placental profiling to identify small genomic imbalances which might contribute to a small proportion of well-characterized, unexplained stillbirths.
doi:10.1002/pd.2817
PMCID: PMC3183137  PMID: 21732394
Stillbirth; copy number variants; deletion syndromes; duplication syndromes; fetal death
21.  White Matter Abnormalities in Major Depression: A Tract-Based Spatial Statistics and Rumination Study 
PLoS ONE  2012;7(5):e37561.
Increasing evidence indicates that major depressive disorder (MDD) is usually accompanied by altered white matter in the prefrontal cortex, the parietal lobe and the limbic system. As a behavioral abnormity of MDD, rumination has been believed to be a substantial indicator of the mental state of the depressive state. So far, however, no report that we are aware of has evaluated the relationship between white matter alterations and the ruminative state. In this study, we first explored the altered white matter using a tract-based spatial statistics (TBSS) method based on diffusion tensor imaging of 19 healthy and 16 depressive subjects. We then investigated correlations between the altered white matter microstructure in the identified altered regions and the severity of ruminations measured by the ruminative response scale. Our results demonstrated altered white matter microstructure in circuits connecting the prefrontal lobe, the parietal lobe and the limbic system (p<0.005, uncorrected), findings which support previous research. More importantly, the result also indicated that a greater alteration in the white matter is associated with a more ruminative state (p<0.05, Bonferroni corrected). The detected abnormalities in the white matter should be interpreted cautiously because of the small sample size in this study. This finding supports the psychometric significance of white matter deficits in MDD.
doi:10.1371/journal.pone.0037561
PMCID: PMC3364284  PMID: 22666366
22.  Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation 
Biochemical Journal  2012;443(Pt 3):821-828.
RNAa (RNA activation) is a mechanism by which small dsRNA (double-stranded RNA), termed saRNA (small activating RNA), target promoter sequences to induce gene expression. This technique represents a novel approach to gene overexpression without the use of exogenous DNA. In the present study, we investigated whether RNAa can modulate expression of the development-related gene NANOG and manipulate cell fate. Using a lentivirus-based reporter system as a screening tool, we identified synthetic saRNAs that stimulate NANOG expression in human NCCIT embryonic carcinoma cells. Mismatch mutations to saRNA duplexes define sequence requirement for gene activation. Functional analysis of NANOG induction reveals saRNA treatment predictably modulates the expression of several known downstream target genes, including FOXH1 (forkhead box H1), REST (RE1-silencing transcription factor), OCT4 (octamer-binding protein 4) and REX1 (reduced expression protein 1). Treatment with RA (retinoic acid) triggers NCCIT cell differentiation, reducing NANOG and OCT4 expression and up-regulating several neural markers [i.e. ASCL1 (achaete-scute complex homologue 1), NEUROD1 (neuronal differentiation 1) and PAX6 (paired box 6)]. However, co-treatment with saRNA antagonizes NANOG down-regulation and RA-induced differentiation. Ectopic overexpression of NANOG via lentiviral transduction further recapitulates saRNA results, providing proof-of-concept that RNAa may be utilized to activate development-related genes and manipulate cell fate.
doi:10.1042/BJ20111491
PMCID: PMC3327998  PMID: 22339500
cell fate; differentiation; induced pluripotent stem cell (iPS cell); NANOG; RNA activation (RNAa); ASCL1, achaete-scute complex homologue 1; dsRNA, double-stranded RNA; ES, embryonic stem; FOXH1, forkhead box H1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HEK, human embryonic kidney; Hsp70, heat-shock protein 70; iPS, induced pluripotent stem; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NEUROD1, neuronal differentiation 1; OCT4, octamer-binding protein 4; PAX6, paired box 6; RA, retinoic acid; REST, RE1-silencing transcription factor; REX1, reduced expression protein 1; RNAa, RNA activation; RT, reverse transcription; saRNA, small activating RNA; SSEA, stage-specific embryonic antigen; TSS, transcription start site
23.  Deletion of Porcn in Mice Leads to Multiple Developmental Defects and Models Human Focal Dermal Hypoplasia (Goltz Syndrome) 
PLoS ONE  2012;7(3):e32331.
Background
Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.
Results
We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.
Conclusions
These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.
doi:10.1371/journal.pone.0032331
PMCID: PMC3295752  PMID: 22412863
24.  Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression 
Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease.
doi:10.1038/mtna.2012.5
PMCID: PMC3381590  PMID: 23343884
CDKN1A; delivery; gene activation; gene therapy; lipid nanoparticles; prostate cancer; saRNA; siRNA
25.  Leukocyte Telomere Length in Healthy White and Black Adolescents: Relations to Race, Sex, Adiposity, Adipokines and Physical Activity 
The Journal of pediatrics  2010;158(2):215-220.
Objectives
To examine the relations of race, sex, adiposity, adipokines and physical activity to telomere length in adolescents.
Study design
Leukocyte telomere length (T/S ratio) was assessed cross-sectionally in 667 adolescents (aged 14–18 years, 48% blacks, 51% girls) using a quantitative PCR method. Generalized Estimating Equations analyses were performed.
Results
Black adolescents had longer telomeres than white adolescents (age and sex adjusted T/S ratio ± SE: 1.32 ± 0.01 vs. 1.27 ± 0.01, p=0.014) and girls had longer telomeres than boys (age and race adjusted T/S ratio ± SE: 1.31 ± 0.01 vs. 1.27 ± 0.01, p=0.007). None of the adiposity or adipokine measures explained a significant proportion of the variance in telomere length. Vigorous physical activity was positively associated with telomere length (adjusted R2=0.019, p=0.009) and accounted for 1.9% of the total variance only in girls.
Conclusion
This study, conducted in a biracial adolescent cohort, demonstrated that: (1) race and sex differences in telomere length have already emerged during adolescence; (2) adiposity and adipokines are not associated with telomere length at this age; and (3) the anti-aging effect of vigorous physical activity may begin in youth especially in girls.
doi:10.1016/j.jpeds.2010.08.007
PMCID: PMC3010324  PMID: 20855079
Telomere length; race; sex; adiposity; adipokines; physical activity; adolescents

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