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2.  Evaluating the Role of Macrocycles in the Susceptibility of Hepatitis C Virus NS3/4A Protease Inhibitors to Drug Resistance 
ACS chemical biology  2013;8(7):1469-1478.
The hepatitis C virus (HCV) infects an estimated 150 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. The available antiviral therapies, which include pegylated-interferon, ribavirin and one of the HCV NS3/4A protease inhibitors telaprevir or boceprevir, are ineffective for some patients and cause severe side effects. More potent NS3/4A protease inhibitors are in clinical development, but the long-term effectiveness of these drugs is challenged by the development of drug resistance. Here, we investigated the role of macrocycles in the susceptibility of NS3/4A protease inhibitors to drug resistance in asunaprevir, danoprevir, vaniprevir, and MK-5172, with similar core structures but varied P2 moieties and macrocyclizations. Linear and macrocyclic analogues of these drugs were designed, synthesized and tested against wild-type and drug-resistant variants R155K, V36M/R155K, A156T, and D168A in enzymatic and antiviral assays. Macrocyclic inhibitors were generally more potent, but the location of the macrocycle was critical for retaining activity against drug-resistant variants – the P1–P3 macrocyclic inhibitors were less susceptible to drug resistance than the linear and P2–P4 macrocyclic analogues. In addition, the heterocyclic moiety at P2 largely determined the inhibitor resistance profile, susceptibility to drug resistance, and the extent of modulation by the helicase domain. Our findings suggest that to design robust inhibitors that retain potency to drug resistant NS3/4A protease variants, inhibitors should combine P1–P3 macrocycles with flexible P2 moieties that optimally contact with the invariable catalytic triad of this enzyme.
PMCID: PMC3884027  PMID: 23594083
Hepatitis C; HCV NS3/4A inhibitors; drug resistance
3.  CARM1 Methylates Chromatin Remodeling Factor BAF155 to Enhance Tumor Progression and Metastasis 
Cancer cell  2014;25(1):21-36.
Coactivator-associated arginine methyltransferase 1 (CARM1), a coactivator for various cancer-relevant transcription factors, is overexpressed in breast cancer. To elucidate the functions of CARM1 in tumorigenesis, we knocked out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology, which resulted in drastic phenotypic and biochemical changes. The CARM1 KO cell lines enabled identification of CARM1 substrates, notably the SWI/SNF core subunit BAF155. Methylation of BAF155 at R1064 was found to be an independent prognostic biomarker for cancer recurrence and to regulate breast cancer cell migration and metastasis. Furthermore, CARM1-mediated BAF155 methylation affects gene expression by directing methylated BAF155 to unique chromatin regions (e.g., c-Myc pathway genes). Collectively, our studies uncover a mechanism by which BAF155 acquires tumorigenic functions via arginine methylation.
PMCID: PMC4004525  PMID: 24434208
4.  Parallel Synthesis of 1,6-Disubstituted-1,2,4-Triazin-3-Ones on Solid-Phase 
ACS combinatorial science  2013;15(7):335-339.
A parallel solid-phase synthesis of 1,6-disubstituted-1,2,4-triazin-3-ones from MBHA resin is described. The reduction of resin-bound nitrosamino acids provides hydrazines efficiently without affecting the amide bond. The trityl protected hydrazine is then reduced with borane, and cyclized with 1,1-carbonyldiimidazole. The desired products are cleaved from their solid support and obtained in good yield and purity. This methodology is of value for the rapid parallel preparation of these potentially bioactive molecules.
PMCID: PMC3875618  PMID: 23750635
solid-phase; 1,2,4-triazinones; N-nitroso; hydrazine; reduction; cyclization
5.  Cardiorespiratory Biomarker Responses in Healthy Young Adults to Drastic Air Quality Changes Surrounding the 2008 Beijing Olympics 
Associations between air pollution and cardiorespiratory mortality and morbidity have been well established, but data to support biologic mechanisms underlying these associations are limited. We designed this study to examine several prominently hypothesized mechanisms by assessing Beijing residents’ biologic responses, at the biomarker level, to drastic changes in air quality brought about by unprecedented air pollution control measures implemented during the 2008 Beijing Olympics.
