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1.  Leaking Underground Storage Tanks and Environmental Injustice: Is There a Hidden and Unequal Threat to Public Health in South Carolina? 
Environmental justice (Print)  2013;6(5):175-182.
There are approximately 590,000 underground storage tanks (USTs) nationwide that store petroleum or hazardous substances. Many of these tanks are leaking, which may increase the risk of exposure to contaminants that promote health problems in host neighborhoods. Within this study, we assessed disparities in the spatial distribution of leaking underground storage tanks (LUSTs) based on socioeconomic status (SES) and race/ethnicity in South Carolina (SC). Chi-square tests were used to evaluate the difference in the proportion of populations who host a LUST compared to those not hosting a LUST for all sociodemographic factors. Linear regression models were applied to examine the association of distance to the nearest LUST with relevant sociodemographic measures. As percent black increased, the distance (both in kilometers and miles) to the nearest LUST decreased. Similar results were observed for percent poverty, unemployment, persons with less than a high school education, blacks in poverty, and whites in poverty. Furthermore, chi-square tests indicated that blacks or non-whites or people with low SES were more likely to live in LUST host areas than in non-host areas. As buffer distance increased, percent black and non-white decreased. SES variables demonstrated a similar inverse relationship. Overall, burden disparities exist in the distribution of LUSTs based on race/ethnicity and SES in SC.
doi:10.1089/env.2013.0019
PMCID: PMC3980862
2.  Characterization of Constitutive Promoters for piggyBac Transposon-Mediated Stable Transgene Expression in Mesenchymal Stem Cells (MSCs) 
PLoS ONE  2014;9(4):e94397.
Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.
doi:10.1371/journal.pone.0094397
PMCID: PMC3979777  PMID: 24714676
3.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids 
PLoS ONE  2014;9(4):e93608.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
doi:10.1371/journal.pone.0093608
PMCID: PMC3973564  PMID: 24695466
4.  Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene 
PLoS ONE  2014;9(3):e92908.
Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.
doi:10.1371/journal.pone.0092908
PMCID: PMC3962475  PMID: 24658746
5.  Assessment of Particulate Matter Levels in Vulnerable Communities in North Charleston, South Carolina prior to Port Expansion 
INTRODUCTION
The Port of Charleston, one of the busiest US ports, currently operates five terminals. The fifth terminal is being planned for expansion to accommodate container ships from the proposed Panama Canal expansion. Such expansion is expected to increase traffic within local vulnerable North Charleston neck communities by at least 7,000 diesel truck trips per day, more than a 70% increase from the present average rate of 10,000 trucks per day. Our objective was to measure the current particulate matter (PM) concentrations in North Charleston communities as a baseline to contrast against future air pollution after the proposed port expansion.
METHODS
Saturation study was performed to determine spatial variability of PM in local Charleston neck communities. In addition, the temporal trends in particulate air pollution within the region were determined across several decades. With the BGI sampler, PM samples were collected for 24 hours comparable to the federal reference method protocol. Gravimetric analysis of the PM filter samples was conducted following EPA protocol.
RESULTS
The range of the PM10 annual average across the region from 1982 to 2006 was 17.0–55.0 μg/m3. On only two occasions were the records of PM10 averaged above the 50.0 μg/m3 national standard. In the case of PM2.5, the annual average for 1999–2006 ranged from 11.0 to 13.5 μg/m3 and no annual average exceeded the 15.0 μg/m3 PM2.5 annual standard.
CONCLUSIONS
Although ambient PM levels have fallen in the Charleston region since the 1960s due to aggressive monitoring by the stakeholders against air pollution, local air pollution sources within the North Charleston neck communities have consistently contributed to the PM levels in the region for several decades. This baseline assessment of ambient PM will allow for comparisons with future assessments to ascertain the impact of the increased truck and port traffic on PM concentrations.
doi:10.4137/EHI.S12814
PMCID: PMC3956811  PMID: 24653648
air pollution; air quality; urban; community; port; particulate matter; environmental justice
6.  Novel P2 tris-tetrahydrofuran group in antiviral compound 1 (GRL-0519) fills the S2 binding pocket of selected mutants of HIV-1 protease 
Journal of medicinal chemistry  2013;56(3):1074-1083.
