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1.  Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma 
Molecular Cancer  2013;12:53.
Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.
Immunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).
The positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.
Our study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.
PMCID: PMC3698204  PMID: 23758864
Nasopharyngeal carcinoma; BCAT1; c-Myc; Proliferation; Migration; Invasion; Gene amplification; Gene regulation
2.  Effects of Insufficient Physical Activity on Mortality and Life Expectancy in Jiangxi Province of China, 2007-2010 
PLoS ONE  2014;9(10):e109826.
Physical inactivity remains an under-researched field in terms of studying burden of disease at provincial level, and no studies have examined the effects of inactivity on life expectancy (LE) in China. The purpose of this study was to estimate mortality risk and LE effects associated with insufficient levels of physical activity in Jiangxi province.
Prevalence of risk factors and mortality counts were extracted from Chronic Diseases and Risk Factors Surveillance Survey (CDRFSS) and Disease Surveillance Points system (DSP), respectively. Insufficient physical activity (IPA) was defined as less than 150 minutes of moderate-intensity physical activity or 60 minutes of vigorous-intensity physical activity per week, accumulated across work, home, transport and discretionary domains. Population-attributable fractions (PAF) were used to calculate the mortality attributable to risk factors, and life table methods were used to estimate the LE gains and LE shifts. Monte Carlo simulation techniques were used for uncertainty analysis. Overall, 5 885 (95% uncertainly interval (UI), 5 047–6 506) and 8 578 (95% UI, 8 227–9 789) deaths in Jiangxi province were attributable to IPA in 2007 and 2010, respectively. The LE gains for elimination of attributable deaths were 0.68 (95% UI, 0.61–076) in 2007, and increased to 0.91 (95% UI, 0.81–1.10) in 2010. If the prevalence of IPA in 2010 had been decreased by 50% or 30%, 3 678 (95% UI, 3 220–4 229) or 2 090 (95% UI, 1 771–2 533) deaths would be avoided, and 0.40 (95% UI, 0.34–0.53) or 0.23 (95% UI, 0.16–0.31) years of LE gained, respectively.
Adults in Jiangxi province of China have a high and increasing prevalence of IPA. Due to the deaths and potential LE gains associated with IPA, there is an urgent need to promote physical activity, one of the most modifiable risk factors, within China's health care reform agenda.
PMCID: PMC4197026  PMID: 25314595
3.  Clinical significance of UGT1A1 gene polymorphisms on irinotecan-based regimens as the treatment in metastatic colorectal cancer 
OncoTargets and therapy  2014;7:1653-1661.
The primary aim of this research was to investigate the association between uridine diphosphate glucuronosyltransferase (UGT)1A1 gene polymorphisms and the toxicities of irinotecan-based regimens in Chinese patients with metastatic colorectal cancer.
The study analyzed the distribution of UGT1A1*28/*6 gene polymorphisms by polymerase chain reaction amplification and pyrosequencing. The adverse reactions and tumor response were evaluated according to National Cancer Institute Common Toxicity Criteria for Adverse Events, Version 3.0, and Response Evaluation Criteria In Solid Tumors, Version 1.0, criteria, respectively. The correlation between UGT1A1 gene polymorphisms and severe delayed diarrhea or neutropenia was analyzed. The influences of UGT1A1*6/*28 polymorphisms on response rate and progression-free survival were also analyzed. Survival analysis was performed by the Kaplan–Meier method, and we used the log-rank test to analyze the effect of genotypes on progression-free survival, the logistic regression model for multivariate analysis, and the Cox regression model for multivariate survival analysis.