To test the hypothesis that changes in air pollution levels are associated with changes in biomarker levels reflecting inflammation, hemostasis, oxidative stress, and autonomic tone, we recruited and retained 125 nonsmoking adults (19 to 33 years old) free of cardiorespiratory and other chronic diseases. Using the combination of a quasi-experimental design and a panel-study approach, we measured biomarkers of autonomic dysfunction (heart rate [HR*] and heart rate variability [HRV]), of systemic inflammation and oxidative stress (plasma C-reactive protein [CRP], fibrinogen, blood cell counts and differentials, and urinary 8-hydroxy-2′-deoxyguanosine [8-OHdG]), of pulmonary inflammation and oxidative stress (fractional exhaled nitric oxide [FeNO], exhaled breath condensate [EBC] pH, EBC nitrate, EBC nitrite, EBC nitrite+nitrate [sum of the concentrations of nitrite and nitrate], and EBC 8-isoprostane), of hemostasis (platelet activation [plasma sCD62P and sCD40L], platelet aggregation, and von Willebrand factor [vWF]), and of blood pressure (systolic blood pressure [SBP] and diastolic blood pressure [DBP]). These biomarkers were measured on each subject twice before, twice during, and twice after the Beijing Olympics. For each subject, repeated measurements were separated by at least one week to avoid potential residual effects from a prior measurement. We measured a large suite of air pollutants (PM2.5 [particulate matter ≤ 2.5 μm in aerodynamic diameter] and constituents, sulfur dioxide [SO2], carbon monoxide [CO], nitrogen dioxide [NO2], and ozone [O3]) throughout the study at a central Beijing site near the residences and workplaces of the subjects on a daily basis. Total particle number (TPN) was also measured at a separate site. We used a time-series analysis to assess changes in pollutant concentration by period (pre-, during-, and post-Olympics periods). We used mixed-effects models to assess changes in biomarker levels by period and to estimate changes associated with increases in pollutant concentrations, controlling for ambient temperature, relative humidity (RH), sex, and the day of the week of the biomarker measurements. We conducted sensitivity analyses to assess the impact of potential temporal confounding and exposure misclassification.
We observed reductions in mean concentrations for all measured pollutants except O3 from the pre-Olympics period to the during-Olympics period. On average, elemental carbon (EC) changed by −36%, TPN by −22%, SO2 by −60%, CO by −48%, and NO2 by −43% (P < 0.05 for all these pollutants). Reductions were observed in mean concentrations of PM2.5 (by −27%), sulfate (SO42−) (by −13%), and organic carbon (OC) (by −23%); however, these values were not statistically significant. Both 24-hour averages and 1-hour maximums of O3 increased (by 20% and 17%, respectively) from the pre-Olympics to the during-Olympics period. In the post-Olympics period after the pollution control measures were relaxed, mean concentrations of most pollutants (with the exception of SO42− and O3) increased to levels similar to or higher than pre-Olympics levels.
Concomitantly and consistent with the hypothesis, we observed, from the pre-Olympics to the during-Olympics period, statistically significant (P ≤ 0.05) or marginally significant (0.05 < P < 0.1) decreases in HR (−1 bpm or −1.7% [95% CI, −3.4 to −0.1]), SBP (−1.6 mmHg or − 1.8% [95% CI, −3.9 to 0.4]), 8-OHdG (−58.3% [95% CI, −72.5 to −36.7]), FeNO (−60.3% [95% CI, −66.0 to −53.6]), EBC nitrite (−30.0% [95% CI, −39.3 to −19.3]), EBC nitrate (−21.5% [95% CI, −35.5 to −4.5]), EBC nitrite+nitrate (−17.6% [95% CI, −28.4 to −5.1]), EBC hydrogen ions (−46% [calculated from EBC pH], or +3.5% in EBC pH [95% CI, 2.2 to 4.9]), sCD62P (−34% [95% CI, −38.4 to −29.2]), sCD40L (−5.7% [95% CI, −10.5 to −0.7]), and vWF (−13.1% [95% CI, −18.6 to −7.5]). Moreover, the percentages of above-detection values out of all observations were significantly lower for plasma CRP and EBC 8-isoprostane in the during-Olympics period compared with the pre-Olympics period. In the post-Olympics period, the levels of the following biomarkers reversed (increased, either with or without statistical significance) from those in the during-Olympics period: SBP (10.7% [95% CI, 2.8 to 18.6]), fibrinogen (4.3% [95% CI, −1.7 to 10.2), neutrophil count (4.7% [95% CI, −7.7 to 17.0]), 8-OHdG (315% [95% CI, 62.0 to 962]), FeNO (130% [95% CI, 62.5 to 225]), EBC nitrite (159% [95% CI, 71.8 to 292]), EBC nitrate (161% [95% CI, 48.0 to 362]), EBC nitrite+nitrate (124% [95% CI, 50.9 to 233]), EBC hydrogen ions (146% [calculated from EBC pH] or −4.8% in EBC pH [95% CI, −9.4 to −0.2]), sCD62P (33.7% [95% CI, 17.7 to 51.8]), and sCD40L (9.1% [95% CI, −3.7 to 23.5]).
