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1.  In Depth Analysis on the Binding Sites of Adamantane Derivatives in HCV (Hepatitis C Virus) p7 Channel Based on the NMR Structure 
PLoS ONE  2014;9(4):e93613.
Background
The recently solved solution structure of HCV (hepatitis C virus) p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine) was determined in a hydrophobic pocket. However the pharmocophore (−NH2) of the ligand was not assigned a specific binding site.
Results
The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T)/6–311+G(d,p) and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed.
Conclusions
Some useful viewpoints are concluded as follows. (1) The amino group (−NH2) of adamantane derivatives is protonated (−NH3+), and the positively charged cation may form cation-π interactions with aromatic amino acids. (2) The aromatic amino acids (His17, Phe20, and Trp21) are the possible binding sites for the protonated amino group (−NH3+) of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3) The higher inhibition potent of rimantadine than amantadine probably because of its higher pKa value (pKa = 10.40) and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed.
doi:10.1371/journal.pone.0093613
PMCID: PMC3979700  PMID: 24714586
2.  Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube 
PLoS ONE  2014;9(4):e91824.
Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.
doi:10.1371/journal.pone.0091824
PMCID: PMC3976262  PMID: 24705376
3.  Cryo-EM Structure of a Molluscan Hemocyanin Suggests its Allosteric Mechanism 
Summary
Hemocyanins are responsible for transporting O2 in the arthropod and molluscan hemolymph. Haliotis diversicolor molluscan hemocyanin isoform 1 (HdH1) is an 8 MDa oligomer. Each subunit is made up of eight functional units (FUs). Each FU contains two Cu ions, which can reversibly bind an oxygen molecule. Here we report a ~4.5 Å cryo-EM structure of HdH1. The structure clearly shows 10 asymmetric units arranged with D5 symmetry. Each asymmetric unit contains two structurally distinct but chemically identical subunits. The map is sufficiently resolved to trace the entire subunit Cα backbone, and to visualize densities corresponding to some large sidechains, Cu ion pairs, and interaction networks of adjacent subunits. A FU topology path intertwining between the two subunits of the asymmetric unit is unambiguously determined. Our observations suggest a structural mechanism for the stability of the entire hemocyanin didecamer and 20 “communication clusters” across asymmetric units responsible for its allosteric property upon oxygen binding.
doi:10.1016/j.str.2013.02.018
PMCID: PMC3657317  PMID: 23541894
4.  BRAFV600E Mutation and Its Association with Clinicopathological Features of Colorectal Cancer: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(3):e90607.
Background
Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC.
Methods
We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model.
Results
25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation.
Conclusions
This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.
doi:10.1371/journal.pone.0090607
PMCID: PMC3940924  PMID: 24594804
5.  High-Resolution Melting Analysis for accurate detection of BRAF mutations: a systematic review and meta-analysis 
Scientific Reports  2014;4:4168.
The high-resolution melting curve analysis (HRMA) might be a good alternative method for rapid detection of BRAF mutations. However, the accuracy of HRMA in detection of BRAF mutations has not been systematically evaluated. We performed a systematic review and meta-analysis involving 1324 samples from 14 separate studies. The overall sensitivity of HRMA was 0.99 (95% confidence interval (CI) = 0.75–0.82), and the overall specificity was very high at 0.99 (95% CI = 0.94–0.98). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 68.01 (95% CI = 25.33–182.64), 0.06 (95% CI = 0.03–0.11), and1263.76 (95% CI = 393.91–4064.39), respectively. The summary receiver operating characteristic curve for the same data shows an area of 1.00 and a Q* value of 0.97. The high sensitivity and specificity, simplicity, low cost, less labor or time and rapid turnaround make HRMA a good alternative method for rapid detection of BRAF mutations in the clinical practice.
doi:10.1038/srep04168
PMCID: PMC3933866  PMID: 24566771
6.  Indices of von Willebrand Factor as Biomarkers of Aortic Stenosis Severity: (From the Biomarkers of Aortic Stenosis Severity [BASS] Study) 
The American journal of cardiology  2012;111(3):374-381.