A total of 167 patients with metastatic colorectal cancer who were treated with irinotecan-based regimens and with detected UGT1A1 gene polymorphisms were enrolled in this research. The rate of UGT1A1*28 homozygous wild-type TA6/6, heterozygous mutant-type TA6/7, and homozygous mutant-type TA7/7 was 65.3% (109/167), 32.3% (54/167), and 2.4% (4/167), respectively; the incidence of UGT1A1*6 wild-type G/G was 67.1% (112/167), heterozygous mutant-type G/A accounted for 28.7% (48/167), and seven cases were homozygous mutant-type A/A (4.2%; 7/167). The incidence of grade 3 or 4 delayed diarrhea in patients carrying UGT1A1*6 (G/A and A/A) was higher than that in the wild-type (G/G) (P=0.021). The rate was significantly lower in patients with the UGT1A1*28 TA6/6 wide-type genotype than those with TA6/7 and TA7/7 mutant-type genotypes (P=0.027). However, neither UGT1A1*6 (P=0.34) nor UGT1A1*28 (P=0.232) variants were significantly associated with severe neutropenia. Our study found no significant differences of severe neutropenia in patients with different numbers of mutational alleles (P=0.354), but patients with two alleles or single allele variants had more chances to develop severe diarrhea than patients with wild-type (P=0.027). No significant differences of either response rate or progression-free survival were found among different genotypes (P>0.05).
For irinotecan-based regimens in metastatic colorectal cancer, the UGT1A1*28 and UGT1A1*6 locus mutations can be regarded as predictors for irinotecan-associated severe delayed diarrhea, whereas no association between UGT1A1 gene polymorphisms and severe neutropenia was observed. We also found that neither clinical response nor prognosis were significantly associated with UGT1A1 gene polymorphisms.
PMCID: PMC4181635  PMID: 25285015
uridine diphosphate glucuronosyltransferase 1A1; gene polymorphism; metastatic colorectal cancer; irinotecan
4.  Dihydromyricetin inhibits migration and invasion of hepatoma cells through regulation of MMP-9 expression 
World Journal of Gastroenterology : WJG  2014;20(29):10082-10093.
AIM: To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells.
METHODS: The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student’s t tests in GraphPad prism 5.0 software and are presented as mean ± SD.
RESULTS: DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 65 ± 10 μmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 μmol/L vs 100 μmol/L DHM, 24 h: 74 ± 6 μmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 μmol/L vs 100 μmol/L DHM, 24 h: 9 ± 3 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 45 ± 2 μmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 μmol/L vs 100 μmol/L DHM, 24 h: 100 ± 6 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 μmol/L vs 100 μmol/L DHM 24 h: 156 ± 10 μmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38, ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels. In addition, PKC-δ protein, a key protein in the regulation of MMP family protein expression, was up-regulated with DHM treatment.
CONCLUSION: These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.
PMCID: PMC4123337  PMID: 25110435
Dihydromyricetin; Migration; Invasion; Hepatic cancer; Matrix metalloproteinase-9
5.  Correlation between EGFR mutation status and response to first-line platinum-based chemotherapy in patients with advanced non-small cell lung cancer 
OncoTargets and therapy  2014;7:1185-1193.
The purpose of this research was to investigate the relationship between epidermal growth factor receptor (EGFR) mutations and the response to first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC).
A total of 266 patients with stage IIIB or IV NSCLC who received platinum-based doublet therapies as first-line chemotherapy were investigated retrospectively, and their clinical data were assessed according to EGFR mutation.
EGFR mutations were identified in 45.5% of patients. There was no significant difference in response rate between EGFR mutation carriers and EGFR wild-type carriers (P=0.484). Among the patients with Kirsten rat sarcoma viral oncogene homolog (KRAS) wild-type, however, those with EGFR mutations responded better to treatment than EGFR wild-type patients (46.2% versus 20.8%, P=0.043). The disease control rate associated with pemetrexed-based treatments was higher than for vinorelbine-based therapies in EGFR mutation patients (P=0.001). EGFR mutation was found in patients with longer progression-free survival and median survival time, and improved 1-year and 2-year overall survival when compared with EGFR wild-type patients (6.1 versus 5.0 months, P=0.004; 18.9 versus 13.8 months, P=0.001; 81.0% versus 63.4%, P=0.002; and 33.9% versus 22.8% P=0.044, respectively). Patients with the EGFR exon 19 mutation had longer progression-free survival than those with EGFR exon 21 mutation (P=0.007). Multivariate analysis showed that the response to first-line chemotherapy and the presence of EGFR mutations were independent prognostic factors in patients with advanced NSCLC.