Furthermore, these biomarkers also showed statistically significant associations with multiple pollutants across different lags after adjusting for meteorologic parameters. The associations were in the directions hypothesized and were consistent with the findings from the comparisons between periods, providing further evidence that the period effects were due to changes in air quality, independent of season and meteorologic conditions or other potential confounders. Contrary to our hypothesis, however, we observed increases in platelet aggregation, red blood cells (RBCs) and white blood cells (WBCs) associated with the during-Olympics period, as well as significant negative associations of these biomarkers with pollutant concentrations. We did not observe significant changes in any of the HRV indices and DBP by period. However, we observed associations between a few HRV indices and pollutant concentrations.
Changes in air pollution levels during the Beijing Olympics were associated with acute changes in biomarkers of pulmonary and systemic inflammation, oxidative stress, and hemostasis and in measures of cardiovascular physiology (HR and SBP) in healthy, young adults. These changes support the prominently hypothesized mechanistic pathways underlying the cardiorespiratory effects of air pollution.
PMCID: PMC4086245  PMID: 23646463
6.  Post-laparoscopic oral contraceptive combined with Chinese herbal mixture in treatment of infertility and pain associated with minimal or mild endometriosis: a randomized controlled trial 
Endometriosis affects fertility negatively. The study aims to evaluate whether laparoscopic surgery combined with oral contraceptive or herbs were more effective than laparoscopic alone in improving fecundity and pelvic pain in women with minimal/mild endometriosis.
A randomized controlled trial (RCT) was conducted in 156 infertile women with minimal/mild endometriosis. After laparoscopic surgery, patients were randomized to three groups: in Group A (n = 52) oral contraceptive (OC) was administered one pill a day, continuous for 63 days without intervals, in Group B (n = 52) OC was administered as above and then Dan’e mixture was added 30 g/day for the latter 30 days, and in control Group C (n = 52) patients tried to get pregnant after surgery without complementary treatment. The follow-up periods were 12 months in Group C and 14 months in complementary medical treatment Group A and B. The pregnant women were further followed up, and labor and pregnancy outcomes were assessed. Primary outcome was pregnancy rate (PR) and live birth rate (LBR). Secondary outcomes included changes of pelvic pain visual analog scale scores and side effects. Analyses were done as intention-to-treat.
The PR was 46.80% (73/156), and the LBR was 69.86% (51/73). Of the 73 pregnancies, 60 occurred within 12 months of follow-up and 7 of the remaining 13 patients underwent assisted reproductive technology for >1 year. No significant difference was observed in PR and LBR among the three groups. Patients given medical treatment (OCs or OCs plus herbal medicine) had significantly decreased pain scores compared with the laparoscopy alone group.
Combination of laparoscopy with OCs or OCs and herbal medicine does not have more advantages than laparoscopy alone in improving fertility of women with minimal/mild endometriosis.
Trial registration
PMCID: PMC4087198  PMID: 24996447
Endometriosis; Infertility; Oral contraceptive pills; Herbal medicine
7.  Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice 
BMC Plant Biology  2014;14:177.
Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown.
We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions.
Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.
