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author:("Li, xiaoyan")
1.  The impact of histological types on the efficacy of angiogenesis inhibitors in the treatment of advanced NSCLC: a meta-analysis of randomized controlled trials 
OncoTargets and therapy  2015;8:2375-2382.
We aimed at assessing the overall efficacy of angiogenesis inhibitor (AI)-containing regimens in the treatment of advanced non-small-cell lung cancer (NSCLC) according to histological types.
Studies from PubMed and Web of Science, and abstracts presented at American Society of Clinical Oncology (ASCO) meeting up to October 31, 2014 were searched to identify relevant studies. Eligible studies included prospective randomized controlled trials (RCTs) evaluating AIs in advanced NSCLC with survival data according to patients’ histologies. The endpoints were overall survival (OS) and progression-free survival (PFS). Statistical analyses were conducted by using either random effects or fixed effect models according to the heterogeneity of included studies.
A total of 10,035 patients with advanced NSCLC from 13 RCTs were identified for analysis. The pooled results demonstrated that AI-containing regimens significantly improved the PFS (HR, 0.84, 95% confidence interval (CI): 0.78–0.91, P<0.001) and OS (HR, 0.92, 95% CI: 0.85–0.99, P=0.017) in lung adenocarcinoma when compared to non-AI-containing regimens. Additionally, there was a significantly improved PFS (HR, 0.87, 95% CI: 0.77–0.98, P=0.027) for AI-containing regimens in squamous cell lung carcinoma, but it did not translated into OS benefit (HR, 1.02, 95% CI: 0.92–1.15, P=0.68). For NSCLC patients with other histological types, the use of AIs did not significantly improve PFS (HR, 0.90, 95% CI: 0.75–1.09, P=0.27) and OS (HR, 0.90, 95% CI: 0.76–1.08, P=0.19).
The findings of this study suggest that the addition of AIs to the treatment therapies for patients with lung adenocarcinoma offers improved survival benefits. Prospective clinical trials investigating the role of AIs in this setting are recommended.
PMCID: PMC4562761  PMID: 26366091
non-small-cell lung cancer; histological types; randomized controlled trials; angio-genesis inhibitors; meta-analysis
2.  Curcumin inhibits lung cancer invasion and metastasis by attenuating GLUT1/MT1-MMP/MMP2 pathway 
Glucose transporter (GLUT) 1 is found highly expressed in malignant tumors and considered a mediator inducing cancer metastasis. Curcumin is a natural product which exerts anti-invasion and metastasis effects in cancer. This study aimed at evaluating whether attenuating GLUT1 was involved in curcumin’s anti-invasion and metastasis effects. In the in vitro part, constricted pcDNA3.1-GLUT1 vector was transfected into A549 cells. MTT assay was used to assess the curcumin’s effects on proliferation in lung cancer A549 cells. Transwell assay was used to evaluate the anti-invasion effect of curcumin on A549 cells. Real-time PCR and Western-blotting were employed to examine the expression levels of GLUT1, membrane type 1-MMP (MT1-MMP) and matrix metalloproteinase (MMP) 2 in curcumin- incubated A549 cells. In the in vivo part, tumor weight and metastatic rate were assessed in nude mice bearing untransfected, empty vector transfected and pcDNA3.1-GLUT1 transfected A549 cells originated tumors. In this study, we found that curcumin began to show significant cytotoxicity against proliferation effect at 45 μmol/L. Curcumin inhibited invasion and expressions of GLUT1, MT1-MMP and MMP2 untransfected A549 cells in a concentration-dependent manner. pcDNA3.1-GLUT1 transfected A549 cells exhibited resistance to curcumin’s anti-invasion effect by up-regulating expressions of GLUT2, MT1-MMP and MMP2. Furthermore, curcumin failed to decrease the metastatic rate in nude mice bearing pcDNA3.1-GLUT1 transfected A549 cells originated tumors. These results suggested that curcumin inhibit lung cancer invasion and metastasis by attenuating GLUT1/MT1-MMP/MMP2 pathway.