We correlated von Willebrand factor activity indices and brain natriuretic peptide (BNP) with measures of aortic stenosis (AS) severity, bleeding, symptoms, and freedom from death or aortic valve replacement. Patients with AS (n=66 [16 mild, 20 moderate, 30 severe]) and aortic valve replacement (n=21) were assessed with VWF antigen (VWF:Ag), VWF latex agglutination immunoturbidic activity (VWF:Ltx), platelet function analyzer collagen plus adenosine diphosphate (PFA-CADP), VWF multimer ratio (VWF:mult ratio), and BNP level after echocardiography. In AS patients, mean gradient correlated with BNP (Spearman r=0.29, P=.02), VWF:Ltx/VWF:Ag (r=–0.41, P<.001), PFA-CADP (r=0.49, P<.001), and VWF:mult ratio (r=–0.76, P<.001). The area under the curve (95% CI) for detection of severe AS was 0.62 (0.48-0.77) by elevated BNP, 0.81 (0.69-0.92) by PFA-CADP closure time, 0.69 (0.55-0.82) by VWF:Ltx/VWF:Ag ratio, and 0.86 (0.76-0.95) by VWF:mult ratio. For VWF:mult ratio, a threshold of 0.15 yielded sensitivity and specificity for severe AS of 77% and positive predictive value of 74%. Bleeding (in 14%) was associated with prolonged PFA-CADP and reduced VWF:Ltx/VWF:Ag. Symptoms were associated with elevated BNP and low Duke Activity Status Index score. In 66 AS patients, freedom from death (n=4) or aortic valve replacement (n=22) was associated with PFA-CADP (P=.003), VWF high molecular weight multimers (HMWM) (P=.009), and VWF:Ltx/VWF:Ag (P<.001), but not BNP (P=.32). In severe AS vs aortic valve replacement, PFA-CADP and VWF:mult ratio differed (P<.001), but BNP and VWF:Ltx/VWF:Ag did not. In conclusion, VWF activity indices are associated with AS severity and bleeding, and predictive of cardiovascular outcome.
doi:10.1016/j.amjcard.2012.10.015
PMCID: PMC3932487  PMID: 23168287
aortic stenosis; von Willebrand factor; brain natriuretic peptide; aortic valve replacement
7.  Chinese Herbal Medicine for Aspirin Resistance: A Systematic Review of Randomized Controlled Trials 
Aspirin resistance (AR) is a prevalent phenomenon and leads to significant clinical consequences, but the current evidence for effective interventional strategy is insufficient. The objective of this systematic review is thus to assess the efficacy and safety of Chinese herbal medicine (CHM) for AR. A systematical literature search was conducted in 6 databases until December 2012 to identify randomized controlled trials (RCTs) of CHM for AR. As a result, sixteen RCTs with a total of 1011 subjects were identified, suggesting that the interests of the medical profession and the public in the use of CHM for AR have grown considerably in the recent years. Tongxinluo capsule and Danshen-based prescriptions were the most frequently used herbal prescriptions, while danshen root, milkvetch root, Leech, and Rosewood were the most frequently used single herbs. Despite the apparent reported positive findings, it is premature to determine the efficacy and safety of CHM for the treatment of AR due to poor methodological quality and insufficient safety data. However, CHMs appeared to be well tolerated in all included studies. Thus, CHM as a promising candidate is worthy of improvement and development for further clinical AR trials. Large sample-size and well-designed rigorous RCTs are needed.
doi:10.1155/2014/890950
PMCID: PMC3950618
8.  Discontinuation of antithrombotic therapy for a year or more in patients with continuous-flow left ventricular assist devices☆ 
The recommended anticoagulation regimen during continuous-flow axial left ventricular assist device (LVAD) support is aspirin and warfarin with a targeted international normalized ratio of 2.0–3.0. We report two patients in whom recurrent gastrointestinal bleeding during LVAD support necessitated discontinuation of this anti-thrombotic regimen for a year or more. Despite this, neither patients developed thrombotic complications during 29 patient-months of follow-up. An acquired von Willebrand factor (VWF) abnormality reflected by the absence or decreased abundance of the highest molecular weight multimers was demonstrated in both patients. The gold standard test for platelet function, light transmission platelet aggregometry was measured in one patient and was normal, indicative that the predominant abnormality in the coagulation profile of these patients is an acquired VWF syndrome. Clinical trials are required to address the question whether it is safe to discontinue anticoagulation in LVAD patients with acquired VWF abnormalities.