PMCID: PMC4083042  PMID: 24980094
Gene expression; Epigenetics; Small RNA; DNA methylation; RDR1; Oryza sativa L
8.  Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment 
Androgens and estrogens, acting via their respective receptors, are important in benign prostatic hyperplasia (BPH). The goal of this study was to quantitatively characterize the tissue distribution and staining intensity of androgen receptor (AR) and estrogen receptor-alpha (ERα), and assess cells expressing both AR and ERα, in human BPH compared to normal prostate. A tissue microarray composed of normal prostate and BPH tissue was used and multiplexed immunohistochemistry was performed to detect AR and ERα. We used a multispectral imaging platform for automated scanning, tissue and cell segmentation and marker quantification. BPH specimens had an increased number of epithelial and stromal cells and increased percentage of epithelium. In both stroma and epithelium, the mean nuclear area was decreased in BPH relative to normal prostate. AR expression and staining intensity in epithelial and stromal cells was significantly increased in BPH compared to normal prostate. ERα expression was increased in BPH epithelium. However, stromal ERα expression and staining intensity was decreased in BPH compared to normal prostate. Double positive (AR & ERα) epithelial cells were more prevalent in BPH, and fewer double negative (AR & ERα) stromal and epithelial negative cells were observed in BPH. These data underscore the importance of tissue layer localization and expression of steroid hormone receptors in the prostate. Understanding the tissue-specific hormone action of androgens and estrogens will lead to a better understanding of mechanisms of pathogenesis in the prostate and may lead to better treatment for BPH.
PMCID: PMC3729928  PMID: 23792768
Benign Prostatic Hyperplasia; Androgen Receptor; Estrogen Receptor alpha
9.  Correlation Analysis of EV71 Detection and Case Severity in Hand, Foot, and Mouth Disease in the Hunan Province of China 
PLoS ONE  2014;9(6):e100003.
An increase in the incidence of hand, foot and mouth disease (HFMD) cases has been observed in the Hunan province of mainland China since 2009 with a particularly higher level of severe cases in 2010–2012. Intestinal viruses of the picornaviridae family are responsible for the human syndrome associated with HFMD with enterovirus 71 (EV71) and Coxsackievirus A16 (Cox A16) being the most common causative strains. HFMD cases associated with EV71 are generally more severe with an increased association of morbidity and mortality. In this study, the etiology surveillance data of HFMD cases in Hunan province from March 2010 to October 2012 were analyzed to determine if there is a statistically relevant linear correlation exists between the detection rate of EV71 in mild cases and the proportion of severe cases among all HFMD patients. As the cases progressed from mild to severe to fatal, the likelihood of EV71 detection increased (25.78%, 52.20% and 84.18%, respectively). For all cases in the timeframe evaluated in this study, the presence of virus was detected in 63.21% of cases; among cases showing positivity for virus, EV71 infection accounted for 50.14%. These results provide evidence to support the observed higher morbidity and mortality associated with this outbreak and emphasizes the importance of early detection in order to implement necessary prevention measures to mitigate disease progression.
PMCID: PMC4062471  PMID: 24941257
10.  Quercetin Significantly Inhibits the Metabolism of Caffeine, a Substrate of Cytochrome P450 1A2 Unrelated to CYP1A2*1C  (−2964G>A) and *1F (734C>A) Gene Polymorphisms 
BioMed Research International  2014;2014:405071.
Background. Quercetin is abundant in plants and human diets. Previous studies indicated that quercetin inhibited the activity of CYP1A2, and the combination of quercetin with the substrates of CYP1A2 might produce herb-drug interactions. This research aims to determine the effects of quercetin and the CYP1A2 gene polymorphisms, namely, CYP1A2*1C  (−2964G>A) and *1F (734C>A), on the metabolism of caffeine. Method. The experiment was designed into two treatment phases separated by a 2-week washout period. Six homozygous individuals for the CYP1A2*1C/*1F (GG/AA) genotype and 6 heterozygous individuals for the CYP1A2*1C/*1F (GA/CA) genotype were enrolled in the study. Quercetin capsules (500 mg) were given to each volunteer once daily for 13 consecutive days, and after that, each subject was coadministrated 100 mg caffeine capsules with 500 mg quercetin on the 14th day. Then a series of venous blood samples were collected for HPLC analysis. Correlation was determined between pharmacokinetics of caffeine and paraxanthine with caffeine metabolite ratio. Results. Quercetin significantly affected the pharmacokinetics of caffeine and its main metabolite paraxanthine, while no differences were found in the pharmacokinetics of caffeine and paraxanthine between GG/AA and GA/CA genotype groups. Conclusion. Quercetin significantly inhibits the caffeine metabolism, which is unrelated to CYP1A2*1C (−2964G>A) and *1F (734C>A) gene polymorphisms.
PMCID: PMC4082882  PMID: 25025048
11.  FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation 
Molecular Cancer  2014;13:150.
Heat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin.
We adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line.
We have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile.
As a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells.
