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1.  Characterization of one sheep border disease virus in China 
Virology Journal  2015;12:15.
Background
Border disease virus (BDV) causes border disease (BD) affecting mainly sheep and goats worldwide. BDV in goat herds suffering diarrhea was recently reported in China, however, infection in sheep was undetermined. Here, BDV infections of sheep herds in Jiangsu, China were screened; a BDV strain was isolated and identified from the sheep flocks in China. The genomic characteristics and pathogenesis of this new isolate were studied.
Results
In 2012, samples from 160 animals in 5 regions of Jiangsu province of China were screened for the presence of BDV genomic RNA and antibody by RT-PCR and ELISA, respectively. 44.4% of the sera were detected positively, and one slowly grown sheep was analyzed to be pestivirus RNA positive and antibody-negative. The sheep kept virus positive and antibody negative in the next 6 months of whole fattening period, and was defined as persistent infection (PI). The virus was isolated in MDBK cells without cytopathic effect (CPE) and named as JSLS12-01. Near-full-length genome sequenced was 12,227 nucleotides (nt). Phylogenetic analysis based on 5'-UTR and Npro fragments showed that the strain belonged to genotype 3, and shared varied homology with the other 3 BDV strains previously isolated from Chinese goats. The genome sequence of JSLS12-01 also had the highest homology with genotype BDV-3 (the strain Gifhorn). Experimental infections of sheep had mild clinical signs as depression and short-period mild fever (5 days). Viremia was detected in 1–7 days post-infection (dpi), and seroconversion began after 14 dpi.
Conclusions
This study reported the genomic and pathogenesis characterizations of one sheep BDV strain, which confirmed the occurrence of BDV infection in Chinese sheep. This sheep derived BDV strain was classified as BDV-3, together with the goat derived strains in China. These results might be helpful for further understanding of BDV infection in China and useful for prevention and control of BDV infections in the future.
doi:10.1186/s12985-014-0217-9
PMCID: PMC4329205
BDV; Complete genome sequence; Phylogenetic analysis; Experimental infection
2.  Rapid and sustained GluA1 S845 phosphorylation in synaptic and extrasynaptic locations in the rat forebrain following amphetamine administration 
Neurochemistry international  2013;64:10.1016/j.neuint.2013.11.001.
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is a major ionotropic glutamate receptor subtype in the mammalian brain. Like other glutamate receptors, the AMPA receptor is regulated by phosphorylation. By phosphorylating specific serine resides in AMPA receptor subunits (GluA1 and GluA2), various protein kinases regulate subcellular/subsynaptic expression and function of the receptor. In this study, we conducted a time course study to evaluate the temporal property of responses of phosphorylation at those sites to dopamine stimulation with the psychostimulant amphetamine in the adult rat striatum and medial prefrontal cortex (mPFC) in vivo. We focused on biochemically-enriched AMPA receptors from synaptic and extrasynaptic compartments. We found that acute injection of amphetamine induced a rapid and relatively sustained increase in GluA1 S845 phosphorylation at both synaptic and extrasynaptic sites in the striatum. Similar results were observed in the mPFC. In contrast to S845, amphetamine did not induce a significant change in GluA1 S831 phosphorylation in synaptic and extrasynaptic pools in the striatum and mPFC. GluA2 S880 phosphorylation in synaptic and extrasynaptic fractions in the two brain regions also remained stable in response to amphetamine. These results support S845 to be a principal site on AMPA receptors sensitive to acute stimulant exposure. Its phosphorylation levels are rapidly upregulated by amphetamine in the two defined subsynaptic microdomains (synaptic versus extrasynaptic locations) in striatal and cortical neurons.
doi:10.1016/j.neuint.2013.11.001
PMCID: PMC3880017  PMID: 24231469
Striatum; basal ganglia; prefrontal cortex; dopamine; glutamate; GluR; excitatory amino acid; addiction
3.  Calcitriol plays a protective role in diabetic nephropathy through anti-inflammatory effects 
Aims: To ascertain the protective role of calcitriol in the development of diabetic nephropathy and unravel the mechanism of the protective effects. Methods: In this prospective study, 69 patients were screened for type 1 diabetes, and 31 patients with type 1 diabetes were enrolled. Among these 31 patients, 24 patients had insufficient or deficient levels of serum vitamin D and 21 patients complied with calcitriol and were followed up. At baseline, these 21 patients who suffered from vitamin D deficiency or insufficiency displayed elevated inflammation markers and urinary albumin excretion in contrast with patients with sufficient vitamin D. Simultaneously, serum 25(OH)D3 level was negatively associated with serum and urinary inflammation markers, such as TNF-α, IL-6, and ICAM-1. Six months later, even though glycol-metabolism was not alleviated, all the serum and urinary inflammation markers decreased significantly. Meanwhile, proteinuria declined with inflammation markers. Results: Calcitriol supplementation alleviated inflammation and proteinuria in patients with type 1 diabetes. Conclusions: Calcitriol might delay the development of diabetic nephropathy through suppressing inflammation.