doi:10.1510/icvts.2010.240747
PMCID: PMC3927556  PMID: 20639306
Heart failure; Ventricular assist device; Anticoagulation; Gastrointestinal bleeding
9.  The impact of heatwaves on mortality in Australia: a multicity study 
BMJ Open  2014;4(2):e003579.
Objectives
To assess the heterogeneity of heatwave-related impacts on mortality across different cities.
Design
A multicity time series study.
Setting
3 largest Australian cities: Brisbane, Melbourne and Sydney.
Participants
All residents living in these cities.
Main outcome measures
Non-external causes mortality data by gender and two age groups (ie, 0–75 and 75+) for these cities during the period 1988–2009 were obtained from relevant government agencies.
Results
Total mortality increased mostly within the same day (lag 0) or a lag of 1 day (lag 1) during almost all heatwaves in three cities. Using the heatwave definition (HWD) as the 95th centile of mean temperature for two or more consecutive days in the summer season, the relative risk for total mortality at lag 1 in Brisbane, Melbourne and Sydney was 1.13 (95% CI 1.08 to 1.19), 1.10 (95% CI 1.06 to 1.14) and 1.06 (95% CI 1.01 to 1.10), respectively. Using the more stringent HWD—the 99th centile of mean temperature for two or more consecutive days, the relative risk of total mortality at the lags of 0–2 days in Brisbane and Melbourne was 1.40 (95% CI 1.29 to 1.51) and 1.47 (95% CI 1.36 to 1.59), respectively. Elderly, particularly females, were more vulnerable to the impact of heatwaves.
Conclusions
A consistent and significant increase in mortality was observed during heatwaves in the three largest Australian cities, but the impacts of heatwave appeared to vary with age, gender, the HWD and geographical area.
doi:10.1136/bmjopen-2013-003579
PMCID: PMC3931989  PMID: 24549159
Climate Changes; Heatwaves; Mean Temperature; Mortality; Time Series Analysis
10.  Prognostic significance of glypican-3 in hepatocellular carcinoma: a meta-analysis 
BMC Cancer  2014;14:104.
Backgrounds
Glypican-3(GPC3) has been implicated in tumor development and progression for several years. However, the prognostic significance of GPC3 expression in patients with hepatocellular carcinoma (HCC) is controversial. We performed a meta-analysis of available studies to assess whether GPC3 can be used as a prognostic factor in patients with HCC.
Methods
We searched PubMed and Ovid EBM Reviews databases and evaluated the reference list of relevant articles for studies that assessed the prognostic relevance of GPC3 in patients with HCC. Meta-analysis was performed using hazard ratio (HR) or odds ratio (OR) and 95% confidence intervals (95% CIs) as effect measures.
Results
A meta-analysis of eight studies included 1070 patients was carried out to evaluate the association between GPC3 and overall survival (OS) and disease-free survival (DFS) in HCC patients. The relation between GPC3 and tumor pathological features was also assessed. Our analysis results indicated that high GPC3 expression predicted poor OS (HR: 1.96, 95% CI: 1.51–2.55) and DFS (HR: 1.99, 95% CI: 1.57-2.51) of patients with HCC. GPC3 overexpression was significantly associated with high tumor grade (OR: 3.30, 95% CI: 2.04–5.33), late TNM stage (OR: 2.26, 95% CI: 1.00–5.12), and the presence of vascular invasion (OR: 2.43, 95% CI: 1.23–4.82).