PMCID: PMC4074137  PMID: 24927996
FW-04-806; Hsp90; Cdc37; HER2; Breast cancer
12.  Development of a Highly Sensitive Glycan Microarray for Quantifying AFP-L3 for Early Prediction of Hepatitis B Virus–Related Hepatocellular Carcinoma 
PLoS ONE  2014;9(6):e99959.
The α-fetoprotein fraction L3 (AFP-L3), which is synthesized by malignant cells and incorporates a fucosylated oligosaccharide, has been investigated as a diagnostic and prognostic marker for hepatocellular carcinoma (HCC). Quantification of AFP-L3 by conventional enzyme-linked immunosorbent assay (ELISA) has not always produced reliable results for serum samples with low AFP, and thus we evaluated the clinical utility of quantifying AFP-L3 using a new and highly sensitive glycan microarray assay. Sera from 9 patients with chronic hepatitis B and 32 patients with hepatitis B virus (HBV)-related HCC were tested for AFP-L3 level using the glycan microarray. Additionally, we compared receiver operator characteristic curves for the ELISA and glycan microarray methods for determination of the AFP-L3: AFP-L1 ratio in patient samples. This ratio was calculated for 8 HCC patients who underwent transarterial embolization therapy pre- or post-treatment with AFP-L3. Glycan microarrays showed that the AFP-L3 ratio of HBV-related HCC patients was significantly higher than that measured for chronic hepatitis B patients. Overall parameters for estimating AFP-L3% in HCC samples were as follows: sensitivity, 53.13%; specificity, 88.89%; and area under the curve, 0.75. The elevated AFP-L3% in the 8 patients with HBV-related HCC was strongly associated with HCC progression. Following one month of transarterial embolization therapy, the relative mean AFP-L3% decreased significantly. In addition, we compared Fut8 gene expression between paired tumor and non-tumor tissues from 24 patients with HBV-related HCC. The Fut8 mRNA expression was significantly increased in tumorous tissues in these patients than that in non-tumor tissue controls. Higher expression of Fut8 mRNA in tumorous tissues in these patients was associated with poor differentiation than well and moderate differentiation. Our results describe a new glycan microarray for the sensitive and rapid quantification of fucosylated AFP; this method is potentially applicable to screening changes in AFP-L3 level for assessment of HCC progression.
PMCID: PMC4057280  PMID: 24927126
13.  Increased Cystic Fibrosis Transmembrane Conductance Regulators Expression and Decreased Epithelial Sodium Channel Alpha Subunits Expression in Early Abortion: Findings from a Mouse Model and Clinical Cases of Abortion 
PLoS ONE  2014;9(6):e99521.
The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR) and epithelial sodium channel alpha subunits (ENaC-α) play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated) had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated). Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.
PMCID: PMC4051784  PMID: 24914548
14.  Association Between Changes in Air Pollution Levels During the Beijing Olympics and Biomarkers of Inflammation and Thrombosis in Healthy Young Adults 
Air pollution is a risk factor for cardiovascular diseases (CVD), but the underlying biological mechanisms are not well understood.
To determine whether markers related to CVD pathophysiological pathways (biomarkers for systemic inflammation and thrombosis, heart rate, and blood pressure) are sensitive to changes in air pollution.
Design, Setting, and Participants
Using a quasi-experimental opportunity offered by greatly restricted air pollution emissions during the Beijing Olympics, we measured pollutants daily and the outcomes listed below in 125 healthy young adults before, during, and after the 2008 Olympics (June 2-October 30). We used linear mixed-effects models to estimate the improvement in outcome levels during the Olympics and the anticipated reversal of outcome levels after pollution controls ended to determine whether changes in outcome levels were associated with changes in pollutant concentrations.
Main Outcome Measures
C-reactive protein (CRP), fibrinogen, von Willebrand factor, soluble CD40 ligand (sCD40L), soluble P-selectin (sCD62P) concentrations; white blood cell count (WBC); heart rate; and blood pressure.