Our data showed that the presence of EGFR mutations meant longer survival times for patients with advanced NSCLC who received platinum-based doublet first-line chemotherapy, especially in those with the exon 19 deletion mutation. Among KRAS wild-type patients, those with EGFR mutation responded better to first-line chemotherapy than EGFR wild-type patients.
PMCID: PMC4085297  PMID: 25061320
non-small cell lung cancer; chemotherapeutic agents; epidermal growth factor receptor mutation; targeted therapy; prognosis
6.  Construction and evaluation of a novel humanized HER2-specific chimeric receptor 
The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed.
HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry.
chA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions.
The results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers.
PMCID: PMC4095682  PMID: 24919843
7.  First Insight into the Genotypic Diversity of Clinical Mycobacterium tuberculosis Isolates from Gansu Province, China 
PLoS ONE  2014;9(6):e99357.
Investigations of Mycobacterium tuberculosis genetic diversity in China have indicated a significant regional distribution. The aim of this study was to characterize the genotypes of clinical M. tuberculosis isolates obtained from Gansu, which has a special geographic location in China.
Methodology/Principal Findings
A total of 467 clinical M. tuberculosis strains isolated in Gansu Province were genotyped by 15-locus mycobacterial interspersed repetitive units–variable number tandem repeats (MIRU-VNTR) and spoligotyping. The results showed that 445 isolates belonged to six known spoligotype lineages, whereas 22 isolates were unknown. The Beijing genotype was the most prevalent (87.58%, n = 409), while the shared type 1 was the dominant genotype (80.94%, n = 378). The second most common lineage was the T lineage, with 25 isolates (5.35%), followed by the H lineage with 5 isolates (1.07%), the MANU family (0.64%, 3 isolates), the U family (0.43%, 2 isolates) and the CAS lineage with 1 isolate (0.21%). By using the VNTR15China method, we observed 15 groups and 228 genotypes among the 467 isolates. We found no association between the five larger groups (including the Beijing genotype) and sex, age, or treatment status, and there was no noticeable difference in the group analysis in different areas. In the present study, seven of the 15 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index.
The Beijing genotype is the predominant genotype in Gansu province. We confirm that VNTR15China is suitable for typing Beijing strains in China and that it has a better discriminatory power than spoligotyping. Therefore, the use of both methods is the most suitable for genotyping analysis of M. tuberculosis.
PMCID: PMC4049826  PMID: 24911588
8.  Expert consensus on acute exacerbation of chronic obstructive pulmonary disease in the People’s Republic of China 
Chronic obstructive pulmonary disease (COPD) is a common disease that severely threatens human health. Acute exacerbation of COPD (AECOPD) is a major cause of disease progression and death, and causes huge medical expenditures. This consensus statement represents a description of clinical features of AECOPD in the People’s Republic of China and a set of recommendations. It is intended to provide clinical guidelines for community physicians, pulmonologists and other health care providers for the prevention, diagnosis, and treatment of AECOPD.
PMCID: PMC4008287  PMID: 24812503
COPD; AECOPD; recommendations; guidelines
9.  Prognosis of 18 H7N9 Avian Influenza Patients in Shanghai 
PLoS ONE  2014;9(4):e88728.
To provide prognosis of an 18 patient cohort who were confirmed to have H7N9 lung infection in Shanghai.
Patients' history, clinical manifestation, laboratory test, treatment strategy and mortality were followed and recorded for data analysis.