PMCID: PMC4307500  PMID: 25664053
Vitamin D; diabetic nephropathy; inflammation
4.  Differential Regulation of CaMKIIα Interactions with mGluR5 and NMDA Receptors by Ca2+ in Neurons 
Journal of neurochemistry  2013;127(5):620-631.
Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5) and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse-enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state-dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca2+/calmodulin binding and activation) loses its affinity for the receptor. Ca2+ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα-binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca2+-dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα-sensitive site. Together, the long intracellular C-terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5-dependent Ca2+ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to crosstalk between the two receptors.
doi:10.1111/jnc.12434
PMCID: PMC3933469  PMID: 24032403
striatum; nucleus accumbens; calcium; calmodulin; glutamate; mGluR; GluN2B; NR2B; G protein-coupled receptor
5.  Phase II Randomized, Placebo-controlled Trial of Green Tea Extract in Patients with High-risk Oral Premalignant Lesions 
Epidemiologic and preclinical data support the oral-cancer prevention potential of green tea extract (GTE). We randomly assigned patients with high-risk oral premalignant lesions (OPLs) to receive GTE at 500 mg/m2, 750 mg/m2, or 1000 mg/m2 or placebo TID for 12 weeks, evaluating biomarkers in baseline and 12-week biopsies. The OPL clinical response rate was higher in all GTE arms (n=28; 50%) versus placebo (n=11; 18.2%; p=0.09) but did not reach statistical significance. However, the 2 higher-dose GTE arms (58.8% [750, 1000 mg/m2], 36.4% [500 mg/m2], and 18.2%, [placebo], p=0.03) had higher responses, suggesting a dose-response effect. GTE treatment also improved histology (21.4% versus 9.1%, p=0.65), though not statistically significant. GTE was well-tolerated although higher doses increased insomnia/nervousness but produced no grade-4 toxicity. Higher mean baseline stromal VEGF correlated with a clinical (p=0.04) but not histologic response. Baseline scores of other biomarkers (epithelial VEGF, p53, Ki-67, cyclin D1, and p16 promoter methylation) were not associated with a response or survival. Baseline p16 promoter methylation (n=5) was associated with a shorter cancer-free survival. Stromal VEGF and cyclin D1 expression were downregulated in clinically responsive GTE patients and upregulated in non-responsive patients at 12 weeks (versus at baseline). An extended (median 27.5 months) follow-up showed a median time to oral cancer of 46.4 months. GTE may suppress OPLs, in part through reducing angiogenic stimulus (stromal VEGF). Higher doses of GTE may improve short-term (12 week) OPL outcome. The present results support longer-term clinical testing of GTE for oral cancer prevention.
doi:10.1158/1940-6207.CAPR-09-0121
PMCID: PMC4243312  PMID: 19892663
green tea extract; oral premalignant lesions; chemoprevention
6.  The BATTLE Trial: Personalizing Therapy for Lung Cancer 
Cancer discovery  2011;1(1):44-53.
Background
Lung cancer is the leading cancer cause of mortality worldwide; large-scale trials have failed to improve clinical outcomes of patients with chemorefractory non-small-cell lung cancer (NSCLC).
Methods
Following an initial equal randomization period, BATTLE adaptively randomized patients with chemorefractory NSCLC to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib based on molecular biomarkers of NSCLC pathogenesis in fresh core needle biopsy specimens. The primary end point was disease control rate (DCR) at 8 weeks.
Results
Of 255 patients randomly assigned to erlotinib (59 patients), vandetanib (54), erlotinib plus bexarotene (37), and sorafenib (105), 244 were eligible for the DCR analysis. Pneumothorax after lung biopsy occurred in 11.5% and treatment-related toxicities grade 3–4 in 6.5% of patients. Overall results were a 46% 8-week DCR, 1.9-month median progression-free survival, 9-month median overall survival, and 35% 1-year survival. Individual markers predicting a significantly superior DCR for a treatment included: epidermal growth factor receptor (EGFR) mutation (P=0.04) for erlotinib; cyclin D1 positivity (P=0.01) or EGFR amplification (P=0.006) for erlotinib plus bexarotene; vascular endothelial growth factor receptor 2 positivity (P=0.05) for vandetanib; and absence of EGFR mutation (P=0.01) or of EGFR high polysomy (P=0.05) for sorafenib. A better 8-week DCR occurred with sorafenib versus all other regimens (64% versus 33%; P<0.001) among EGFR wild-type patients and versus all other regimens (61% versus 32%; P=0.11) among mutant-KRAS patients. The prespecified biomarker groups were less predictive than the individual biomarkers analyzed in this study.