Concentrations of particulate and gaseous pollutants decreased substantially (−13% to −60%) from the pre-Olympic period to the during-Olympic period. Using 2-sided tests conducted at the .003 level, we observed statistically significant improvements in sCD62P levels by −34.0% (95% CI, −38.4% to −29.2%; P<.001) from a pre-Olympic mean of 6.29 ng/mL to a during-Olympic mean of 4.16 ng/mL and von Willebrand factor by −13.1% (95% CI, −18.6% to −7.5%; P<.001) from 106.4% to 92.6%. After adjustments for multiple comparisons, changes in the other outcomes were not statistically significant. In the post-Olympic period when pollutant concentrations increased, most outcomes approximated pre-Olympic levels, but only sCD62P and systolic blood pressure were significantly worsened from the during-Olympic period. The fraction of above-detection-limit values for CRP (percentage ≥0.3 mg/L) was reduced from 55% in the pre-Olympic period to 46% in the during-Olympic period and reduced further to 36% in the post-Olympic period. Interquartile range increases in pollutant concentrations were consistently associated with statistically significant increases in fibrinogen, von Wille-brand factor, heart rate, sCD62P, and sCD40L concentrations.
Changes in air pollution levels during the Beijing Olympics were associated with acute changes in biomarkers of inflammation and thrombosis and measures of cardiovascular physiology in healthy young persons. These findings are of uncertain clinical significance.
PMCID: PMC4049319  PMID: 22665106
15.  Malondialdehyde in exhaled breath condensate and urine as a biomarker of air pollution induced oxidative stress 
Underlying mechanisms by which air pollutants adversely affect human health remain poorly understood. Oxidative stress has been considered as a potential mechanism that may promote lipid peroxidation by reactive oxygen species, leading to the formation of malondialdehyde (MDA) that is excreted in biofluids (e.g., urine and exhaled breath condensate (EBC)). A panel study was conducted to examine whether concentrations of MDA in EBC and urine were associated, respectively, with changes in air pollution levels brought by the Beijing Olympic air pollution control measures. EBC and urine samples from 125 healthy adults were collected twice in each of the pre-, during-, and post-Olympic periods. Period-specific means of MDA and changes in MDA levels associated with increases in 24-h average pollutant concentrations were estimated using linear mixed-effects models. From the pre- to the during-Olympic period, when concentrations of most pollutants decreased, EBC MDA and urinary MDA significantly decreased by 24% (P < 0.0001) and 28% (P = 0.0002), respectively. From the during-Olympic to the post-Olympic period, when concentrations of most pollutants increased, EBC MDA and urinary MDA increased by 28% (P = 0.094) and 55% (P = 0.046), respectively. Furthermore, the largest increases in EBC MDA associated with one interquartile range (IQR) increases in all pollutants but ozone ranged from 10% (95% CI: 2%, 18%) to 19% (95% CI: 14%, 25%). The largest increases in urinary MDA associated with IQR increases in pollutant concentration ranged from 9% (95%: 0.3%, 19%) to 15% (95% CI: 3%, 28%). These findings support the utility of EBC MDA as a biomarker of oxidative stress in the respiratory tract and urinary MDA as a biomarker of systemic oxidative stress in relation to air pollution exposure in healthy young adults. Both EBC and urine samples can be collected noninvasively in the general population.
PMCID: PMC4049321  PMID: 23321859
The Beijing Olympics; lipid peroxidation; malondialdehyde; oxidative stress; exhaled breath condensate
16.  Measurement of inflammation and oxidative stress following drastic changes in air pollution during the Beijing Olympics: a panel study approach 
Ambient air pollution has been linked to cardiovascular and respiratory morbidity and mortality in epidemiology studies. Frequently, oxidative and nitrosative stress are hypothesized to mediate these pollution effects, however precise mechanisms remain unclear. This paper describes the methodology for a major panel study to examine air pollution effects on these and other mechanistic pathways. The study took place during the drastic air pollution changes accompanying the 2008 Olympics in Beijing, China. After a general description of air pollution health effects, we provide a discussion of panel studies and describe the unique features of this study that make it likely to provide compelling results. This study should lead to a clearer and more precise definition of the role of oxidative and nitrosative stress, as well as other mechanisms, in determining acute morbidity and mortality from air pollution exposure.
PMCID: PMC4049322  PMID: 20716299
panel study; oxidative stress; exhaled breath condensate; 2008 Olympics
17.  The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro 
BioMed Research International  2014;2014:684765.
Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.
PMCID: PMC4070544  PMID: 25003128
18.  The Role of Ubiquitination and Sumoylation in Diabetic Nephropathy 
BioMed Research International  2014;2014:160692.