Conclusions
GPC3 overexpression indicates a poor prognosis for patients with HCC, and it may also have predictive potential for HCC invasion and metastasis.
doi:10.1186/1471-2407-14-104
PMCID: PMC3984430  PMID: 24548704
Glypican-3; Hepatocellular carcinoma; Prognosis
11.  Coxsackievirus-induced acute neonatal central nervous system disease model 
Coxsackievirus B (CVB) is a significant pathogen that causes pediatric central nervous system disease with acute syndromes commonly. The onset of its infection was abrupt, and after recovery there usually will be severe mental sequelae. The disease model for research was not established by the way of natural infection, although there are various investigations about the CVB-induced central nervous system (CNS) diseases. Thus, we have established an acute neonatal CNS disease mice model by CVB orally infecting. This model imitated the natural infection route and focuses the onset of CNS disease, inducing severe infection and lesion in the hippocampus and cortex regions, and the stability of the model was demonstrated. A pathology score system was developed for quantitative pathology analysis, which standardizes the CNS pathology analysis by statistics analysis. By this model, the track of CVB penetrating the blood brain barrier in vivo has been captured. One of the experimental strains CVB3/Macocy, as a new variant, was isolated, and its genomic RNA was cloned. According to its nucleotide sequence, we have characterized its genomic structure and defined its genotype. Based on the sequence, some mutations which do not change the CVB-induced CNS damage have been found. The model is an effective tool for studies on CVB-induced CNS diseases.
PMCID: PMC3971288
Coxsackievirus B; acute neonatal central nervous system disease; mouse model; strain Macocy
12.  Visceral pathology of acute systemic injury in newborn mice on the onset of Coxsackie virus infection 
Coxsackievirus B (CVB) is a significant pathogen of neonatal diseases with severe systemic involvement and high mortality. Hence, it is essential to develop a CVB-induced acute systemic disease model on newborn mouse and study the injury at the onset phase. In this work, a clinical strain of CVB3, Nancy, and its variant strain, Macocy, were adopted in 24 hour old neonates by oral infection. The pathological changes in the heart, liver and lung tissues were analyzed by pathology assays. In situ end labeling assay for programmed cell death was carried out for liver tissues. The data on fatality and infection rates and pathology scores were analyzed statistically. The genomic sequences of the two strains were aligned. The model represented the manifest clinical syndromes of hepatitis, pneumonia and myocardial injury at the onset phase, in which massive numbers of hepatocytes had undergone programmed cell death. Statistical and pathological analysis indicated that the myocardial injury was mild, whereas the liver and lung were more severe. The fatality rate, infection and pathology of the two CVB strains were the same. Therefore, two nucleotide mutations in the 5’ UTR and four amino acid mutations in polyprotein, which did not alter virulence, were shown. By peroral CVB infection of neonatal mice, we developed an acute systemic disease model for studying visceral pathology and systemic disease. At the onset of acute neonatal systemic disease, the hepatitis and pneumonia may be the dominant reason of death, as the injury of liver and lung is more severe than that of heart.
PMCID: PMC3971291
Coxsackievirus B; acute neonatal systemic disease; pathological diversity; mutation sites
13.  A comparison of liver protection among 3-D conformal radiotherapy, intensity-modulated radiotherapy and RapidArc for hepatocellular carcinoma 
Purpose
The analysis was designed to compare dosimetric parameters among 3-D conformal radiotherapy (3DCRT), intensity-modulated radiotherapy (IMRT) and RapidArc (RA) to identify which can achieve the lowest risk of radiation-induced liver disease (RILD) for hepatocellular carcinoma (HCC).
Methods
Twenty patients with HCC were enrolled in this study. Dosimetric values for 3DCRT, IMRT, and RA were calculated for total dose of 50 Gy/25f. The percentage of the normal liver volume receiving >40, >30, >20, >10, and >5 Gy (V40, V30, V20, V10 and V5) were evaluated to determine liver toxicity. V5, V10, V20, V30 and Dmean of liver were compared as predicting parameters for RILD. Other parameters included the conformal index (CI), homogeneity index (HI), and hot spot (V110%) for the planned target volume (PTV) as well as the monitor units (MUs) for plan efficiency, the mean dose (Dmean) for the organs at risk (OARs) and the maximal dose at 1% volume (D1%) for the spinal cord.
