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1.  TnBP⁄Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus 
PLoS ONE  2015;10(2):e0117800.
Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.
doi:10.1371/journal.pone.0117800
PMCID: PMC4320006  PMID: 25658612
2.  Effects of pitavastatin versus atorvastatin on the peripheral endothelial progenitor cells and vascular endothelial growth factor in high-risk patients: a pilot prospective, double-blind, randomized study 
Background
Circulating endothelial progenitor cells (EPCs) reflect endothelial repair capacity and may be a significant marker for the clinical outcomes of cardiovascular disease. While some high-dose statin treatments may improve endothelial function, it is not known whether different statins may have similar effects on EPCs.This study aimed to investigate the potential class effects of different statin treatment including pitavastatin and atorvastatin on circulating EPCs in clinical setting.
Methods
A pilot prospective, double-blind, randomized study was conducted to evaluate the ordinary dose of pitavastatin (2 mg daily) or atorvastatin (10 mg daily) treatment for 12 weeks on circulating EPCs in patients with cardiovascular risk such as hypercholesterolemia and type 2 diabetes mellitus (T2DM). Additional in vitro study was conducted to clarify the direct effects of both statins on EPCs from the patients.
Results
A total of 26 patients (19 with T2DM) completed the study. While the lipid-lowering effects were similar in both treatments, the counts of circulating CD34+KDR+EPCs were significantly increased (from 0.021 ± 0.015 to 0.054 ± 0.044% of gated mononuclear cells, P < 0.05) only by pitavastatin treatment. Besides, plasma asymmetric dimethylarginine level was reduced (from 0.68 ± 0.10 to 0.53 ± 0.12 μmol/L, P < 0.05) by atorvastatin, and plasma vascular endothelial growth factor (VEGF) level was increased (from 74.33 ± 32.26 to 98.65 ± 46.64 pg/mL, P < 0.05) by pitavastatin. In the in vitro study, while both statins increased endothelial nitric oxide synthase (eNOS) expression, only pitavastatin increased the phosphorylation of eNOS in EPCs. Pitavastatin but not atorvastatin ameliorated the adhesion ability of early EPCs and the migration and tube formation capacities of late EPCs.
Conclusions
While both statins similarly reduced plasma lipids, only pitavastatin increased plasma VEGF level and circulating EPCs in high-risk patients, which is probably related to the differential pleiotropic effects of different statins.
Trial registration
This trial is registered at ClinicalTrials.gov, NCT01386853.
doi:10.1186/s12933-014-0111-1
PMCID: PMC4223413  PMID: 25027585
Pitavastatin; Atorvastatin; Hypercholesterolemia; Endothelial progenitor cell; Endothelial nitric oxide synthase; Vascular endothelial growth factor
3.  Antihepatoma Activity of Artocarpus communis Is Higher in Fractions with High Artocarpin Content 
The Scientific World Journal  2014;2014:978525.
Extracts from natural plants have been used in traditional medicine for many centuries worldwide. Artocarpus communis is one such plant that has been used to treat liver cirrhosis, hypertension, and diabetes. To our knowledge, this study is the first to investigate the antihepatoma activity of A. communis toward HepG2 and PLC/PRF/5 cells and the first to explore the relationship between antihepatoma activity and the active compound artocarpin content in different fractions of A. communis. A. communis methanol extract and fractions induced dose-dependent reduction of tumor cell viability. DNA laddering analysis revealed that A. communis extract and fractions did not induce apoptosis in HepG2 and PLC/PRF/5 cells. Instead, acridine orange staining revealed that A. communis triggered autophagic cell death in a dose-dependent manner. The antihepatoma activity of A. communis is attributable to artocarpin. The fractions with the highest artocarpin content were also the fractions with the highest antihepatoma activity in the following order: dichloromethane fraction > methanol extract > ethyl acetate fraction > n-butanol fraction > n-hexane fraction. Taken together, A. communis showed antihepatoma activity through autophagic cell death. The effect was related to artocarpin content. Artocarpin could be considered an indicator of the anticancer potential of A. communis extract.
doi:10.1155/2014/978525
PMCID: PMC4123630  PMID: 25133268
4.  In vivo and in vitro evaluation of the cytotoxic effects of Photosan-loaded hollow silica nanoparticles on liver cancer 
Nanoscale Research Letters  2014;9(1):319.