Diabetic nephropathy (DN) is a common and characteristic microvascular complication of diabetes; the mechanisms that cause DN have not been clarified, and the epigenetic mechanism was promised in the pathology of DN. Furthermore, ubiquitination and small ubiquitin-like modifier (SUMO) were involved in the progression of DN. MG132, as a ubiquitin proteasome, could improve renal injury by regulating several signaling pathways, such as NF-κB, TGF-β, Nrf2-oxidative stress, and MAPK. In this review, we summarize how ubiquitination and sumoylation may contribute to the pathology of DN, which may be a potential treatment strategy of DN.
PMCID: PMC4065738  PMID: 24991536
19.  Region-Based Nasopharyngeal Carcinoma Lesion Segmentation from MRI Using Clustering- and Classification-Based Methods with Learning 
Journal of Digital Imaging  2012;26(3):472-482.
In clinical diagnosis of nasopharyngeal carcinoma (NPC) lesion, clinicians are often required to delineate boundaries of NPC on a number of tumor-bearing magnetic resonance images, which is a tedious and time-consuming procedure highly depending on expertise and experience of clinicians. Computer-aided tumor segmentation methods (either contour-based or region-based) are necessary to alleviate clinicians’ workload. For contour-based methods, a minimal user interaction to draw an initial contour inside or outside the tumor lesion for further curve evolution to match the tumor boundary is preferred, but parameters within most of these methods require manual adjustment, which is technically burdensome for clinicians without specific knowledge. Therefore, segmentation methods with a minimal user interaction as well as automatic parameters adjustment are often favored in clinical practice. In this paper, two region-based methods with parameters learning are introduced for NPC segmentation. Two hundred fifty-three MRI slices containing NPC lesion are utilized for evaluating the performance of the two methods, as well as being compared with other similar region-based tumor segmentation methods. Experimental results demonstrate the superiority of adopting learning in the two introduced methods. Also, they achieve comparable segmentation performance from a statistical point of view.
PMCID: PMC3649041  PMID: 22854973
Nasopharyngeal carcinoma; Magnetic resonance image; Tumor segmentation; Spectral clustering; Support vector machine; Metric learning
20.  Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii 
Applied and Environmental Microbiology  2013;79(24):7646-7653.
Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation.
PMCID: PMC3837804  PMID: 24077712
21.  High Glucose Induces Sumoylation of Smad4 via SUMO2/3 in Mesangial Cells 
BioMed Research International  2014;2014:782625.
Recent studies have shown that sumoylation is a posttranslational modification involved in regulation of the transforming growth factor-β (TGF-β) signaling pathway, which plays a critical role in renal fibrosis in diabetic nephropathy (DN). However, the role of sumoylation in the regulation of TGF-β signaling in DN is still unclear. In the present study, we investigated the expression of SUMO (SUMO1 and SUMO2/3) and Smad4 and the interaction between SUMO and Smad4 in cultured rat mesangial cells induced by high glucose. We found that SUMO1 and SUMO2/3 expression was significantly increased in the high glucose groups compared to the normal group (P < 0.05). Smad4 and fibronectin (FN) levels were also increased in the high glucose groups in a dose-dependent manner. Coimmunoprecipitation and confocal laser scanning revealed that Smad4 interacted and colocalized with SUMO2/3, but not with SUMO1 in mesangial cells. Sumoylation (SUMO2/3) of Smad4 under high glucose condition was strongly enhanced compared to normal control (P < 0.05). These results suggest that high glucose may activate TGF-β/Smad signaling through sumoylation of Samd4 by SUMO2/3 in mesangial cells.
PMCID: PMC4058256  PMID: 24971350
23.  Increased expression of the pluripotency markers sex-determining region Y-box 2 and Nanog homeobox in ovarian endometriosis 
The precise etiology of endometriosis is not fully understood; the involvement of stem cells theory is a new hypothesis. Related studies mainly focus on stemness-related genes, and pluripotency markers may play a role in the etiology of endometriosis. We aimed to analyze the transcription pluripotency factors sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and octamer-binding protein 4 (OCT4) in the endometrium of reproductive-age women with and without ovarian endometriosis.
We recruited 26 women with laparoscopy-diagnosed ovarian endometriosis (endometriosis group) and 16 disease-free women (control group) to the study. Endometrial and endometriotic samples were collected. SOX2, NANOG, and OCT4 expression were analyzed with quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry.