PMCID: PMC4538170  PMID: 26309547
Lung cancer; curcumin; glucose transporter
3.  Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay 
PLoS ONE  2015;10(4):e0125701.
The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.
PMCID: PMC4410951  PMID: 25915630
4.  Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease 
Viruses  2015;7(4):1558-1577.
Enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease with especially severe neurologic complications, which mainly account for fatalities from this disease. To date, the pathogenesis of EV71 in the central neurons system has remained unclear. Cytokine-mediated immunopathogenesis and nervous tissue damage by virus proliferation are two widely speculated causes of the neurological disease. To further study the pathogenesis, we identified a common epitope (co-epitope) between EV71 VP1 and human mediator complex subunit 25 (MED25) highly expressed in brain stem. A monoclonal antibody (2H2) against the co-epitope was prepared, and its interaction with MED25 was examined by ELISA, immunofluorescence assay and Western blot in vitro and by live small animal imaging in vivo. Additionally, 2H2 could bind to both VP1 and MED25 with the affinity constant (Kd) of 10−7 M as determined by the ForteBio Octet System. Intravenously injected 2H2 was distributed in brain stem of mice after seven days of EV71 infection. Interestingly, 2H2-like antibodies were detected in the serum of EV71-infected patients. These findings suggest that EV71 infection induces the production of antibodies that can bind to autoantigens expressed in nervous tissue and maybe further trigger autoimmune reactions resulting in neurological disease.
PMCID: PMC4411665  PMID: 25826188
enterovirus 71; common antigen; MED25; neurological disease
5.  Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein 
PLoS ONE  2015;10(2):e0118041.
The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.
Methods and Results
To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.
Conclusions and Significance
We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.
PMCID: PMC4337900  PMID: 25706372
6.  Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction 
PLoS ONE  2014;9(10):e111422.
BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif “KRXK” in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.
PMCID: PMC4210262  PMID: 25347789
7.  Recent advances in renal interstitial fibrosis and tubular atrophy after kidney transplantation 
Although kidney transplantation has been an important means for the treatment of patients with end stage of renal disease, the long-term survival rate of the renal allograft remains a challenge. The cause of late renal allograft loss, once known as chronic allograft nephropathy, has been renamed “interstitial fibrosis and tubular atrophy” (IF/TA) to reflect the histologic pattern seen on biopsy. The mechanisms leading to IF/TA in the transplanted kidney include inflammation, activation of renal fibroblasts, and deposition of extracellular matrix proteins. Identifying the mediators and factors that trigger IF/TA may be useful in early diagnosis and development of novel therapeutic strategies for improving long-term renal allograft survival and patient outcomes. In this review, we highlight the recent advances in our understanding of IF/TA from three aspects: pathogenesis, diagnosis, and treatment.
PMCID: PMC4185272  PMID: 25285155
Interstitial fibrosis; Renal transplantation; Renal allograft loss; Tubular atrophy
8.  Frequency Selectivity in Pulse Responses of Pt/Poly(3-Hexylthiophene-2,5-Diyl)/Polyethylene Oxide + Li+/Pt Hetero-Junction 
PLoS ONE  2014;9(9):e108316.
Pt/poly(3-hexylthiophene-2,5-diyl)/polyethylene oxide + Li+/Pt hetero junctions were fabricated, and their pulse responses were studied. The direct current characteristics were not symmetric in the sweeping range of ±2 V. Negative differential resistance appeared in the input range of 0 to 2 V because of de-doping (or reduction) in the side with the semiconductor layer. The device responded stably to a train of pulses with a fixed frequency. The inverse current after a pulse was related to the back-migrated ions. Importantly, the weight calculated based on the inverse current strength, was depressed during low-frequency stimulations but was potentiated during high-frequency stimulations when pulses were positive. Therefore, frequency selectivity was first observed in a semiconducting polymer/electrolyte hetero junction. Detailed analysis of the pulse response showed that the input frequency could modulate the timing of ion doping, de-doping, and re-doping at the semiconducting polymer/electrolyte interface, which then resulted in the frequency selectivity. Our study suggests that the simple redox process in semiconducting polymers can be modulated and used in signal handling or the simulation of bio-learning.