GRL-0519 (1) is a potent antiviral inhibitor of HIV-1 protease (PR) possessing tris-tetrahydrofuran (tris-THF) at P2. The high resolution X-ray crystal structures of inhibitor 1 in complexes with single substitution mutants PRR8Q, PRD30N, PRI50V, PRI54M, and PRV82A were analyzed in relation to kinetic data. The smaller valine side chain in PRI50V eliminated hydrophobic interactions with inhibitor and the other subunit consistent with 60-fold worse inhibition. Asn30 in PRD30N showed altered interactions with neighboring residues and 18-fold worse inhibition. Mutations V82A and I54M showed compensating structural changes consistent with 6-7-fold lower inhibition. Gln8 in PRR8Q replaced the ionic interactions of wild type Arg8 with hydrogen bond interactions without changing the inhibition significantly. The carbonyl oxygen of Gly48 showed two alternative conformations in all structures likely due to the snug fit of the large tris-THF group in the S2 subsite in agreement with high antiviral efficacy of 1 on resistant virus.
doi:10.1021/jm301519z
PMCID: PMC3574189  PMID: 23298236
HIV / AIDS; aspartic protease; X-ray crystallography; drug resistance
7.  Changes in pulmonary tuberculosis prevalence: evidence from the 2010 population survey in a populous province of China 
Background
This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches.
Methods
A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray (CXRAY), or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.
Results
Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.
Conclusions
The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms.
doi:10.1186/1471-2334-14-21
PMCID: PMC3890533  PMID: 24410932
Tuberculosis; Population based prevalence survey; Case finding; China
8.  Interaction of prenatal maternal smoking, interleukin 13 genetic variants and DNA methylation influencing airflow and airway reactivity 
Clinical Epigenetics  2013;5(1):22.
Background
Asthma is characterized by airflow limitation and airway reactivity (AR). Interleukin-13 (IL-13) is involved in the pathogenesis of asthma. Two functional SNPs, rs20541 and rs1800925, of the IL-13 gene (IL13) have been frequently associated with asthma-related lung functions. However, genetic variation alone does not fully explain asthma risk. DNA-methylation (DNA-M) is an epigenetic mechanism that regulates gene expression and can be influenced by both environment and genetic variants. To explore the interplay of prenatal maternal smoking, genetic variants and DNA-M, we used a two-stage model: (1) identifying cytosine phosphate guanine (CpG) sites where DNA-M is influenced by the interaction between genetic variants and maternal smoking during pregnancy (conditional methQTL (methylation quantitative trait loci)); and (2) determining the effect of the interaction between DNA-M of CpG (from stage 1) and SNPs (modifying genetic variants; modGV) on airflow limitation and AR in 245 female participants of the Isle of Wight birth cohort. DNA-M was assessed using the Illumina Infinium HumanMethylation450 BeadChip.
Findings
Six CpG sites were analyzed in stage 1. DNA-M at cg13566430 was influenced by interaction of maternal smoking during pregnancy and rs20541. In stage 2, genotype at rs1800925 interacted with DNA-M at cg13566430 significantly affecting airflow limitation (P = 0.042) and AR (P = 0.01).
Conclusion
Both genetic variants and environment affect DNA-M. This study supports the proposed two-stage model (methQTL and modGV) to study genetic variants, environment and DNA-M interactions in asthma-related lung function.
doi:10.1186/1868-7083-5-22
PMCID: PMC3892084  PMID: 24314122
Asthma genetics and epigenetics; Airway reactivity; DNA methylation; IL13 gene; Lung functions; Maternal smoking during pregnancy
9.  Crosstalk between Wnt/β-Catenin and Estrogen Receptor Signaling Synergistically Promotes Osteogenic Differentiation of Mesenchymal Progenitor Cells 
PLoS ONE  2013;8(12):e82436.
Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.
doi:10.1371/journal.pone.0082436
PMCID: PMC3855436  PMID: 24340027
10.  Power of a reproducing kernel-based method for testing the joint effect of a set of single-nucleotide polymorphisms 
Genetica  2012;140(10-12):421-427.