Compared to the control group, SOX2 mRNA and protein expression was significantly higher in the eutopic endometrium of participants in the endometriosis group. In the endometriosis group, SOX2 and NANOG mRNA and protein expression were significantly increased in ectopic endometrium compared with eutopic endometrium; there was a trend towards lower OCT4 mRNA expression and higher OCT4 protein expression in ectopic endometrium.
The transcription pluripotency factors SOX2 and NANOG were overexpression in ovarian endometriosis, their role in pathogenesis of endometriosis should be further studied.
PMCID: PMC4031377  PMID: 24884521
Endometriosis; Sex-determining region Y-box 2; NANOG; Octamer-binding protein 4
24.  Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice 
eLife  2014;3:e02265.
All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBötzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b- or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing.
eLife digest
A healthy adult at rest will breathe in and out around 20 times per minute. Each breath requires a complex series of coordinated muscle activity. Inhalation begins with the opening of the airway followed by the contraction of the diaphragm and the intercostal muscles between the ribs, causing the chest cavity to expand. As the lungs increase in volume, the pressure inside them drops and air is drawn in. Relaxation of the diaphragm and intercostal muscles compresses the lungs, causing us to exhale.
Breathing is driven by the brainstem and it cannot be suppressed indefinitely: holding your breath eventually triggers a reflex that forces breathing to resume. The region of the brainstem that controls breathing is called the preBötzinger Complex. However, there is increasing evidence that a second region in the brainstem is also involved. This region, which is called the retrotrapezoid nucleus/parafacial respiratory group, consists of three types of excitatory neurons—Dbx1 neurons, Phox2b neurons, and Atoh1 neurons—but their roles had not been clear. Now, using multiple lines of genetically modified mice, Tupal et al. have teased apart the roles of these three cell types.
These experiments showed that the Dbx1 neurons—which are also found in the preBötzinger Complex—have an essential role in sending the signals from the brain that drive the different muscle activities needed to breathe. The Phox2b neurons modulate breathing based on the level of carbon dioxide in the blood. Atoh1 neurons help control the sequence of respiratory muscle activity during a breath, probably by selectively inhibiting different populations of Dbx1 neurons.
The work of Tupal et al. indicates that distinct populations of neurons within the brainstem independently control two different aspects of breathing: the generation of breathing rhythms, and the coordination of these rhythms. Given that many other physiological processes involve rhythmic activity patterns, this model may help us to understand how the brain generates and controls complex behaviors more generally.
PMCID: PMC4060005  PMID: 24842997
breathing; central pattern generator; PreBötzinger Complex; oscillator; transcription; mouse
25.  Use of lectin microarray to differentiate gastric cancer from gastric ulcer 
AIM: To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer.
METHODS: Twenty cases of human gastric cancer tissue and 20 cases of human gastric ulcer tissue were collected and processed. Protein was extracted from the frozen tissues and stored. The lectins were dissolved in buffer, and the sugar-binding specificities of lectins and the layout of the lectin microarray were summarized. The median of the effective data points for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Formalin-fixed paraffin-embedded gastric cancer tissues and their corresponding gastric ulcer tissues were subjected to Ag retrieval. Biotinylated lectin was used as the primary antibody and HRP-streptavidin as the secondary antibody. The glycopatterns of glycoprotein in gastric cancer and gastric ulcer specimens were determined by lectin microarray, and then validated by lectin histochemistry. Data are presented as mean ± SD for the indicated number of independent experiments.
RESULTS: The glycosylation level of gastric cancer was significantly higher than that in ulcer. In gastric cancer, most of the lectin binders showed positive signals and the intensity of the signals was stronger, whereas the opposite was the case for ulcers. Significant differences in the pathological score of the two lectins were apparent between ulcer and gastric cancer tissues using the same lectin. For MPL and VVA, all types of gastric cancer detected showed stronger staining and a higher positive rate in comparison with ulcer, especially in the case of signet ring cell carcinoma and intra-mucosal carcinoma. GalNAc bound to MPL showed a significant increase. A statistically significant association between MPL and gastric cancer was observed. As with MPL, there were significant differences in VVA staining between gastric cancer and ulcer.
CONCLUSION: Lectin microarray can differentiate the different glycopatterns in gastric cancer and gastric ulcer, and the lectins MPL and VVA can be used as biomarkers.
PMCID: PMC4017062  PMID: 24833877
Gastric cancer; Gastric ulcer; Lectin microarray; Lectin histochemistry; Differentiate

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