Conclusions
The first completed biopsy-mandated study in pretreated NSCLC, BATTLE confirmed our pre-specified hypotheses regarding biomarker and targeted treatment interactions, establishing a new paradigm for personalizing therapy for patients with NSCLC. (ClinicalTrials.gov numbers, NCT00409968, NCT00411671, NCT00411632, NCT00410059, NCT00410189.)
doi:10.1158/2159-8274.CD-10-0010
PMCID: PMC4211116  PMID: 22586319
7.  Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity 
PLoS ONE  2014;9(10):e110826.
Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.
doi:10.1371/journal.pone.0110826
PMCID: PMC4207755  PMID: 25340750
8.  Oral Epithelium as a Surrogate Tissue for Assessing Smoking-Induced Molecular Alterations in Lungs 
Background
Both the lungs and oral cavity are exposed to tobacco carcinogens in smokers. We hypothesized that the oral epithelium undergoes molecular alterations similar to those in lungs and therefore may be used as a surrogate tissue to assess tobacco-induced molecular alterations.
Methods
Promoter methylation of p16 and FHIT genes was analyzed with methylation-specific PCR in 1,774 oral and bronchial brush specimens (baseline and 3 months after intervention) from 127 smokers enrolled in a prospective randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites/total sites per subject) was analyzed blindly.
Results
At baseline, promoter methylation was observed in 23%, 17%, and 35% of the bronchial tissues for p16, FHIT, and either of the two genes, respectively, which were comparable to the 19%, 15%, and 31% observed in the oral tissues. Among the 125 individuals with available data from both oral and bronchial tissues, strong correlations were observed between tissues from the two sites (P<0.0001 for both p16 and FHIT). Among the 39 individuals with oral tissue methylation in either of the two genes, the mean bronchial methylation index was 0.53 (± 0.29) compared with only 0.27 (± 0.26) for the 86 subjects without oral tissue methylation (P<0.0001). Similar correlations were also observed in samples obtained at 3 months after chemopreventive intervention.
Conclusions
The oral epithelium may be used as a surrogate tissue to assess tobacco-induced molecular damage in lungs, which has an important implication in conducting biomarker-based lung cancer prevention trials.
doi:10.1158/1940-6207.CAPR-08-0058
PMCID: PMC4183362  PMID: 19138934
9.  Impact of smoking cessation on global gene expression in the bronchial epithelium of chronic smokers 
Cigarette smoke is the major cause of lung cancer and can interact in complex ways with drugs for lung cancer prevention or therapy. Molecular genetic research promises to elucidate the biologic mechanisms underlying divergent drug effects in smokers versus non-smokers and to help in developing new approaches for controlling lung cancer. The present study compared global gene expression profiles (determined via Affymetrix microarray measurements in bronchial epithelial cells) between chronic smokers, former smokers, and never smokers. Smoking effects on global gene expression were determined from a combined analysis of three independent datasets. Differential expression between current and never smokers occurred in 591 of the 13,902 genes measured on the microarrays (P < 0.01 and >2 fold change; pooled data)—a profound effect. In contrast, differential expression between current and former smokers occurred in only 145 of the measured genes (P < 0.01 and >2 fold change; pooled data). Nine of these 145 genes showed consistent and significant changes in each of the three datasets (P < 0.01 and >2 fold change), with 8 being down-regulated in former smokers. Seven of the 8 down-regulated genes, including CYP1B1 and 3 AKR genes, influence the metabolism of carcinogens and/or therapeutic/chemopreventive agents. Our data comparing former and current smokers allowed us to pinpoint the genes involved in smoking–drug interactions in lung cancer prevention and therapy. These findings have important implications for developing new targeted and dosing approaches for prevention and therapy in the lung and other sites, highlighting the importance of monitoring smoking status in patients receiving oncologic drug interventions.
doi:10.1158/1940-6207.CAPR-07-0017
PMCID: PMC4181408  PMID: 19138944
10.  MicroRNA expression profile in exosome discriminates extremely severe infections from mild infections for hand, foot and mouth disease 
BMC Infectious Diseases  2014;14(1):506.
Background
Changes of miRNAs in exosome have been reported in different disease diagnosis and provided as potential biomarkers. In this study, we compared microRNA profile in exosomes in 5 MHFMD and 5 ESHFMD as well as in 5 healthy children.
Methods
Different expression of miRNAs in exosomes across all the three groups were screened using miRNA microarray method. Further validated test was conducted through quantitative real-time PCR assays with 54 exosome samples (18 ESHFMD, 18 MHFMD, and 18 healthy control). The judgment accuracy was then estimated by the receiver operating characteristic (ROC) curve analysis; and the specificity and sensitivity were evaluated by the multiple logistic regression analysis.