This study aimed to compare the inhibitory effects of photosensitizers loaded in hollow silica nanoparticles and conventional photosensitizers on HepG2 human hepatoma cell proliferation and determine the underlying mechanisms. Photosensitizers (conventional Photosan-II or nanoscale Photosan-II) were administered to in vitro cultured HepG2 hepatoma cells and treated by photodynamic therapy (PDT) with various levels of light exposure. To assess photosensitizers' effects, cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, apoptotic and necrotic cells were measured by flow cytometry and the expression of caspase-3 and caspase-9 evaluated by western blot. Finally, the in vivo effects of nanoscale and conventional photosensitizers on liver cancer were assessed in nude mice. Nanoscale Photosan-II significantly inhibited hepatoma cell viability in a concentration-dependent manner and this effect was more pronounced with high laser doses. Moreover, nanoscale photosensitizers performed better than the conventional ones under the same experimental conditions (p < 0.05). Flow cytometry data demonstrated that laser-induced cell death was markedly increased after treatment with nanoscale Photosan-II in comparison with free Photosan-II (p < 0.05). Activated caspase-3 and caspase-9 levels were significantly higher in cells treated with Photosan-II loaded in silica nanoparticles than free Photosan-II (p < 0.05). Accordingly, treatment with nanoscale photosensitizers resulted in improved outcomes (tumor volume) in a mouse model of liver cancer, in comparison with conventional photosensitizers. Hollow silica nanoparticles containing photosensitizer more efficiently inhibited hepatoma cells than photosensitizer alone, through induction of apoptosis, both in vivo and in vitro.
doi:10.1186/1556-276X-9-319
PMCID: PMC4082675  PMID: 25024681
Nanoscale photosensitizer; Conventional photosensitizer; Photodynamic therapy; Hepatic carcinoma; Cell apoptosis
5.  Oxaliplatin-Based Chemotherapy Is More Beneficial in KRAS Mutant than in KRAS Wild-Type Metastatic Colorectal Cancer Patients 
PLoS ONE  2014;9(2):e86789.
To identify better regimens in currently available chemotherapy would be beneficial to KRAS mutant metastatic colorectal cancer (mCRC) patients because they have fewer treatment options than KRAS wild-type mCRC patients. Clinicopathologic features and overall survival (OS) of KRAS mutant and wild-type mCRC patients who had used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens were compared to those who had never-used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens respectively. Between 2007 and 2012, a total of 394 mCRC patients, in whom 169 KRAS mutant and 225 KRAS wild-type, were enrolled. In KRAS mutant patients who had used oxaliplatin-based regimens (N = 131), the OS was significantly longer than that in KRAS mutant patients who had never-used oxaliplatin-based regimens (N = 38). The OS was 28.8 months [95% confidence interval (CI): 23.2–34.4] in KRAS mutant patients who had used oxaliplatin-based regimens versus 17.8 months [95% CI: 6.5–29.1] in KRAS mutant patients who had never-used oxaliplatin-based regimens (P = 0.026). Notably, OS in KRAS wild-type mCRC patients who had used oxaliplatin-based regimens (N = 185) was not significantly longer than that in KRAS wild-type mCRC patients who had never-used oxaliplatin-based regimens (N = 40) (P = 0.25). Furthermore, the OS in KRAS mutant patients who had used either irinotecan-based, bevacizumab-based or cetuximab-based regimens was not significantly different than that in KRAS mutant patients who had never-used either irinotecan-based, bevacizumab-based or cetuximab-based regimens respectively. In multivariate analyses, patients who had used oxaliplatin-based regimens remains an independent prognostic factor for longer OS in KRAS mutant mCRC patients. In conclusion, oxaliplatin-based regimens are more beneficial in KRAS mutant than in KRAS wild-type mCRC patients.
doi:10.1371/journal.pone.0086789
PMCID: PMC3913571  PMID: 24505265
6.  Diabetes Mellitus and the Risk of Alzheimer’s Disease: A Nationwide Population-Based Study 
PLoS ONE  2014;9(1):e87095.