This study explored a semi-parametric method built upon reproducing kernels for estimating and testing the joint effect of a set of single nucleotide polymorphisms (SNPs). The kernel adopted is the identity-by-state (IBS) kernel that measures SNP similarity between subjects. In this article, through simulations we first assessed its statistical power under different situations. It was found that in addition to the effect of sample size, the testing power was impacted by the strength of association between SNPs and the outcome of interest, and by the SNP similarity among the subjects. A quadratic relationship between SNP similarity and testing power was identified, and this relationship was further affected by sample sizes. Next we applied the method to a SNP-lung function data set to estimate and test the joint effect of a set of SNPs on forced vital capacity, one type of lung function measure. The findings were then connected to the patterns observed in simulation studies and further explored via variable importance indices of each SNP inferred from a variable selection procedure.
doi:10.1007/s10709-012-9690-5
PMCID: PMC3562424  PMID: 23180006
Reproducing kernels; SNP; Mixed linear models; Testing power; Variable selection
11.  Psychometrics of the Short Form 36 Health Survey Version 2 (SF-36v2) and the Quality of Life Scale for Drug Addicts (QOL-DAv2.0) in Chinese Mainland Patients with Methadone Maintenance Treatment 
PLoS ONE  2013;8(11):e79828.
Objective
To test psychometrics of the Short Form 36 Health Survey version 2 (SF-36v2) and the Quality of Life Scale for Drug Addicts (QOL-DAv2.0) in Chinese mainland patients with methadone maintenance treatment (MMT).
Methods
A total of 1,212 patients were recruited from two MMT clinics in Xi’an, China. Reliability was estimated with Cronbach’s α and intra-class correlation (ICC). Convergent and discriminant validity was assessed using multitrait-multimethod correlation matrix. Sensitivity was measured with ANOVA and relative efficiency. Responsiveness was evaluated by pre-post paired-samples t-test and standardized response mean based on the patients’ health status changes following 6-month period.
Results
Cronbach’s α of the SF-36v2 physical and mental summary components were 0.80 and 0.86 (eight scales range 0.73–0.92) and the QOL-DAv2.0 was 0.96 (four scales range: 0.80–0.93). ICC of the SF-36v2 two components were 0.86 and 0.85 (eight scales range: 0.72–0.87) and the QOL-DAv2.0 was 0.94 (four scales range: 0.88–0.92). Convergent validity was lower between the two instruments (γ <0.70) while discriminant validity was acceptable within each instrument. Sensitivity was satisfied in self-evaluated health status (both instruments) and average daily methadone dose (SF-36v2 physical functioning and vitality scales; QOL-DAv2.0 except psychology scale). Responsiveness was acceptable in the improved health status change (SF-36v2 except vitality scale; QOL-DAv2.0 except psychology and symptoms scales) and deteriorated health status change (SF-36v2 except vitality, social functioning and mental health scales; QOL-DAv2.0 except society scale).
Conclusions
The SF-36v2 and the QOL-DAv2.0 are valid tools and can be used independently or complementary according to different emphases of health-related quality of life evaluation in patients with MMT.
doi:10.1371/journal.pone.0079828
PMCID: PMC3835928  PMID: 24278188
12.  Epidemiologic Methods of Assessing Asthma and Wheezing Episodes in Longitudinal Studies: Measures of Change and Stability 
Journal of Epidemiology  2013;23(6):399-410.
Background
In settings in which diseases wax and wane, there is a need to measure disease dynamics in longitudinal studies. Traditional measures of disease occurrence (eg, cumulative incidence) do not address change or stability or are limited to stable cohorts (eg, incidence) and may thus lead to erroneous conclusions. To illustrate how different measures can be used to detect disease dynamics, we investigated sex differences in the occurrence of asthma and wheezing, using a population-based study cohort that covered the first 18 years of life.
Methods
In the Isle of Wight birth cohort (n = 1456), prevalence, incidence, cumulative incidence, positive and negative transitions, and remission were determined at ages 1 or 2, 4, 10, and 18 years. Latent transition analysis was used to simultaneously identify classes of asthma and wheezing (related phenotypes) and characterize transition probabilities over time. Trajectory analysis was used to characterize the natural history of asthma and wheezing.
Results
Regarding time-specific changes, positive and negative transition probabilities were more informative than other measures of associations because they revealed a sex switchover in asthma prevalence (P < 0.05). Transition probabilities were able to identify the origin of a sex-specific dynamic; in particular, prior wheezing transitioned to asthma at age 18 years among girls but not among boys. In comparison with latent transition analysis, trajectory analysis did not directly identify a switchover in prevalence among boys and girls.
Conclusions
In longitudinal analyses, transition analyses that impose minimal restrictions on data are needed in order to produce appropriate information on disease dynamics.
doi:10.2188/jea.JE20120201
PMCID: PMC3834276  PMID: 23994864
asthma; cohort study; incidence; prevalence; trajectory; transition probability
13.  Five-year Prognosis after Mild to Moderate Ischemic Stroke by Stroke Subtype: A Multi-Clinic Registry Study 
PLoS ONE  2013;8(11):e75019.