A total of 18 patients had been admitted into Shanghai Public Health Clinical Center from April 8th to July 29, 2013. 22.2% of the patients were found to have live poultry contact history and 80% were aged male patients with multiple co-morbidities including diabetes, hypertension and/or chronic obstructive pulmonary disease (COPD). This group of patients was admitted to the clinical center around 10 days after disease onset. According to laboratory examinations, increased C reactive protein (CRP), Procalcitonin (PCT), Plasma thromboplastin antecedent (PTA) and virus positive time (days) were indicative of patients' mortality. After multivariate analysis, only CRP level showed significant prediction of mortality (P = 0.013) while results of prothrombin time (PT) analysis almost reached statistical significance (P = 0.056).
H7N9 infection induced pneumonia of different severity ranging from mild to severe pneumonia or acute lung injury/acute respiratory distress syndrome to multiple organ failure. Certain laboratory parameters such as plasma CRP, PCT, PTA and virus positive days predicted mortality of H7N9 infection and plasma CRP is an independent predictor of mortality in these patients.
PMCID: PMC3973662  PMID: 24695420
10.  Normobaric hyperoxia combined with minocycline provides greater neuroprotection than either alone in transient focal cerebral ischemia 
Experimental neurology  2012;240:9-16.
Normobaric hyperoxia (NBO), which maintains penumbral oxygenation, reduces brain injury during cerebral ischemia, and minocycline, a tetracycline derivative, reduces reperfusion injury, including inflammation, apoptosis and matrix metalloproteinases (MMPs) activation. Since they have different mechanisms of action, we hypothesized that combining them would provide greater neuroprotection. To test the hypothesis, we evaluated the neuroprotective effects of the combination of NBO with minocycline. Male Sprague Dawley rats were exposed to NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery, followed by 48 hrs of reperfusion. Minocycline (3 mg/kg) or vehicle was intravenously administered to rats 15 min after reperfusion onset. Treatment with NBO and minocycline alone resulted in 36% and 30% reductions in infarction volume, respectively. When the two treatments were combined, there was a 68% reduction in infarction volume. The combination therapy also significantly reduced hemispheric swelling, which was absent with monotherapy. In agreement with its greater neuro-and vasoprotection, the combination therapy showed greater inhibitory effects on MMP-2/9 induction, occludin degradation, caspase-3 and -9 activation and apoptosis inducing factor (AIF) induction in ischemic brain tissue. Our results show that NBO plus minocycline effectively reduces brain injury in transient focal cerebral ischemia with protection due to inhibition on MMP-2/9-mediated occludin degradation and attenuation of caspase-dependent and independent apoptotic pathways.
PMCID: PMC3563388  PMID: 23195595
oxygen; minocycline; stroke; infarction; hemispheric swelling
11.  High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus 
Nature Communications  2013;4:2673.
Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.
Stripe rust is one of the most destructive wheat diseases. Here, Zheng and colleagues report a draft genome sequence of wheat stripe rust fungus, generated using a fosmid-to-fosmid approach, and provide insight into its race evolution and pathogenesis.
PMCID: PMC3826619  PMID: 24150273
12.  Differential Gene Expression Profiling and Biological Process Analysis in Proximal Nerve Segments after Sciatic Nerve Transection 
PLoS ONE  2013;8(2):e57000.
After traumatic injury, peripheral nerves can spontaneously regenerate through highly sophisticated and dynamic processes that are regulated by multiple cellular elements and molecular factors. Despite evidence of morphological changes and of expression changes of a few regulatory genes, global knowledge of gene expression changes and related biological processes during peripheral nerve injury and regeneration is still lacking. Here we aimed to profile global mRNA expression changes in proximal nerve segments of adult rats after sciatic nerve transection. According to DNA microarray analysis, the huge number of genes was differentially expressed at different time points (0.5 h–14 d) post nerve transection, exhibiting multiple distinct temporal expression patterns. The expression changes of several genes were further validated by quantitative real-time RT-PCR analysis. The gene ontology enrichment analysis was performed to decipher the biological processes involving the differentially expressed genes. Collectively, our results highlighted the dynamic change of the important biological processes and the time-dependent expression of key regulatory genes after peripheral nerve injury. Interestingly, we, for the first time, reported the presence of olfactory receptors in sciatic nerves. Hopefully, this study may provide a useful platform for deeply studying peripheral nerve injury and regeneration from a molecular-level perspective.