Results
There were 11 different miRNAs in exosomes of MHFMD and ESHFMD compared to healthy children, of which 4 were up-regulated and 7 were down-regulated. Further validation indicated that the 4 significant differentially expressed candidate miRNAs (miR-671-5p, miR-16-5p, miR-150-3p, and miR-4281) in exosome showed the same changes as in the microarray analysis, and the expression level of three miRNAs (miR-671-5p, miR-16-5p, and miR-150-3p) were significantly different between MHFMD or ESHFMD and the healthy controls. The accuracy of the test results were high with the under curve (AUC) value range from 0.79 to 1.00. They also provided a specificity of 72%-100% and a sensitivity of 78%-100%, which possessed ability to discriminate ESHFMD from MHFMD with the AUC value of 0.76-0.82.
Conclusions
This study indicated that the exosomal miRNA from patients with different condition of HFMD express unique miRNA profiles. Exosomal miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping HFMD infections.
doi:10.1186/1471-2334-14-506
PMCID: PMC4262082  PMID: 25231540
Exosomal microRNA Profile; HFMD; Diagnosis; Biomarker
11.  Regulation of phosphorylation of synaptic and extrasynaptic GluA1 AMPA receptors in the rat forebrain by amphetamine 
European journal of pharmacology  2013;715(0):164-171.
The AMPA receptor is regulated by phosphorylation. Two major phosphorylation sites (S831 and S845) are located in the intracellular C-terminal tail of GluA1 subunits. The phosphorylation on these sites controls receptor expression and function and is subject to the regulation by psychostimulants. In this study, we further characterized the regulation of S831 and S845 phosphorylation by amphetamine (AMPH) in the adult rat striatum and medial prefrontal cortex (mPFC) in vivo. We focused on the specific fraction of GluA1/AMPA receptors enriched from synaptic and extrasynaptic membranes, using a pre-validated biochemical fractionation procedure. We found that acute AMPH administration elevated GluA1 S845 phosphorylation in the defined synaptic membrane from the striatum in a dose-dependent manner. AMPH also induced a comparable increase in S845 phosphorylation in the extrasynaptic fraction of striatal GluA1. Similar increases in S845 phosphorylation in both synaptic and extrasynaptic pools were observed in the mPFC. In contrast, S831 phosphorylation was not altered in synaptic and extrasynaptic GluA1 in striatal neurons and synaptic GluA1 in mPFC neurons in response to AMPH, although a moderate increase in S831 phosphorylation was seen in extrasynaptic GluA1 after an AMPH injection at a high dose. Total synaptic and extrasynaptic GluA1 expression remained stable in the two regions after AMPH administration. Our data demonstrate the differential sensitivity of S845 and S831 phosphorylation to dopamine stimulation. S845 is a primary site where phosphorylation of GluA1 is upregulated by AMPH in striatal and mPFC neurons at both synaptic and extrasynaptic compartments.
doi:10.1016/j.ejphar.2013.05.027
PMCID: PMC3763922  PMID: 23747591
Striatum; basal ganglia; striatum; prefrontal cortex; dopamine; glutamate; GluR1; excitatory amino acid; addiction
12.  Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance 
Purpose
Salivary duct carcinoma over-expresses epidermal growth factor receptor (EGFR) and HER-2 though underlying mechanisms remain undefined. Because of potential utilization of these markers as treatment targets, we evaluated protein and gene status by several techniques to determine complementary value.
Experimental Design
A tissue microarray of 66 salivary duct carcinomas was used for immunohistochemical analysis of HER-2 and EGFR expression (semiquanititively evaluated into a 3-tiered system), and fluorescence in situ hybridization for gene copy number, and chromosomes 7 and 17 ploidy status. Sequencing of Exons 18, 19 and 21 of the EGFR gene for mutations was performed.
Results
EGFR Forty-six (69.7%) of the 66 tumors showed some form of EGFR expression (17,3+; 17,2+; 12,1+) but none gene amplification. Five (9.4%) of 53 tumors showed mutations in exon 18 (3) and exon 19 (2). Polysomy of chromosome 7(average >2.5 copies per cell) was detected in 15 (25.0%) of 60 tumors (6,3+; 5,2+; 2,1+; 2,0+ expression) and correlated with poor 3-year survival (p=0.015). HER-2: Seventeen (25.8%) of 66 tumors expressed HER-2 (10,3+; 3,2+; 4,1+). Eight tumors showed HER-2 gene amplification (6,3+; 1,1+; 1,0+ protein expression). Chromosome 17 polysomy was found in eight (15.7%) of 51 tumors; two with HER-2 expression (3+; 1+).
Conclusion
Our study shows that salivary duct carcinomas: 1) harbor EGFR gene mutations in a subset of tumors which may guide therapy, 2) pursue aggressive clinical course in cases with Chromosome 7 polysomy and high EGFR expression, and 3) with HER-2 gene amplification and protein high-expression maybe selected for targeted therapy.
doi:10.1158/1078-0432.CCR-09-0238
PMCID: PMC4152860  PMID: 20371674
Salivary duct carcinoma; epidermal growth factor receptor; HER-2; Fluorescence in situ hybridization; polysomy; mutation
13.  Dynamic downregulation of Nogo receptor expression in the rat forebrain by amphetamine 
Neurochemistry international  2013;63(3):195-200.