PMCID: PMC4171527  PMID: 25244151
9.  Correlation of ADC value with pathologic indexes in colorectal tumor homografts in Balb/c mouse 
Noninvasive diffusion-weighted magnetic resonance imaging (DWI) is a well-studied MR imaging technique for quantifying water diffusion especially in tumor area. The correlation between apparent diffusion coefficient (ADC) value and apoptosis or proliferation is not clear by now. This study aimed to investigate whether DWI-ADC value could be used as an imaging marker related with pathologic indexes of tumors.
A total of 30 Balb/c mice with HT29 colorectal carcinoma were subjected to DWI and histologic analysis. The percentage of ADC changes and the apoptotic and proliferating indexes were calculated at predefined time points. Kolmogorov-Smirnov distances were considered to determine whether the percentage of ADC changes, and the apoptotic and proliferating indexes were normally distributed. An independent-samples t-test was used to analyze the difference between apoptotic and proliferating indexes in the two groups.
There was a statistically significant difference in proliferating index between the radiotherapy and control groups (mean proliferating index: 49.27% vs. 83.09%), and there was a statistically significant difference in apoptotic index between the two groups (mean apoptotic index: 37.7% vs. 2.71%). A significant positive correlation was found between the percentage of ADC changes of the viable tissue and apoptotic index. Pearson’s correlation coefficient was 0.655 (P=0.015). A significant negative correlation was found between the percentage of ADC changes of the viable tissue and ki-67 proliferation index. Pearson’s correlation coefficient was 0.734 (P<0.001).
Our results suggest that ADC value may be used in measurement of cell apoptotic and proliferating indexes in colorectal carcinoma.
PMCID: PMC4153920  PMID: 25232218
Diffusion-weighted magnetic resonance imaging (DWI); apparent diffusion coefficient (ADC); apoptosis; proliferation; HT29
10.  Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening 
Viruses  2014;6(7):2778-2795.
Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.
PMCID: PMC4113793  PMID: 25036464
enterovirus 71; coxsackievirus A16; luteolin; reporter virus; high‑throughput assay; antiviral drug discovery
11.  Analysis of Cross-Reactive Neutralizing Antibodies in Human HFMD Serum with an EV71 Pseudovirus-Based Assay 
PLoS ONE  2014;9(6):e100545.
Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial ( Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.
PMCID: PMC4070950  PMID: 24964084
12.  A RG-II Type Polysaccharide Purified from Aconitum coreanum Alleviates Lipopolysaccharide-Induced Inflammation by Inhibiting the NF-κB Signal Pathway 
PLoS ONE  2014;9(6):e99697.
Korean mondshood root polysaccharides (KMPS) isolated from the root of Aconitum coreanum (Lévl.) Rapaics have shown anti-inflammatory activity, which is strongly influenced by their chemical structures and chain conformations. However, the mechanisms of the anti-inflammatory effect by these polysaccharides have yet to be elucidated. A RG-II polysaccharide (KMPS-2E, Mw 84.8 kDa) was isolated from KMPS and its chemical structure was characterized by FT-IR and NMR spectroscopy, gas chromatography–mass spectrometry and high-performance liquid chromatography. The backbone of KMPS-2E consisted of units of [→6) -β-D-Galp (1→3)-β-L-Rhap-(1→4)-β-D-GalpA-(1→3)-β-D-Galp-(1→] with the side chain →5)-β-D-Arap (1→3, 5)-β-D-Arap (1→ attached to the backbone through O-4 of (1→3,4)-L-Rhap. T-β-D-Galp is attached to the backbone through O-6 of (1→3,6)-β-D-Galp residues and T-β-D-Ara is connected to the end group of each chain. The anti-inflammatory effects of KMPS-2E and the underlying mechanisms using lipopolysaccharide (LPS) - stimulated RAW 264.7 macrophages and carrageenan-induced hind paw edema were investigated. KMPS-2E (50, 100 and 200 µg/mL) inhibits iNOS, TLR4, phospho-NF-κB–p65 expression, phosphor-IKK, phosphor-IκB-α expression as well as the degradation of IκB-α and the gene expression of inflammatory cytokines (TNF-α, IL-1β, iNOS and IL-6) mediated by the NF-κB signal pathways in macrophages. KMPS-2E also inhibited LPS-induced activation of NF-κB as assayed by electrophorectic mobility shift assay (EMSA) in a dose-dependent manner and it reduced NF-κB DNA binding affinity by 62.1% at 200µg/mL. In rats, KMPS-2E (200 mg/kg) can significantly inhibit carrageenan-induced paw edema as ibuprofen (200 mg/kg) within 3 h after a single oral dose. The results indicate that KMPS-2E is a promising herb-derived drug against acute inflammation.