PMCID: PMC3578805  PMID: 23437294
13.  Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Cellular Uptake 
PLoS ONE  2012;7(12):e50902.
Two ruthenium(II) complexes, Λ-[Ru(phen)2(p-HPIP)]2+ and Δ-[Ru(phen)2(p-HPIP)]2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)2(p-HPIP)]2+ against telomerase, suggesting that Λ-[Ru(phen)2(p-HPIP)]2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)2(p-HPIP)]2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.
PMCID: PMC3517606  PMID: 23236402
14.  Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage 
The Journal of Neuroscience  2012;32(9):3044-3057.
Blood brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran, and promoted the secretion of metalloproteinase-2 and 9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2-h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.
PMCID: PMC3339570  PMID: 22378877
Cerebral ischemia; oxygen-glucose deprivation; blood brain barrier; matrix metalloproteinase; caveolin-1; tight junction proteins
15.  Investigation on Mycobacterium tuberculosis Diversity in China and the Origin of the Beijing Clade 
PLoS ONE  2011;6(12):e29190.
Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB) epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods.
Methodology/Principal Findings
We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2%) of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD) 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region.
This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion.
PMCID: PMC3248407  PMID: 22220207
16.  Normobaric hyperoxia protects the blood brain barrier through inhibiting Nox2 containing NADPH oxidase in ischemic stroke 
Medical Gas Research  2011;1:22.
Normobaric hyperoxia (NBO) has been shown to be neuro- and vaso-protective during ischemic stroke. However, the underlying mechanisms remain to be fully elucidated. Activation of NADPH oxidase critically contributes to ischemic brain damage via increase in ROS production. We herein tested the hypothesis that NBO protects the blood-brain barrier (BBB) via inhibiting gp91phox (or called Nox2) containing NADPH oxidase in a mouse model of middle cerebral artery occlusion (MCAO). Wild-type C57/BL6 mice and gp91phoxknockout mice were given NBO (95% O2) or normoxia (21% O2) during 90-min MCAO, followed by 22.5 hrs of reperfusion. BBB damage was quantified by measuring Evans blue extravasation. The protein levels of matrix metalloproteinase-9 (MMP-9), tight junction protein occludin and gp91phox were assessed with western blot. Gel zymography was used to assess the gelatinolytic activity of MMP-9. In the wild type mice, cerebral ischemia and reperfusion led to remarkable Evans blue extravasation, significantly increased gp91phox and MMP-9 levels and decreased occludin levels in the ischemic brain tissue. In gp91phox knockout mice, the changes in Evans blue extravasation, MMP-9 and occludin were at much smaller magnitudes when compared to the wild type. Importantly, NBO treatment significantly reduced the changes in all measured parameters in wild type mice, while did not cause additional reductions in these changes when gp91phox was knocked out. These results indicate that activation of Nox2 containing NADPH oxidase is implicated in the induction of MMP-9, loss of occludin and BBB disruption in ischemic stroke, and inhibition of Nox2 may be an important mechanism underlying NBO-afforded BBB protection.
PMCID: PMC3231818  PMID: 22146586
17.  Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China▿ †  
Journal of Clinical Microbiology  2010;48(11):4102-4106.
A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.
PMCID: PMC3020837  PMID: 20739484
18.  Association of interleukin-10 gene polymorphisms with breast cancer in a Chinese population 
Interleukin-10(IL-10) is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions. Polymorphisms in the IL-10 gene promoter genetically determine interindividual differences in IL-10 production. This study was performed to determined whether polymorphisms in the IL-10 gene promoter were associated with breast cancer in a Chinese Han population.