Nogo receptors (NgR) are a family of cell surface receptors that are broadly expressed in the mammalian brain. These receptors could serve as an inhibitory element in the regulation of activity-dependent axonal growth and spine and synaptic formation in the adult animal brain. Thus, through balancing the structural response to changing cellular and synaptic inputs, NgRs participate in constructing activity-dependent morphological plasticity. Psychostimulants have been well documented to induce morphological plasticity critical for addictive properties of stimulants, although underlying molecular mechanisms are poorly understood. In this study, we initiated a study to investigate the response of NgRs to a stimulant. We tested the effect of acute administration of amphetamine on protein expression of two principal NgR subtypes (NgR1 and NgR2) in the rat striatum, medial prefrontal cortex (mPFC) and hippocampus. We found that a single injection of amphetamine induced a rapid and time-dependent decrease in NgR1 and NgR2 expression in the striatum and mPFC. A relatively delayed and time-dependent decrease in expression of the two receptors was seen in the hippocampus. The drug-induced decrease in NgR1 and NgR2 expression in the three forebrain regions was dose-dependent. A behaviorally active dose of the drug was required to trigger a significant reduction in NgR1 and NgR2 expression. These data indicate that NgRs are subject to the regulation by the stimulant. Amphetamine exposure exerts the inhibitory modulation of basal NgR1 and NgR2 expression in the key structures of reward circuits in vivo.
doi:10.1016/j.neuint.2013.06.006
PMCID: PMC3735811  PMID: 23770273
NgR; reticulon 4 receptor; Nogo-66; stimulant; addiction; spine density; striatum; prefrontal cortex; hippocampus
14.  Zn-Responsive Proteome Profiling and Time-Dependent Expression of Proteins Regulated by MTF-1 in A549 Cells 
PLoS ONE  2014;9(8):e105797.
Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways.
doi:10.1371/journal.pone.0105797
PMCID: PMC4146543  PMID: 25162517
15.  Novel dimensions of piRNAs in cancer 
Cancer letters  2013;336(1):46-52.
Piwi-interacting RNAs (piRNAs), a newly identified class of small non-coding RNAs, direct the Piwi-dependent transposon silencing, heterochromatin modification and germ cell maintenance. Owing to our limited knowledge regarding their biogenesis, piRNAs are considered as the most mysterious class of small regulatory RNAs, particularly in pathogenesis such as tumorigenesis. Recently, several lines of evidence have emerged to suggest that piRNAs may be dis-regulated and play crucial roles in tumorigenesis in previously unsuspected ways. In this prospective piece, we will discuss the emerging insights into the potential novel roles of piRNAs in carcinogenesis and highlight their potential implications in cancer detection, classification and therapy.
doi:10.1016/j.canlet.2013.04.008
PMCID: PMC3707114  PMID: 23603435
PIWI-interacting RNAs; cancer; diagnosis; therapy
16.  The rice endophyte Harpophora oryzae genome reveals evolution from a pathogen to a mutualistic endophyte 
Scientific Reports  2014;4:5783.
The fungus Harpophora oryzae is a close relative of the pathogen Magnaporthe oryzae and a beneficial endosymbiont of wild rice. Here, we show that H. oryzae evolved from a pathogenic ancestor. The overall genomic structures of H. and M. oryzae were found to be similar. However, during interactions with rice, the expression of 11.7% of all genes showed opposing trends in the two fungi, suggesting differences in gene regulation. Moreover, infection patterns, triggering of host defense responses, signal transduction and nutritional preferences exhibited remarkable differentiation between the two fungi. In addition, the H. oryzae genome was found to contain thousands of loci of transposon-like elements, which led to the disruption of 929 genes. Our results indicate that the gain or loss of orphan genes, DNA duplications, gene family expansions and the frequent translocation of transposon-like elements have been important factors in the evolution of this endosymbiont from a pathogenic ancestor.
doi:10.1038/srep05783
PMCID: PMC4105740  PMID: 25048173
17.  Anti-HDGF targets cancer and cancer stromal stem cells resistant to chemotherapy 
Purpose
Approximately 1/3 of the patients with advanced non-small cell lung cancer (NSCLC) will initially respond to platinum-based chemotherapy, but virtually all tumors will progress (acquired resistance). The remainder will progress during initial treatment (primary resistance). In this study, we test whether the treatment can be improved by inhibiting hepatoma-derived growth factor (HDGF).
Methods
Thirteen primary NSCLC hetero-transplant models were used to test four treatment regimens including platinum-based chemotherapy with and without bevacizumab (VEGF neutralizing antibody) or HDGF-H3 (HDGF neutralizing antibody) and chemotherapy with bevacizumab and HDGF-H3. Expression of stem-cell related genes was measured using quantitative RT-PCR and immunohistochemistry.