PMCID: PMC4057409  PMID: 24927178
13.  Conditional Activin Receptor Type IB (Acvr1b) Knockout Mice Reveal Hair Loss Abnormality 
The in vivo functions of the activin A receptor type 1b (Acvr1b) have been difficult to study because Acvr1b−/− mice die during embryogenesis. To investigate the roles of Acvr1b in the epithelial tissues, we created mice with a conditional disruption of Acvr1b (Acvr1bflox/flox) and crossed them with K14-Cre mice. Acvr1bflox/flox; K14-Cre mice displayed various degrees of hairlessness at postnatal day 5, and the phenotype is exacerbated by age. Histological analyses showed that those hair follicles that developed during morphogenesis were later disrupted by delays in hair cycle reentry. Failure in cycling of the hair follicles and regrowth of the hair shaft and the inner root sheath resulted in subsequent severe hair loss. Apart from previous reports of other members of the transforming growth factor-β/activin/bone morphogenic protein pathways, we demonstrate a specialized role for Acvr1b in hair cycling in addition to hair follicle development. Acvr1bflox/flox; K14-Cre mice also had a thicker epidermis than did wild-type mice, which resulted from persistent proliferation of skin epithelial cells; however, no tumor formation was observed by 18 months of age. Our analysis of this Acvr1b knockout mouse line provides direct genetic evidence that Acvr1b signaling is required for both hair follicle development and cycling.
PMCID: PMC4049458  PMID: 21191412
14.  Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS 
Journal of Thoracic Disease  2014;6(5):524-533.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK® MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples.
We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK® MS system. Peptide spectra acquired by the system were compared with the VITEK® MS IVD database Version 2.0, and the identification scores were recorded.
Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK® MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database.
MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK® MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic identification for most bacterial and yeast strains routinely isolated in clinical microbiology laboratories.
PMCID: PMC4015010  PMID: 24822114
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS); VITEK-MS; bacteria; yeasts; identification
15.  Mutational Analyses of Multiple Oncogenic Pathways in Intraductal Papillary Mucinous Neoplasms of the Pancreas 
Pancreas  2008;36(2):168-172.
There is much accumulated evidence that EGFR, HER2, and their downstream signaling pathway members such as KRAS, BRAF, and PIK3CA are strongly implicated in cancer development and progression. Recently, mutations in the kinase domains of EGFR and HER2, associated with increased sensitivity to tyrosine kinase inhibitors, have been described.
To evaluate the mutational status of these genes in intraductal papillary mucinous neoplasm (IPMN)/intraductal papillary mucinous carcinoma (IPMC), EGFR and HER2 were analyzed in 36 IPMN/IPMC, and the results were correlated to the mutational status of the KRAS, BRAF, and PIK3CA genes in the samples.
Together, we identified 1 silent mutation of HER2, 17 (43%) KRAS mutations, 1 (2.7%) BRAF mutation, and 4 (11%) mutations of PIK3CA in the IPMN/IPMC samples.