We genotyped 315 patients with breast cancer and 322 healthy control subjects for -1082A/G, -819T/C and -592A/C single nucleotide polymorphisms in the promoter region of the IL-10 gene by polymerase chain reactionerestriction fragment length polymorphism (PCR-RFLP).
There were no significant differences in genotype, allele, or haplotype frequencies in all three loci between patients and healthy controls. Analysis of breast cancer prognostic and predictive factors revealed that the -1082AA genotype was associated with a significantly increased risk of lymph node (LN) involvement (P = 0.041) and larger tumor size (P = 0.039) at the time of diagnosis. Furthermore, in the haplotype analysis of IL-10 gene, we found that patients carrying ATA haplotype were in higher LN involvement (p = 0.022) and higher tumor stage(p = 0.028) of breast cancer at the time of diagnosis compared with others.
Our findings suggest that IL-10 promoter polymorphisms participate in the progression of breast cancer rather than in its initial development in Chinese Han women.
PMCID: PMC2907337  PMID: 20553628
19.  Transcriptome Analysis of a cDNA Library from Adult Human Epididymis 
Mammalian Gene Collection (MGC) verified over 9000 human full-ORF genes and FLJ Program reported 21 243 cDNAs of which 14 409 were unique ones and 5416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20 000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website
PMCID: PMC2650638  PMID: 18390568
human epididymis cDNA library; transcriptomes for human epididymis; sperm maturation
21.  Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response 
PLoS ONE  2015;10(2):e0116697.
Salinity is a major abiotic stress that limits plant productivity and quality throughout the world. Roots are the sites of salt uptake. To better understand salt stress responses in maize, we performed a comparative proteomic analysis of seedling roots from the salt-tolerant genotype F63 and the salt-sensitive genotype F35 under 160 mM NaCl treatment for 2 days. Under salinity conditions, the shoot fresh weight and relative water content were significantly higher in F63 than in F35, while the osmotic potential was significantly lower and the reduction of the K+/Na+ ratio was significantly less pronounced in F63 than in F35. Using an iTRAQ approach, twenty-eight proteins showed more than 2.0- fold changes in abundance and were regarded as salt-responsive proteins. Among them, twenty-two were specifically regulated in F63 but remained constant in F35. These proteins were mainly involved in signal processing, water conservation, protein synthesis and biotic cross-tolerance, and could be the major contributors to the tolerant genotype of F63. Functional analysis of a salt-responsive protein was performed in yeast as a case study to confirm the salt-related functions of detected proteins. Taken together, the results of this study may be helpful for further elucidating salt tolerance mechanisms in maize.
PMCID: PMC4320067  PMID: 25659111
22.  Mechanisms of Gefitinib-mediated reversal of tamoxifen resistance in MCF-7 breast cancer cells by inducing ERα re-expression 
Scientific Reports  2015;5:7835.
Estrogen receptor (ER)-positive breast cancer patients may turn ER-negative and develop acquired drug resistance, which compromises the efficacy of endocrine therapy. By investigating the phenomenon that gefitinib can re-sensitise tamoxifen (TAM)-resistant MCF-7 breast cancer cells (MCF-7/TAM) to TAM, the present study verified that gefitinib could reverse the acquired drug resistance in endocrine therapy and further explored the underlying mechanism.ERα-negative MCF-7/TAM cells were established. Upon treating the cells with gefitinib, the mRNA and protein levels of ERα and ERβ, as well as the expression of molecules involved in the MAPK pathway, were examined using the RT-PCR and immunocytochemistry. The RT-PCR results showed that the mRNA levels of ERα and ERβ in MCF-7/TAM cells were up-regulated following gefitinib treatment; specifically, ERα was re-expressed, and ERβ expression was up-regulated. The expression of molecules involved in the MAPK pathway, including RAS, MEK1/2, and p-ERK1/2, in MCF-7/TAM cells was significantly up-regulated, compared with MCF-7 cells. After the gefitinib treatment, the expression levels of MEK1/2 and p-ERK1/2 were significantly down-regulated. ERα loss is the primary cause for TAM resistance. Gefitinib reverses TAM resistance primarily by up-regulating the ERα mRNA level and inducing the re-expression of ERα. The MAPK pathway plays a key role in ERα re-expression.