Background and Purpose
Mild to moderate ischemic stroke is a common presentation in the outpatient setting. Among the various subtypes of stroke, lacunar infarction (LI) is generally very common. Currently, little is known about the long-term prognosis and factors associated with the prognosis between LI and non-LI. This study aims to compare the risk of death and acute cardiovascular events between patients with LI and non-LI, and identify potential risk factors associated with these outcomes.
Methods
A total of 710 first-ever ischemic stroke patients (LI: 474, non-LI: 263) from 18 clinics were recruited consecutively from 2003 to 2004. They were prospectively followed-up until the end of 2008. Hazard ratios and 95% confidence intervals were calculated using multivariable Cox proportional hazards regression.
Results
After a 5-year follow up, 54 deaths and 96 acute cardiovascular events occurred. Recurrent stroke was the most common cause of death (19 cases, 35.18%) and new acute cardiovascular events (75 cases, 78.13%). There were no significant differences between patients with LI and non-LI in their risks of death, new cardiovascular events, and recurrent stroke after adjusting for age, sex, hypertension, diabetes, cardiac diseases, body mass index, dyslipidemia, smoking, alcohol consumption, ADL dependence, and depressive symptoms. Among the modifiable risk factors, diabetes, hypertension, ADL dependency, and symptoms of depression were independent predictors of poor outcomes in patients with LI. In non-LI patients, however, no modifiable risk factors were detected for poor outcomes.
Conclusion
Long-term outcomes did not differ significantly between LI and non-LI patients. Detecting and managing vascular risk factors and depression as well as functional rehabilitation may improve the prognoses of LI patients.
doi:10.1371/journal.pone.0075019
PMCID: PMC3817184  PMID: 24223696
14.  ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1 
Nucleic Acids Research  2013;41(22):10241-10253.
The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.
doi:10.1093/nar/gkt837
PMCID: PMC3905876  PMID: 24049075
15.  Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2013;8(9):e73086.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
doi:10.1371/journal.pone.0073086
PMCID: PMC3760886  PMID: 24019898
16.  Effect of GSTM2-5 polymorphisms in relation to tobacco smoke exposures on lung function growth: a birth cohort study 
Background
Genetic variation within GSTM2-5 genes may interfere with detoxification of environmental compounds, thereby having a detrimental effect on lung function following exposures such as tobacco smoke. We aim to investigate the influence of variants and associated methylation in the GSTM gene cluster with changes in lung function growth during adolescence.
Methods
Growth in forced expiratory volume (FEV1), forced vital capacity (FVC), and change in FEV1/FVC ratio measures were obtained from children in the Isle of Wight birth cohort at ages 10 and 18. Illumina GoldenGate assays were used to genotype 10 tagging polymorphisms from GSTM2 (rs574344 and rs12024479), GSTM3 (rs1537236, rs7483, and rs10735234), GSTM4 (rs668413, rs560018, and rs506008), and GSTM5 (rs929166 and rs11807) genes. Diplotypes were generated in the software Phase 3.0.2. DNA methylation was measured in over 450,000 CpG sites using the Infinium HumanMethylation450 BeadChip (Illumina 450K) in a subsample of 245 18-year olds from the Isle of Wight birth cohort. Gender, age, in utero smoke exposure, secondhand smoke exposure (SHS), and current smoking status were assessed via questionnaire; smoke exposures were validated with urine cotinine. We used linear mixed models to estimate the effect of GSTM diplotypes on lung function across time and examine interactions with tobacco smoke.
Results
1,121 (77%) out of 1,456 children had information on lung function at ages 10 or 18. After adjustment for false discovery rate, one diplotype in GSTM3 had a detrimental effect on changes in FEV1 (p=0.03), and another diplotype in GSTM3 reduced FVC (p=0.02) over time. No significant interactions with smoking were identified. SHS significantly modified the relationship between diplotypes and methylation levels in one GSTM2 CpG site; however, this site did not predict lung function outcomes at age 18. Joint effects of GSTM loci and CpG sites located within these loci on adolescent lung growth were detected.