The EGFR and ERBB2 (HER2) mutations are very infrequent in IPMN/IPMC, suggesting the limited possibility of targeting mutated ERBB2 and EGFR for therapy for these lesions. The KRAS, BRAF, and PIK3CA, however, could represent interesting targets for future therapies in these lesions.
PMCID: PMC3915029  PMID: 18376308
16.  Establishment of cell lines with increased susceptibility to EV71/CA16 by stable overexpression of SCARB2 
Virology Journal  2013;10:250.
Human enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly.
In this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry.
Levels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2 × 102 times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 102–103-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells.
We established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.
PMCID: PMC3765843  PMID: 23919614
SCARB2; EV71/CA16; 293 cells; RD cells; Vero cells
17.  Caffeine-Induced Activated Glucocorticoid Metabolism in the Hippocampus Causes Hypothalamic-Pituitary-Adrenal Axis Inhibition in Fetal Rats 
PLoS ONE  2012;7(9):e44497.
Epidemiological investigations have shown that fetuses with intrauterine growth retardation (IUGR) are susceptible to adult metabolic syndrome. Clinical investigations and experiments have demonstrated that caffeine is a definite inducer of IUGR, as children who ingest caffeine-containing food or drinks are highly susceptible to adult obesity and hypertension. Our goals for this study were to investigate the effect of prenatal caffeine ingestion on the functional development of the fetal hippocampus and the hypothalamic-pituitary-adrenal (HPA) axis and to clarify an intrauterine HPA axis-associated neuroendocrine alteration induced by caffeine. Pregnant Wistar rats were intragastrically administered 20, 60, and 180 mg/kg·d caffeine from gestational days 11–20. The results show that prenatal caffeine ingestion significantly decreased the expression of fetal hypothalamus corticotrophin-releasing hormone. The fetal adrenal cortex changed into slight and the expression of fetal adrenal steroid acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc), as well as the level of fetal adrenal endogenous corticosterone (CORT), were all significantly decreased after caffeine treatment. Moreover, caffeine ingestion significantly increased the levels of maternal and fetal blood CORT and decreased the expression of placental 11β-hydroxysteroid dehydrogenase-2 (11β-HSD-2). Additionally, both in vivo and in vitro studies show that caffeine can downregulate the expression of fetal hippocampal 11β-HSD-2, promote the expression of 11β-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor (GR), and enhance DNA methylation within the hippocampal 11β-HSD-2 promoter. These results suggest that prenatal caffeine ingestion inhibits the development of the fetal HPA axis, which may be associated with the fetal overexposure to maternal glucocorticoid and activated glucocorticoid metabolism in the fetal hippocampus. These results will be beneficial in elucidating the developmental toxicity of caffeine and in exploring the fetal origin of adult HPA axis dysfunction and metabolic syndrome susceptibility for offspring with IUGR induced by caffeine.
PMCID: PMC3435254  PMID: 22970234
18.  Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis 
PLoS ONE  2012;7(9):e40363.
To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820–1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.
PMCID: PMC3433482  PMID: 22962577
19.  Complete Genomic Sequence of Duck Flavivirus from China 
Journal of Virology  2012;86(6):3398-3399.
We report here the complete genomic sequence of the Chinese duck flavivirus TA strain. This work is the first to document the complete genomic sequence of this previously unknown duck flavivirus strain. The sequence will help further relevant epidemiological studies and extend our general knowledge of flaviviruses.
PMCID: PMC3302339  PMID: 22354941
20.  CD8+ T Cells Mediate the Athero-Protective Effect of Immunization with an ApoB-100 Peptide 
PLoS ONE  2012;7(2):e30780.