Results
Among 13 primary NSCLC hetero-transplant models, 3 (23%) responded to chemotherapy but all relapsed within 20 days. The residual tumors after response to the chemotherapy exhibited an increased expression in 51 (61%) of 84 genes related with stem-cell proliferation and maintenance, particularly those in Notch and Wnt pathways, suggesting an enrichment for stem-cell populations in the residual tumors. Interestingly, tumors from 2 of the 3 models treated with HDGF-H3, bevacizumab, and chemotherapy combination did not relapse during 6 months of post treatment observation. Importantly, this treatment combination substantially down-regulated expression levels in 57 (68%) of the 84 stem-cell related genes, including 34 (67%) of the 51 genes up-regulated after the chemotherapy.
Conclusion
These data support the hypothesis that cancer stem-cells (CSCs) are a mechanism for chemotherapy resistance and suggest HDGF may be a target for repressing CSCs to prevent relapse of NSCLC sensitive to chemotherapy.
doi:10.1158/1078-0432.CCR-12-3478
PMCID: PMC3707124  PMID: 23695169
HDGF; antibody; NSCLC; cancer stem-cell; therapy
18.  Methylenetetrahydrofolate reductase C677T gene polymorphism and essential hypertension: A meta-analysis of 10,415 subjects 
Biomedical Reports  2014;2(5):699-708.
The methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism has been suggested to be associated with the risk of essential hypertension (EH), however, results remain inconclusive. To investigate this association, the present meta-analysis of 27 studies including 5,418 cases and 4,997 controls was performed. The pooled odds ratio (OR) and its corresponding 95% confidence interval were calculated using the random-effects model. A significant association between the MTHFR C677T gene polymorphism and EH was found under the allelic (OR, 1.32; 95% CI, 1.20–1.45; P=0.000), dominant (OR, 1.39; 95% CI, 1.25–1.55; P=0.000), recessive (OR, 1.38; 95% CI, 1.18–1.62; P=0.000), homozygote (OR, 1.59; 95% CI, 1.32–1.92; P=0.000), and heterozygote (OR, 1.32; 95% CI, 1.20–1.45; P=0.000) genetic models. A strong association was also revealed in subgroups, including Asian, Caucasian and Chinese. The Japanese subgroup did not show any significant association under all models. Meta-regression analyses suggested that the study design was a potential source of heterogeneity, whereas the subgroup analysis additionally indicated that the population origin may also be an explanation. Another subgroup analysis revealed that hospital-based studies have a stronger association than population-based studies, however, the former suffered a greater heterogeneity. Funnel plot and Egger’s test manifested no evidence of publication bias. In conclusion, the present study supports the evidence for the association between the MTHFR C677T gene polymorphism and EH in the whole population, as well as in subgroups, such as Asian, Caucasian and Chinese. The carriers of the 677T allele are susceptible to EH.
doi:10.3892/br.2014.302
PMCID: PMC4106611  PMID: 25054014
methylenetetrahydrofolate reductase; meta-analysis; essential hypertension; gene polymorphism
19.  Biological Activity of Celecoxib in the Bronchial Epithelium of Current and Former Smokers 
Purpose
Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death in Western countries. One important approach taken to address this problem is the development of effective chemoprevention strategies. In this study, we examined whether the cyclooxygenase-2 (COX-2) inhibitor celecoxib, as evidenced by decreased cell proliferation, is biologically active in the bronchial epithelium of current and former smokers.
Patients and Methods
Current or former smokers with at least a 20 pack-year (pack-year = number of packs of cigarettes per day times number of years smoked) smoking history were randomized into one of four treatment arms (3-month intervals of celecoxib then placebo, celecoxib then celecoxib, placebo then celecoxib, or placebo then placebo) and underwent bronchoscopies with biopsies at baseline, 3 months, and 6 months. The 204 patients were primarily (79.4%) current smokers; 81 received either low-dose celecoxib or placebo and 123 received either high-dose celecoxib or placebo. Celecoxib was originally administered orally at 200 mg twice daily and the protocol subsequently increased the dose to 400 mg twice daily. The primary endpoint was change in Ki-67 labeling (from baseline to 3 months) in bronchial epithelium.
Results
No cardiac toxicities were observed in the participants. Although the effect of low-dose treatment was not significant, high-dose celecoxib decreased Ki-67 labeling by 3.85% in former smokers and by 1.10% in current smokers—a significantly greater reduction (P = 0.02) than that seen with placebo after adjusting for metaplasia and smoking status.
Conclusion
A 3–6-month celecoxib regimen proved safe to administer. Celecoxib 400 mg bid was biologically active in the bronchial epithelium of current and former smokers; additional studies on the efficacy of celecoxib in NSCLC chemoprevention may be warranted.
doi:10.1158/1940-6207.CAPR-09-0233
PMCID: PMC4028718  PMID: 20103722
20.  Exosomal Proteome Profiling: A Potential Multi-Marker Cellular Phenotyping Tool to Characterize Hypoxia-Induced Radiation Resistance in Breast Cancer 
Proteomes  2013;1(2):87-108.