Conclusions
Diplotypes within GSTM2-5 genes are associated with lung function growth across adolescence, but do not appear to modify the effect of tobacco smoke exposures on adolescent lung growth. Interactions between DNA methylation and diplotypes should be taken into account to gain further understanding on lung function in adolescence.
doi:10.1186/1471-2466-13-56
PMCID: PMC3846453  PMID: 24004509
Smoking; Lung function; Diplotype; Human; Longitudinal study; Epigenetics; Methylation quantitative trait loci
17.  Tyrosinase Inhibitory Effects and Antioxidative Activities of Saponins from Xanthoceras Sorbifolia Nutshell 
PLoS ONE  2013;8(8):e70090.
Certain saponins are bioactive compounds with anticancer, antivirus and antioxidant activities. This paper discussed inhibitory effects of saponins from Xanthoceras Sorbifolia on tyrosinase, through the research of the rate of tyrosinase catalyzed L-DOPA oxidation. The inhibition rate of tyrosinase activity presented non-linear changes with the saponins concentration. The rate reached 52.0% when the saponins concentration was 0.96 mg/ml. Antioxidant activities of saponins from Xanthoceras Sorbifolia were evaluated by using hydroxyl and superoxide radical scavenging assays. The hydroxyl radical scavenging effects of the saponins were 15.5–68.7%, respectively at the concentration of 0.18–2.52 mg/ml. The superoxide radical scavenging activity reduced from 96.6% to 7.05% with the time increasing at the concentration of 1.44 mg/ml. All the above antioxidant evaluation indicated that saponins from Xanthoceras Sorbifolia exhibited good antioxidant activity in a concentration- dependent manner.
doi:10.1371/journal.pone.0070090
PMCID: PMC3749133  PMID: 23990897
18.  Correction: Pyrolytic and Kinetic Characteristics of the Thermal Decomposition of Perilla frutescens Polysaccharide 
PLoS ONE  2013;8(8):10.1371/annotation/f4052f66-23a5-4736-9ad7-b984e6cfb28b.
doi:10.1371/annotation/f4052f66-23a5-4736-9ad7-b984e6cfb28b
PMCID: PMC3742800
19.  The effect of parental allergy on childhood allergic diseases depends on the sex of the child 
Background
Parent of origin effect is important in understanding the genetic basis of childhood allergic diseases and to improve our ability to identify high risk children.
Objective
To investigate parent of origin effect in childhood allergic diseases.
Methods
The Isle of Wight Birth Cohort (n=1,456) has been examined at 1, 2, 4, 10 and 18-years. Information on prevalence of asthma, eczema, rhinitis and environmental factors was obtained using validated questionnaires. Skin prick tests were carried out at ages 4, 10 and 18 year, and total IgE at 10 and 18 years. Parental history of allergic disease was assessed soon after the birth of the child when maternal IgE was also measured. Prevalence ratios (PR) and their 95% confidence intervals (CI) were estimated, applying log-linear models, adjusted for confounding variables.
Results
When stratified for sex of the child, maternal asthma was associated with asthma in girls [PR:1.91 (CI:1.34–2.72), p=0.0003], but not in boys [PR:1.29 (CI:0.85–1.96), p=0.23), while paternal asthma was associated with asthma in boys [PR:1.99 (CI:1.42–2.79), p<0.0001], but not in girls [PR: 1.03 (0.59–1.80) p=0.92). Maternal eczema increased the risk of eczema in girls [PR: 1.92 (CI: 1.37–2.68); p=0.0001] only, while paternal eczema did the same for boys (PR: 2.07 (CI:1.32–3.25); P=0.002). Similar trends were observed when the effect of maternal and paternal allergic disease was assessed for childhood atopy and when maternal total IgE was related to total IgE in children at age 10 and 18 years.
Conclusions
The current study indicates a sex dependent association of parental allergic conditions with childhood allergies; maternal allergy increasing the risk in girls and paternal allergy in boys. This has implications for childhood allergy prediction and prevention.
doi:10.1016/j.jaci.2012.03.042
PMCID: PMC3409323  PMID: 22607991
maternal; paternal; sex; cohort; parent of origin; atopy; asthma; eczema; rhinitis; allergy; IgE
20.  Interactive effect of STAT6 and IL13 gene polymorphisms on eczema status: results from a longitudinal and a cross-sectional study 
BMC Medical Genetics  2013;14:67.