Results
The Dmean of IMRT was higher than 3DCRT (p = 0.045). For V5, there was a significant difference: RA > IMRT >3DCRT (p <0.05). 3DCRT had a lower V10 and higher V20, V30 values for liver than RA (p <0.05). RA and IMRT achieved significantly better CI and lower V110% values than 3DCRT (p <0.05). RA had better HI, lower MUs and shorter delivery time than 3DCRT or IMRT (p <0.05).
Conclusion
For right lobe tumors, RapidArc may have the lowest risk of RILD with the lowest V20 and V30 compared with 3DCRT or IMRT. For diameters of tumors >8 cm in our study, the value of Dmean for 3DCRT was lower than IMRT or RapidArc. This may indicate that 3DCRT is more suitable for larger tumors.
doi:10.1186/1748-717X-9-48
PMCID: PMC3922419  PMID: 24502643
Hepatocellular carcinoma; Radiotherapy; Dosimetry; Radiation-induced liver disease; Liver protection
14.  Ephrin-A3 Suppresses Wnt Signaling to Control Retinal Stem Cell Potency 
Stem cells (Dayton, Ohio)  2013;31(2):349-359.
The ciliary epithelium (CE) of adult mammals has been reported to provide a source of retinal stem cells (RSCs) that can give rise to all retinal cell types in vitro. A recent study, however, suggests that CE-derived cells possess properties of pigmented ciliary epithelial cells and display little neurogenic potential. Here we show that the neurogenic potential of CE-derived cells is negatively regulated by ephrin-A3, which is upregulated in the CE of postnatal mice and presents a strong prohibitory niche for adult RSCs. Addition of ephrin-A3 inhibits proliferation of CE-derived RSCs and increases pigment epithelial cell fate. In contrast, absence of ephrin-A3 promotes proliferation and increases expression of neural progenitor cell markers and photoreceptor progeny. The negative effects of ephrin-A3 on CE-derived RSCs are mediated through activation of an EphA4 receptor and suppression of Wnt3a/β-catenin signaling. Together, our data suggest that CE-derived RSCs contain the intrinsic machinery to generate photoreceptors and other retinal neurons, while the CE of adult mice expresses negative regulators that prohibit the proliferation and neural differentiation of RSCs. Manipulating ephrin and Wnt/β-catenin signaling may, thus, represents a viable approach to activating the endogenous neurogenic potential of CE-derived RSCs for treating photoreceptor damage and retinal degenerative disorders.
doi:10.1002/stem.1283
PMCID: PMC3572266  PMID: 23165658
Adult stem cells; cell signaling; neural differentiation; retina; stem cell plasticity
15.  SiO2 nanoparticles change colour preference and cause Parkinson's-like behaviour in zebrafish 
Scientific Reports  2014;4:3810.
With advances in the development of various disciplines, there is a need to decipher bio-behavioural mechanisms via interdisciplinary means. Here, we present an interdisciplinary study of the role of silica nanoparticles (SiO2-NPs) in disturbing the neural behaviours of zebrafish and a possible physiological mechanism for this phenomenon. We used adult zebrafish as an animal model to evaluate the roles of size (15-nm and 50-nm) and concentration (300 μg/mL and 1000 μg/mL) in SiO2-NP neurotoxicity via behavioural and physiological analyses. With the aid of video tracking and data mining, we detected changes in behavioural phenotypes. We found that compared with 50-nm nanosilica, 15-nm SiO2-NPs produced greater significant changes in advanced cognitive neurobehavioural patterns (colour preference) and caused potentially Parkinson's disease-like behaviour. Analyses at the tissue, cell and molecular levels corroborated the behavioural results, demonstrating that nanosilica acted on the retina and dopaminergic (DA) neurons to change colour preference and to cause potentially Parkinson's disease-like behaviour.