Immunization of hypercholesterolemic mice with selected apoB-100 peptide antigens reduces atherosclerosis but the precise immune mediators of athero-protection remain unclear. In this study we show that immunization of apoE (-/-) mice with p210, a 20 amino acid apoB-100 related peptide, reduced aortic atherosclerosis compared with PBS or adjuvant/carrier controls. Immunization with p210 activated CD8+ T cells, reduced dendritic cells (DC) at the site of immunization and within the plaque with an associated reduction in plaque macrophage immunoreactivity. Adoptive transfer of CD8+ T cells from p210 immunized mice recapitulated the athero-protective effect of p210 immunization in naïve, non-immunized mice. CD8+ T cells from p210 immunized mice developed a preferentially higher cytolytic response against p210-loaded dendritic cells in vitro. Although p210 immunization profoundly modulated DCs and cellular immune responses, it did not alter the efficacy of subsequent T cell dependent or independent immune response to other irrelevant antigens. Our data define, for the first time, a role for CD8+ T cells in mediating the athero-protective effects of apoB-100 related peptide immunization in apoE (-/-) mice.
PMCID: PMC3276497  PMID: 22347402
21.  Ghrelin Inhibits Visceral Afferent Activation of Catecholamine Neurons in the Solitary Tract Nucleus 
Brainstem A2/C2 catecholamine (CA) neurons in the solitary tract nucleus (NTS) are thought to play an important role in the control of food intake and other homeostatic functions. We have previously demonstrated that these neurons, which send extensive projections to brain regions involved in the regulation of appetite, are strongly and directly activated by solitary tract (ST) visceral afferents. Ghrelin, a potent orexigenic peptide released from the stomach, is proposed to act in part through modulating NTS CA neurons but the underlying cellular mechanisms are unknown. Here we identified CA neurons using transgenic mice that express enhanced green florescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). We then determined how ghrelin modulates TH-EGFP neurons using patch clamp techniques in a horizontal brain slice preparation. Ghrelin inhibited the frequency of spontaneous glutamate inputs (sEPSCs) onto TH-EGFP neurons, including cholecystokinin-sensitive neurons, an effect blocked by the GHSR1 antagonist, D-Lys-3-GHRP-6. This resulted in a decrease in the basal firing rate of NTS TH-EGFP neurons, an effect blocked by the glutamate antagonist NBQX. Ghrelin also dose-dependently inhibited the amplitude of ST afferent evoked EPSCs (ST-EPSCs) in TH-EGFP NTS neurons, decreasing the success rate for ST-evoked action potentials. In addition, ghrelin decreased the frequency of mini-EPSCs suggesting its actions are pre-synaptic to reduce glutamate release. Lastly, ghrelin’s inhibition of the ST-EPSCs was significantly increased by an 18 hour fast. These results demonstrate a potential mechanism by which ghrelin inhibits NTS TH neurons through a pathway whose responsiveness is increased during fasting.
PMCID: PMC3163901  PMID: 21368060
NTS; ghrelin; catecholamine; fasted; visceral; CCK
22.  New monoclonal anti-mouse DC-SIGN antibodies reactive with acetone-fixed cells 
Journal of immunological methods  2010;360(1-2):66-75.
Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN.. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.
PMCID: PMC2924951  PMID: 20558171
Monoclonal Antibody; Polyclonal Antibody; DC-SIGN; CD209a; Dendritic Cells
23.  Reduced expression of N-Myc downstream-regulated gene 2 in human thyroid cancer 
BMC Cancer  2008;8:303.
NDRG2 (N-Myc downstream-regulated gene 2) was initially cloned in our laboratory. Previous results have shown that NDRG2 expressed differentially in normal and cancer tissues. Specifically, NDRG2 mRNA was down-regulated or undetectable in several human cancers, and over-expression of NDRG2 inhibited the proliferation of cancer cells. NDRG2 also exerts important functions in cell differentiation and tumor suppression. However, it remains unclear whether NDRG2 participates in carcinogenesis of the thyroid.
In this study, we investigated the expression profile of human NDRG2 in thyroid adenomas and carcinomas, by examining tissues from individuals with thyroid adenomas (n = 40) and carcinomas (n = 35), along with corresponding normal tissues. Immunohistochemistry, quantitative RT-PCR and western blot methods were utilized to determine both the protein and mRNA expression status of Ndrg2 and c-Myc.