Background
Eczema is a prevalent skin disease that is mainly characterized by systemic deviation of immune response and defective epidermal barrier. Th2 cytokines, such as IL-13 and transcription factor STAT6 are key elements in the inflammatory response that characterize allergic disorders, including eczema. Previous genetic association studies showed inconsistent results for the association of single nucleotide polymorphisms (SNPs) with eczema. Our aim was to investigate whether SNPs in IL13 and STAT6 genes, which share a biological pathway, have an interactive effect on eczema risk.
Methods
Data from two independent population-based studies were analyzed, namely the Isle of Wight birth cohort study (IOW; n = 1,456) and for the purpose of replication the Swansea PAPA (Poblogaeth Asthma Prifysgol Abertawe; n = 1,445) cross-sectional study. Log-binomial regressions were applied to (i) account for the interaction between IL13 (rs20541) and STAT6 (rs1059513) polymorphisms and (ii) estimate the combined effect, in terms of risk ratios (RRs), of both risk factors on the risk of eczema.
Results
Under a dominant genetic model, the interaction term [IL13 (rs20541) × STAT6 (rs1059513)] was statistically significant in both studies (IOW: adjusted Pinteraction = 0.046; PAPA: Pinteraction = 0.037). The assessment of the combined effect associated with having risk genotypes in both SNPs yielded a 1.52-fold increased risk of eczema in the IOW study (95% confidence interval (CI): 1.05 – 2.20; P = 0.028) and a 2.01-fold higher risk of eczema (95% CI: 1.29 – 3.12; P = 0.002) in the PAPA study population.
Conclusions
Our study adds to the current knowledge of genetic susceptibility by demonstrating for the first time an interactive effect between SNPs in IL13 (rs20541) and STAT6 (rs1059513) on the occurrence of eczema in two independent samples. Findings of this report further support the emerging evidence that points toward the existence of genetic effects that occur via complex networks involving gene-gene interactions (epistasis).
doi:10.1186/1471-2350-14-67
PMCID: PMC3700873  PMID: 23815671
Eczema; Gene-gene Interaction; Epistasis; STAT6; IL13; Genetic Association Study
21.  Inhibition of multiple pathogenic pathways by histone deacetylase inhibitor SAHA in a corneal alkali-burn injury model 
Molecular pharmaceutics  2012;10(1):307-318.
Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.
doi:10.1021/mp300445a
PMCID: PMC3697033  PMID: 23186311
corneal neovascularization; HDAC; HDAC inhibitor; hemangiogenesis; lymphangiogenesis; inflammation; alkali-burn injury
22.  The methylation of the LEPR/LEPROT genotype at the promoter and body regions influence concentrations of leptin in girls and BMI at age 18 years if their mother smoked during pregnancy 
To determine whether DNA methylation (DNA-M) of the leptin receptor genotype (LEPR/LEPROT) links gestational smoking and leptin serum levels and BMI later in life, we focused on female offspring, 18 years of age, from the Isle of Wight Birth Cohort (IOWBC). Leptin binds to the leptin receptor encoded by the LEPR/LEPROT genotype. Using general linear models, we tested a two-stage model. First, we investigated whether single nucleotide polymorphisms (SNPs) acting as methylation quantitative trait loci (methQTLs) depending on gestational smoking were related to differentially methylated cytosine-phosphate-guanine (CpG) sites. In stage 2, we tested whether the selected CpG sites, in interaction with other SNPs (modifiable genetic variants, modGV), are associated with serum leptin and BMI (stage 2). Children from the IOWBC were followed from birth to age 18. Information on gestational smoking was gathered upon delivery. SNPs tagging LEPR and LEPROT genes were genotyped. Data on LEPR/LEPROT DNA-M and leptin were obtained from blood samples drawn at age 18; to determine BMI, height and weight were ascertained. Blood samples were provided by 238 girls. Of the 21 CpG sites, interactions between gestational smoking and SNPs were detected for 16 CpGs. Methylation of seven of the 16 CpGs were, in interaction with modGVs, associated with leptin levels at age 18 years. Two CpGs survived a multiple testing penalty and were also associated with BMI. This two-stage model may explain why maternal smoking has a long-term effect on leptin levels and BMI in girls at age 18 years.
PMCID: PMC3709113  PMID: 23875062
LEPR; LEPROT; leptin; CpG sites; in utero smoking exposure; rs12059300; BMI
23.  Increased invasion and tumorigenicity capacity of CD44+/CD24- breast cancer MCF7 cells in vitro and in nude mice 
Background
Identification of cancer stem cells (CSCs) and their behaviors will provide insightful information for the future control of human cancers. This study investigated CD44 and CD24 cell surface markers as breast cancer CSC markers in vitro and in vivo.