PMCID: PMC4314651  PMID: 25644501
23.  The miR9863 Family Regulates Distinct Mla Alleles in Barley to Attenuate NLR Receptor-Triggered Disease Resistance and Cell-Death Signaling 
PLoS Genetics  2014;10(12):e1004755.
Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.
Author Summary
Plants rely on cell-surface and intracellular immune receptors to sense pathogen invasion and to mediate defense responses. However, uncontrolled activation of immune responses is harmful to plant growth and development. Small RNAs have recently been shown to fine-tune the expression of intracellular immune receptors and contribute to the regulation of defense signaling in dicot plants, while similar processes have not been well documented in monocot grain crops, such as barley and wheat. Here, we show that, in barley, some members of the miR9863 family target a subset of Mla alleles that confer race-specific disease resistance to the powdery mildew fungus. These miRNAs act on Mla transcripts by cleavage and translational repression. Production of a type of trans-acting small RNAs, designated as phasiRNAs, enhances the effects of miRNA regulation on Mla targets. We propose that Mla-mediated immune signaling is fine-tuned by the miRNAs at later stage of MLA activation to avoid overloading of immune responses in barley cells.
PMCID: PMC4263374  PMID: 25502438
24.  Emerging Applications of Conjugated Polymers in Molecular Imaging 
In recent years, conjugated polymers have attracted considerable attention from the imaging community as a new class of contrast agent due to their intriguing structural, chemical, and optical properties. Their size and emission wavelength tunability, brightness, photostability, and low toxicity have been demonstrated in a wide range of in vitro sensing and cellular imaging applications, and have just begun to show impact in in vivo settings. In this Perspective, we summarize recent advances in engineering conjugated polymers as imaging contrast agents, their emerging applications in molecular imaging (referred to as in vivo uses in this paper), as well as our perspectives on future research.
PMCID: PMC3785565  PMID: 23860904
25.  miRNA-124 down-regulates SOX8 expression and suppresses cell proliferation in non-small cell lung cancer 
Non-small lung cell carcinoma (NSCLC) is a leading lethal disease and a global health burden. The function of the Sex determining region Y (SRY)-related high mobility group box (SOX) family gene in cancer has attracted the attention of more and more scientists recently, yet there are few reports regarding the role of SOX in NSCLC. Our study aimed to investigate the expression of SOX8, a protein belonging to the E group of the SOX family, as well as SOX9, in non-small cell lung cancer (NSCLC) and the relationship of gene expression to clinicopathological factors and prognosis in patients. Immunohistochemical analysis was used to measure the expression of SOX8 in 80 NSCLC and 7 adjacent normal tissues. SOX8 expression was detected as elevated in tumor samples and correlated to tumor size (P < 0.001), lymph node metastasis (P = 0.001), differentiation classification (P = 0.015), and clinical stage (P = 0.013) significantly. Moreover, Kaplan-Meier survival analysis demonstrated that shorter survival time for patients who had higher SOX8 expression (P < 0.001). In addition, our experiments indicate that miRNA-124 functions as a tumor suppressor in NSCLC. We also demonstrate miRNA-124 directly targeted and decreased SOX8 in NSCLC cell lines, suggesting smiRNA-124 may regulate NSCLC cell proliferation via decreasing SOX8 (oncogenicity of biomarker in NSCLC).
PMCID: PMC4270566  PMID: 25550787
NSCLC; miRNA-124; Sox8; IHC; lung cancer; survival analysis; cell proliferation

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