The immunostaining analysis revealed a decrease of Ndrg2 expression in thyroid carcinomas. When comparing adenomas or carcinomas with adjacent normal tissue from the same individual, the mRNA expression level of NDRG2 was significantly decreased in thyroid carcinoma tissues, while there was little difference in adenoma tissues. This differential expression was confirmed at the protein level by western blotting. However, there were no significant correlations of NDRG2 expression with gender, age, different histotypes of thyroid cancers or distant metastases.
Our data indicates that NDRG2 may participate in thyroid carcinogenesis. This finding provides novel insight into the important role of NDRG2 in the development of thyroid carcinomas. Future studies are needed to address whether the down-regulation of NDRG2 is a cause or a consequence of the progression from a normal thyroid to a carcinoma.
PMCID: PMC2576469  PMID: 18940011
24.  BRAF and KRAS Gene Mutations in Intraductal Papillary Mucinous Neoplasm/Carcinoma (IPMN/IPMC) of the Pancreas 
Cancer letters  2006;249(2):242-248.
The Raf/MEK/ERK (MAPK) signal transduction is an important mediator of a number of cellular fates including growth, proliferation and survival. The BRAF gene is activated by onogenic RAS, leading to cooperative effects in cells responding to growth factor signals. Our study was performed to elucidate a possible role of BRAF in the development of IPMN (Intraductal Papillary Mucinous Neoplasm) and IPMC (Intraductal Papillary Mucinous Carcinoma) of the pancreas. Mutations of BRAF and KRAS were evaluated in 36 IPMN/IPMC samples and two mucinous cystadenomas by direct genomic sequencing. Exons 1 for KRAS, and 5, 11, and 15 for BRAF were examined. Totally we identified 17 (47%) KRAS mutations in exon 1, codon 12 and one missense mutation (2.7%) within exon 15 of BRAF. The mutations appear to be somatic since the same alterations were not detected in the corresponding normal tissues. Our data provide evidence that oncogenic properties of BRAF contribute to the tumorigenesis of IPMN/IPMC, but at a lower frequency than KRAS.
PMCID: PMC1865507  PMID: 17097223
Intraductal papillary mucinous neoplasm; pancreas; KRAS; BRAF
25.  PIK3CA Mutations in Intraductal Papillary Mucinous Neoplasm/Carcinoma (IPMN/IPMC) of the Pancreas 
Recent studies have reported high frequencies of somatic mutations in the phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene in various human solid tumors. More than 75% of those somatic mutations are clustered in the helical (exon 9) and kinase domains (exon 20). The three hot-spot mutations, E542K, E545K, and H1047R, have been proven to elevate the lipid kinase activity of PIK3CA and activate the Akt signaling pathway. The mutational status of PIK3CA in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMNC) has not been evaluated previously.
To evaluate a possible role for PIK3CA in the tumorigenesis of IPMN and IPMNC, exons 1, 4, 5, 6, 7, 9, 12, 18, and 20 were analyzed in 36 IPMN/IPMC and two mucinous cystadenoma specimens by direct genomic DNA sequencing.
We identified four missense mutations in the nine screened exons of PIK3CA from 36 IPMN/IPMC specimens (11%). One of the four mutations, H1047R, has been previously reported as a hot-spot mutation. The remaining three mutations, T324I, W551G, and S1015F, were novel and somatic.
This is the first report of PIK3CA mutation in pancreatic cancer. Our data provide evidence that oncogenic properties of PIK3CA contribute to the tumorigenesis of IPMN/IPMC.
PMCID: PMC1780026  PMID: 16778113
IPMN/IPMC, intraductal papillary mucinous neoplasm/carcinoma; PCR, polymerase chain reaction; PIK3CA, phosphoinositide-3-kinase, catalytic, alpha; PIP3, phosphatidylinositol-3,4,5-triphosphate; LOH, loss of heterozygosity

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