doi:10.1038/srep03810
PMCID: PMC3898208  PMID: 24448416
16.  Expression analysis of BMP2, BMP5, BMP10 in human colon tissues from Hirschsprung disease patients 
Objective: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF β) superfamily. BMP2, BMP5 and BMP10 exert their biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family. Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. However, putative Notch function in enteric nervous system (ENS) development and the etiology of HSCR is unknown. Methods: Aganglionic and ganglionic colon segment tissues of 50 HSCR patients were investigated for the expression pattern of BMP2, BMP5 and BMP10 using real-time RT-PCR, Western blot analysis and immunohistochemical staining. Results: The mRNA levels of BMP2, BMP5 and BMP10 in the stenotic colon segment from HSCR patients were significantly higher than those in the normal ones. Similar increased expressions of them in the stenotic colon segments were detected by Western blotting coupled with densitometry analysis. Lastly, immunohistologicl stain showed significant BMP2, 5 and 10 increases in mucous and muscular layers from stenotic colon segments compared to normal segments. Conclusions: BMP2, BMP5 and BMP10 are elevated in the stenotic colon segment of HSCR, and BMPs signaling plays a pivotal role in the development of HSCR.
PMCID: PMC3925897  PMID: 24551273
Bone morphogenetic proteins (BMPs); Hirschsprung disease (HSCR); enteric nervous system (ENS); stenotic colon; ganglion cell
17.  Adjuvant chemotherapy, p53, carcinoembryonic antigen expression and prognosis after D2 gastrectomy for gastric adenocarcinoma 
AIM: To investigate adjuvant chemotherapy, p53 and carcinoembryonic antigen (CEA) expression and prognosis after D2 gastrectomy for stage II/III gastric adenocarcinoma.
METHODS: A total of 286 patients with stage II or III gastric adenocarcinoma who underwent D2 radical gastrectomy between May 2007 and December 2010 were enrolled into this study. One hundred and sixty-nine of these patients received surgery plus adjuvant chemotherapy, and 117 patients received surgery alone. Tumor expression of p53 and CEA proteins in all patients was evaluated immunohistochemically and correlated with clinicopathological parameters. The Kaplan-Meier curves for overall survival (OS) and disease-free survival (DFS) with log-rank testing were used to compare the survival difference. A Cox proportional hazard regression model was used for multivariate analysis.
RESULTS: Patients with adjuvant chemotherapy had a significantly better median OS (50.87 mo vs 30.73 mo, P = 0.000) and median DFS (36.30 mo vs 25.60 mo, P = 0.001) than patients with surgery alone in the entire cohort. Consistent results with the entire cohort were found in stage II (P = 0.006 and P = 0.047), stage III (P = 0.005 and P = 0.030), and stage IIIB/IIIC patients (P = 0.000 and P = 0.001). The median OS and DFS advantages were confirmed by multivariate analysis (P = 0.000 and P = 0.008) and maintained when the analyses were restricted to fluoropyrimidine monotherapy (P = 0.003 and P = 0.001) and fluoropyrimidine plus platinum regimen (P = 0.001 and P = 0.007), however, not the fluoropyrimidine plus taxane (P = 0.198 and P = 0.777) or platinum plus taxane (P = 0.666 and P = 0.687) regimens. Median OS and median DFS did not differ significantly between the patients with p53(+) and p53(-) tumors (P = 0.608 and P = 0.064), or between patients with CEA(+) and CEA(-) tumors (P = 0.052 and P = 0.989), which were maintained when the analyses were restricted to surgery alone (p53: P = 0.864 and P = 0.431; CEA: P = 0.142 and P = 0.948), adjuvant chemotherapy (p53: P = 0.802 and P = 0.091; CEA: P = 0.223 and P = 0.946) and even different chemotherapy regimens (P > 0.05).