Radiation and drug resistance are significant challenges in the treatment of locally advanced, recurrent and metastatic breast cancer that contribute to mortality. Clinically, radiotherapy requires oxygen to generate cytotoxic free radicals that cause DNA damage and allow that damage to become fixed in the genome rather than repaired. However, approximately 40% of all breast cancers have hypoxic tumor microenvironments that render cancer cells significantly more resistant to irradiation. Hypoxic stimuli trigger changes in the cell death/survival pathway that lead to increased cellular radiation resistance. As a result, the development of noninvasive strategies to assess tumor hypoxia in breast cancer has recently received considerable attention. Exosomes are secreted nanovesicles that have roles in paracrine signaling during breast tumor progression, including tumor-stromal interactions, activation of proliferative pathways and immunosuppression. The recent development of protocols to isolate and purify exosomes, as well as advances in mass spectrometry-based proteomics have facilitated the comprehensive analysis of exosome content and function. Using these tools, studies have demonstrated that the proteome profiles of tumor-derived exosomes are indicative of the oxygenation status of patient tumors. They have also demonstrated that exosome signaling pathways are potentially targetable drivers of hypoxia-dependent intercellular signaling during tumorigenesis. This article provides an overview of how proteomic tools can be effectively used to characterize exosomes and elucidate fundamental signaling pathways and survival mechanisms underlying hypoxia-mediated radiation resistance in breast cancer.
doi:10.3390/proteomes1020087
PMCID: PMC4029595  PMID: 24860738
hypoxia; radiation; breast cancer; tumor microenvironment; exosomes; proteomics
21.  Cardioprotective activity of placental growth factor in a rat model of acute myocardial infarction: nanoparticle-based delivery versus direct myocardial injection 
Background
To comparatively evaluate the cardioprotective activity of placental growth factor (PGF) delivered through direct injection and a nanoparticle-based system respectively and to study the underlying mechanisms in a rat model of acute myocardial infarction (AMI).
Methods
Poly lactic-co-glycolic acid (PLGA)-based PGF-carrying nanoparticles (PGF-PLGANPs) were created. The mean size and morphology of particles were analyzed with particle size analyzer and transmission electronic microscopy (TEM). Encapsulation efficiency and sustained-release dose curve were analyzed by ELISA. Sprague-Dawley rats were randomized into four groups (n = 10). While animals in the first group were left untreated as controls, those in the other 3 groups underwent surgical induction of AMI, followed by treatment with physiological saline, PGF, and PGF-PLGANPs, respectively. Cardiac function was evaluated by transthoracic echocardiography at 4 weeks after treatment. At 6 weeks, rats were sacrificed, infarction size was analyzed with Masson trichrome staining, and protein contents of TIMP-2, MT1-MMP and MMP-2 at the infarction border were determined by immunohistochemistry and western blotting analysis.
Results
PGF was released for at least 15 days, showing successful preparation of PGF-PLGANPs. Coronary artery ligation successfully induced AMI. Compared to physiological saline control, PGF, injected to the myocardium either as a nude molecule or in a form of nanoparticles, significantly reduced infarction size, improved cardiac function, and elevated myocardial expression of TIMP-2, MT1-MMP, and MMP-2 (P < 0.05). The effect of PGF-PLGANPs was more pronounced than that of non-encapsulated PGF (P < 0.05).
Conclusion
Target PGF delivery to myocardium may improve cardiac function after AMI in rats. PLGA-based nanoparticles appear to be a better approach to delivery PGF. PGF exerts its cardioprotective effect at least partially through regulating metalloproteinase-mediated myocardial tissue remodeling.
doi:10.1186/1471-2261-14-53
PMCID: PMC4014437  PMID: 24742302
Placental growth factor; Acute myocardial infarction; Cardiac function; Nanoparticles-mediated drug delivery; Vicious ventricular remodeling
22.  Smurf1-Mediated Lys29-Linked Nonproteolytic Polyubiquitination of Axin Negatively Regulates Wnt/β-Catenin Signaling 
Molecular and Cellular Biology  2013;33(20):4095-4105.