Methods
Flow cytometry with CD44 and CD24 markers was used to sort breast cancer MCF7 cells for scanning electron microscopy (SEM), tumor cell invasion assay, and nude mouse xenograft assay.
Results
Flow cytometry assay using CD44 and CD24 markers sorted MCF7 cells into four subsets, i.e., CD44+/CD24-/low, CD44-/CD24+, CD44+/CD24+, and CD44-/CD24-. The SEM data showed that there were many protrusions on the surface of CD44+/CD24-/low cells. CD44+/CD24-/low cells had many microvilli and pseudopodia. The CD44+/CD24-/low cells had a higher migration and invasion abilities than that of the other three subsets of the cells. The in vivo tumor formation assay revealed that CD44+/CD24- cells had the highest tumorigenic capacity compared to the other three subsets.
Conclusion
CD44 and CD24 could be useful markers for identification of breast CSCs because CD44+/CD24-/low cells had unique surface ultrastructures and the highest tumorigenicity and invasive abilities.
doi:10.1186/1475-2867-13-62
PMCID: PMC3717045  PMID: 23799994
Breast cancer stem cells; Ultrastructure; Tumorigenicity; Invasive ability
24.  Shiftwork Duration and the Awakening Cortisol Response Among Police Officers 
Chronobiology international  2011;28(5):446-457.
Police officers are required to work irregular hours, which induces stress, fatigue, and sleep disruption, and they have higher rates of chronic disease and mortality. Cortisol is a well-known “stress hormone” produced via activation of the hypothalamic-pituitary-adrenal axis. An abnormal secretion pattern has been associated with immune system dysregulation and may serve as an early indicator of disease risk. This study examined the effects of long- and short-term shiftwork on the cortisol awakening response among officers (n = 68) in the Buffalo Cardio-Metabolic Occupational Police Stress (BCOPS) pilot study (2001–2003). The time each officer spent on day (start time: 04:00–11:59 h), afternoon (12:00–19:59 h), or night (20:00–03:59 h) shifts was summarized from 1994 to examination date to characterize long-term (mean: 14 ± 9 yrs) and short-term (3, 5, 7, or 14 days prior to participation) shiftwork exposures. The cortisol awakening response was characterized by summarizing the area under the curve (AUC) for samples collected on first awakening, and at 15-, 30-, and 45-min intervals after waking. Data were collected on a scheduled training or off day. The cortisol AUC with respect to ground (AUCG) summarized total cortisol output after waking, and the cortisol AUC with respect to increase (AUCI) characterized the waking cortisol response. Officers also completed the Center for Epidemiologic Studies Depression scale. Waking cortisol AUC values were lower among officers working short-term night or afternoon shifts than day shifts, with maximal differences occurring after 5 days of shiftwork. The duration of long-term shiftwork was not associated with the cortisol awakening response, although values were attenuated among officers with more career shift changes.
doi:10.3109/07420528.2011.573112
PMCID: PMC3655701  PMID: 21721860
Cortisol awakening response; Police; Shiftwork; Stress
25.  Association of the Period3 clock gene length polymorphism with salivary cortisol secretion among police officers 
Neuro endocrinology letters  2013;34(1):27-37.
OBJECTIVE
This study evaluated whether measures of waking or diurnal cortisol secretion, or self-reported psychological disturbances differed among police officers with a Period3 (PER3) clock gene length polymorphism.
METHODS
The cortisol awakening response was characterized via the area under the salivary cortisol curve with respect to the increase (AUCI) or total waking cortisol (AUCG). Diurnal cortisol measures included the slope of diurnal cortisol and the diurnal AUCG. Psychological disturbances were characterized using the Center for Epidemiologic Studies Depression Scale, Impact of Events Scale, and Life Events Scale.
RESULTS
Officers with a 4/5 or 5/5 genotype had higher awakening AUCG and greater diurnal cortisol AUCG levels compared to officers with the 4/4 genotype. Among those working more afternoon or night shifts, waking AUCI and AUCG were greater among officers with a 4/5 or 5/5 genotype compared to the 4/4 referents.
CONCLUSION
Cortisol secretion was modified among police officers with different PER3 VNTR clock gene variants.
PMCID: PMC3655703  PMID: 23524621
PER3 VNTR; circadian; cortisol; shiftwork; police

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