CONCLUSION: Patients after D2 gastrectomy for stage II/III gastric adenocarcinoma had significantly better survival after fluoropyrimidine monotherapy and fluoropyrimidine plus platinum. p53 and CEA were not prognostic.
doi:10.3748/wjg.v20.i1.264
PMCID: PMC3886018  PMID: 24415881
Gastric adenocarcinoma; Adjuvant chemotherapy; p53; Carcinoembryonic antigen; Immunohistochemistry
18.  Potential biomarkers for paclitaxel sensitivity in hypopharynx cancer cell 
Paclitaxel has been proved to be active in treatment and larynx preservation of HNSCC, however, the fact that about 20-40% patients do not respond to paclitaxel makes it urgent to figure out the biomarkers for paclitaxel-based treatment in Hypopharynx cancer (HPC) patients to improve the therapy effect. In this work, Fadu cells, treated or untreated with low dose of paclitaxel for 24 h, were applied to DNA microarray chips. The differential expression in mRNAs and miRs was analyzed and the network between expression-altered mRNAs and miRs was constructed. Differentially expressed genes were mainly enriched in superpathway of cholesterol biosynthesis (ACAT2, MSMO1, LSS, FDFT1 and FDPS etc.), complement system (C3, C1R, C1S, CFR and CFB etc.), interferon signaling (IFIT1, IFIT3, IFITM1 and MX1 etc.), mTOR signaling (MRAS, PRKAA2, PLD1, RND3 and EIF4A1 etc.) and IGF1 signaling (MRAS, IGFBP7, JUN and FOS etc.), most of these pathways are implicated in tumorigenesis or chemotherapy resistance. The first three pathways were predicted to be suppressed, while the last two pathways were predicted to be induced by paclitaxel, suggesting the combination therapy with mTOR inhibition and paclitaxel might be better than single one. The dramatically expression-altered miRs were miR-112, miR-7, miR-1304, miR-222*, miR-29b-1* (these five miRs were upregulated) and miR-210 (downregulated). The 26 putative target genes mediated by the 6 miRs were figured out and the miR-gene network was constructed. Furthermore, immunoblotting assay showed that ERK signaling in Fadu cells was active by low dose of paclitaxel but repressed by high dose of paclitaxel. Collectively, our data would provide potential biomarkers and therapeutic targets for paclitaxel-based therapy in HPC patients.
PMCID: PMC3843255  PMID: 24294361
Paclitaxel; hypopharynx cancer; DNA microarray; mTOR signaling
19.  Astrocyte elevated gene-1 (AEG-1) promotes hepatocarcinogenesis: novel insights from a mouse model 
Hepatology (Baltimore, Md.)  2012;56(5):1782-1791.
Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not Wild type (WT) mice, with N-nitrosodiethylamine (DEN), resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance towards senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media (CM) from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated association of Factor XII (FXII) mRNA with polysomes resulting in increased translation. siRNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis.
Conclusion
We uncover novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence and activation of coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.
doi:10.1002/hep.25868
PMCID: PMC3449036  PMID: 22689379
Astrocyte elevated gene-1 (AEG-1); transgenic; hepatocellular carcinoma (HCC); senescence; angiogenesis
20.  Insulin receptor substrate signaling suppresses neonatal autophagy in the heart 
The Journal of Clinical Investigation  2013;123(12):5319-5333.
The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.
doi:10.1172/JCI71171
PMCID: PMC3859408  PMID: 24177427
21.  Is Household Air Pollution a Risk Factor for Eye Disease? 
In developing countries, household air pollution (HAP) resulting from the inefficient burning of coal and biomass (wood, charcoal, animal dung and crop residues) for cooking and heating has been linked to a number of negative health outcomes, mostly notably respiratory diseases and cancers. While ocular irritation has been associated with HAP, there are sparse data on adverse ocular outcomes that may result from acute and chronic exposures. We consider that there is suggestive evidence, and biological plausibility, to hypothesize that HAP is associated with some of the major blinding, and painful, eye conditions seen worldwide. Further research on this environmental risk factor for eye diseases is warranted.
doi:10.3390/ijerph10115378
PMCID: PMC3863851  PMID: 24284355
biomass; blindness; cataract; trachoma; dry eye disease; household air pollution
22.  Optimization of Storage Temperature for Cultured ARPE-19 Cells 
Journal of Ophthalmology  2013;2013:216359.
Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C–37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.
doi:10.1155/2013/216359
PMCID: PMC3819763  PMID: 24251032
23.  A Randomized Controlled Study on the Effects of Bisoprolol and Atenolol on Sympathetic Nervous Activity and Central Aortic Pressure in Patients with Essential Hypertension 
PLoS ONE  2013;8(9):e72102.
Objective
β-blockers (BBs) with different pharmacological properties may have heterogeneous effects on sympathetic nervous activity (SNA) and central aortic pressure (CAP), which are independent cardiovascular factors for hypertension. Hence, we analyzed the effects of bisoprolol and atenolol on SNA and CAP in hypertensive patients.
Methods
This was a prospective, randomized, controlled study in 109 never-treated hypertensive subjects randomized to bisoprolol (5 mg) or atenolol (50 mg) for 4–8 weeks. SNA, baroreflex sensitivity (BRS) and heart rate (HR) variability (HRV) were measured using power spectral analysis using a Finometer. CAP and related parameters were determined using the SphygmoCor device (pulse wave analysis).
Results
Both drugs were similarly effective in reducing brachial BP. However, central systolic BP (−14±10 mm Hg vs −6±9 mm Hg; P<0.001) and aortic pulse pressure (−3±10 mm Hg vs +3±8 mm Hg; P<0.001) decreased more significantly with bisoprolol than with atenolol. The augmentation index at a HR of 75 bpm (AIxatHR75) was significantly decreased (29%±11% to 25%±12%; P = 0.026) in the bisoprolol group only. Furthermore, the change in BRS in the bisoprolol group (3.99±4.19 ms/mmHg) was higher than in the atenolol group (2.66±3.78 ms/mmHg), although not statistically significant (P>0.05). BRS was stable when RHR was controlled (RHR≤65 bpm), and the two treatments had similar effects on the low frequency/high frequency (HF) ratio and on HF.
Conclusion
BBs seem to have different effects on arterial distensibility and compliance in hypertensive subjects. Compared with atenolol, bisoprolol may have a better effect on CAP.
Trial Registration
ClinicalTrials.gov NCT01762436
doi:10.1371/journal.pone.0072102
PMCID: PMC3769307  PMID: 24039738
24.  The anticancer properties of iron core–gold shell nanoparticles in colorectal cancer cells 
Previously, iron core–gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells’ relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the “active” component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core–shell nanoparticles are essential for the anticancer properties observed in CRC cells.
doi:10.2147/IJN.S47742
PMCID: PMC3771749  PMID: 24039416
cancer therapy; Fe; gold-coated iron; nanoparticles; differential cytotoxicity
25.  Rotenone Directly Induces BV2 Cell Activation via the p38 MAPK Pathway 
PLoS ONE  2013;8(8):e72046.
Parkinson’s disease (PD) is the second most common neurodegenerative disease. Although its pathogenesis is still unclear, increasing evidence suggests that mitochondrial dysfunction induced by environmental toxins, such as mitochondrial complex I inhibitors, plays a significant role in the disease process. The microglia in PD brains are highly activated, and inflammation is also an essential element in PD pathogenesis. However, the means by which these toxins activate microglia is still unclear. In the present study, we found that rotenone, a mitochondrial complex I inhibitor, could directly activate microglia via the nuclear factor kappa B (NF-κB) signaling pathway, thereby inducing significantly increased expression of inflammatory cytokines. We further observed that rotenone induced caspase-1 activation and mature IL-1β release, both of which are strictly dependent on p38 mitogen-activated protein kinase (MAPK). The activation of p38 is associated with the presence of reactive oxygen species (ROS) produced by rotenone. Removal of these ROS abrogated the activation of the microglia. Therefore, our data suggest that the environmental toxin rotenone can directly activate microglia through the p38 MAPK pathway.
doi:10.1371/journal.pone.0072046
PMCID: PMC3748029  PMID: 23977201

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