Ubiquitination plays important and diverse roles in modulating protein functions. As a C2-WW-HECT-type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Here, we report a novel function of Smurf1 in regulating Wnt/β-catenin signaling through targeting axin for nonproteolytic ubiquitination. Our data unambiguously demonstrate that Smurf1 ubiquitinates axin through Lys 29 (K29)-linked polyubiquitin chains. Unexpectedly, Smurf1-mediated axin ubiquitination does not lead to its degradation but instead disrupts its interaction with the Wnt coreceptors LRP5/6, which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling. The inhibitory function of Smurf1 on Wnt/β-catenin signaling is further evidenced by analysis with Smurf1 knockout murine embryonic fibroblasts. We next identified K789 and K821 in axin as the ubiquitination sites by Smurf1. Consistently, Smurf1 could neither disrupt the interaction of an axinK789/821R double mutant with LRP5/6 nor attenuate the phosphorylation of LRP6 in axinK789/821R-expressing cells. Collectively, our studies uncover Smurf1 as a new regulator for the Wnt/β-catenin signaling pathway via modulating the activity of axin.
doi:10.1128/MCB.00418-13
PMCID: PMC3811687  PMID: 23959799
23.  Effect of salidroside on lung injury by upregulating peroxisome proliferator-activated receptor γ expression in septic rats 
Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. Thus, the aim of the present study was to investigate the beneficial effects of salidroside on ameliorating cecal ligation and puncture (CLP)-induced lung inflammation. Rats underwent CLP surgery to induce ALI and 800 mg/kg salidroside (i.v.) was administered 24 h after the CLP challenge. Subsequently, biochemical changes in the blood and lung tissues, as well as morphological and histological alterations in the lungs, that were associated with inflammation and injury were analysed. CLP was shown to significantly increase the serum levels of plasma tumour necrosis factor-α and interleukin-6, -1β and-10. In addition, CLP increased pulmonary oedema, thickened the alveolar septa and caused inflammation in the lung cells. These changes were ameliorated by the administration of 800 mg/kg salidroside (i.v.) 24 h after the CLP challenge. This post-treatment drug administration also significantly attenuated the lipopolysaccharide-induced activation of nuclear factor-κβ and increased the release of peroxisome proliferator-activated receptor γ in the lung tissue. Therefore, salidroside administered following the induction of ALI by CLP significantly prevented and reversed lung tissue injuries. The positive post-treatment effects of salidroside administration indicated that salidroside may be a potential candidate for the management of lung inflammation in CLP-induced endotoxemia and septic shock.
doi:10.3892/etm.2014.1629
PMCID: PMC4043580  PMID: 24926325
sepsis; peroxisome proliferator-activated receptor γ; salidroside; rat; cecal ligation and puncture; lung injury
24.  Plasminogen Activator Inhibitor-1 Is Involved in Impaired Bone Repair Associated with Diabetes in Female Mice 
PLoS ONE  2014;9(3):e92686.
Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1+/+) and PAI-1-deficient (PAI-1−/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1+/+ and PAI-1−/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1+/+ mice, this delayed bone repair was blunted in PAI-1−/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1−/− mice compared to PAI-1+/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1+/+ mice, were partially protected in PAI-1−/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.
doi:10.1371/journal.pone.0092686
PMCID: PMC3961397  PMID: 24651693
25.  Effect of melilotus extract on lung injury by upregulating the expression of cannabinoid CB2 receptors in septic rats 
Background
M. Suaveolens Ledeb has long been used in China to treat inflammatory infectious diseases. Melilotus is extracted from Melilotus Suaveolens Ledeb and its therapeutic potential is associated with its anti-inflammatory activity. However, the precise mechanisms underlying its effects are unknown. This study was conducted to evaluate the protective effects of melilotus extract in a rat cecal ligation and puncture (CLP)-induced animal model of acute lung injury (ALI).
Methods
A sepsis model was induced by CLP-like lung inflammation. Two hours prior to CLP administration, the treatment group was administered melilotus extract via oral injection. RT-PCR and Western blotting were used to test the expression of cannabinoid receptor (CB)2, NF-κβ and IκB from single peripheral blood mononuclear cells and lung tissues respectively. Enzyme linked immune sorbent assay was used to detect serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and IL-12. The numbers of neutrophils, lymphocytes, macrophages and total cells in the bronchoalveolar lavage (BAL) fluid were counted. For histologic analysis, hematoxylin and eosin (H&E) stains were evaluated.
Results
After inducing ALI by CLP for 24 hours, melilotus extract up-regulated peripheral blood mononuclear cell CB2 expression, blocked the activity of NF-κβ65, and the number of neutrophils, lymphocytes and total cells were significantly lower in the melilotus extract group than the control group. In addition, TNF-α and IL-6 levels were significantly decreased in the melilotus extract group. Histological results demonstrated the attenuation effect of melilotus extract on CLP-induced lung inflammation. CB2 was negatively correlated to NF-κβ mRNA and proteins, respectively (r = -0.377, P < 0.05; r = -0.441, P < 0.05).
Conclusion
The results of this study indicated melilotus extract significantly reduced CLP-induced lung inflammation by up-regulating CB2 expression. The remarkable protective effects of melilotus extract suggest its therapeutic potential in CLP induced-acute lung injury treatment.
doi:10.1186/1472-6882-14-94
PMCID: PMC3995869  PMID: 24612782
Sepsis; Melilotus extract; Cannabinoid receptors; Cecal ligation and puncture; Rats

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