Objectives
Possible association between diabetes mellitus (DM) and Alzheimer’s disease (AD) has been controversial. This study used a nationwide population-based dataset to investigate the relationship between DM and subsequent AD incidence.
Methods
Data were collected from Taiwan’s National Health Insurance Research Database, which released a cohort dataset of 1,000,000 randomly sampled people and confirmed it to be representative of the Taiwanese population. We identified 71,433 patients newly diagnosed with diabetes (age 58.74±14.02 years) since January 1997. Using propensity score, we matched them with 71,311 non-diabetic subjects by time of enrollment, age, gender, hypertension, hyperlipidemia, and previous stroke history. All the patients were followed up to December 31, 2007. The endpoint of the study was occurrence of AD.
Results
Over a maximum 11 years of follow-up, diabetic patients experienced a higher incidence of AD than non-diabetic subjects (0.48% vs. 0.37%, p<0.001). After Cox proportional hazard regression model analysis, DM (hazard ratio [HR], 1.76; 95% confidence interval [CI], 1.50–2.07, p<0.001), age (HR, 1.11; 95% CI, 1.10–1.12, p<0.001), female gender (HR, 1.24; 95% CI, 1.06–1.46, p = 0.008), hypertension (HR, 1.30; 95% CI, 1.07–1.59, p = 0.01), previous stroke history (HR, 1.79; 95% CI, 1.28–2.50, p<0.001), and urbanization status (metropolis, HR, 1.32; 95% CI, 1.07–1.63, p = 0.009) were independently associated with the increased risk of AD. Neither monotherapy nor combination therapy with oral antidiabetic medications were associated with the risk of AD after adjusting for underlying risk factors and the duration of DM since diagnosis. However, combination therapy with insulin was found to be associated with greater risk of AD (HR, 2.17; 95% CI, 1.04–4.52, p = 0.039).
Conclusion
Newly diagnosed DM was associated with increased risk of AD. Use of hypoglycemic agents did not ameliorate the risk.
doi:10.1371/journal.pone.0087095
PMCID: PMC3906115  PMID: 24489845
7.  Overexpression of TRIM24 Is Associated with the Onset and Progress of Human Hepatocellular Carcinoma 
PLoS ONE  2014;9(1):e85462.
The survival and colonization of tumor cells at new locations involve a variety of complex genetic, epigenetic, and microenvironmental factors. TRIM24 was originally named transcription intermediary factor 1-alpha (TIF1α), which was associated with cellular proliferation and was an oncogene in tumor development. Here we provide the first evidence of the expression profile and clinicopathological significance of TRIM24 in patients with hepatocellular carcinoma (HCC). Immunohistochemistry was employed to determine the expression level of TRIM24 in HCC tissues and noncancerous liver tissues. Elevated TRIM24 level was found in 61.4% HCC samples (51/83) correlating with AFP (P = 0.036), poor differentiation (P = 0.004), intrahepatic metastasis (P = 0.004), recurrence (P = 0.000006), and shorter tumor-free survival time (P = 0.002). Small interfering RNA induced down-regulation of TRIM24 promoted apoptosis in HCC cell line HepG2. Moreover, western blotting analysis revealed that knockdown of TRIM24 increased the protein levels of p53, Bax, and Caspase-8, and decreased Bcl-2, Survivin, Cyclin D1, and CDK4. Depletion of TRIM24 decreased Snail, Slug, β-catenin, and Vimentin, and increased E-cadherin expression, which suggested the involvement of TRIM24 in EMT. These results indicated that TRIM24 plays an important role in the pathogenesis of human HCC.
doi:10.1371/journal.pone.0085462
PMCID: PMC3883694  PMID: 24409330
8.  High-Dose Amoxicillin with Clavulanate for the Treatment of Acute Otitis Media in Children 
The Scientific World Journal  2014;2014:965096.
Objective. This study uses the acute otitis media clinical practice guideline proposed in 2004 as a reference to evaluate whether antibiotics doses that are in line with the recommendations lead to better prognosis. The study also attempts to clarify possible factors that influence the outcome. Study Design. Retrospective cohort study. Subjects and Methods. A total of 400 children with acute otitis media were enrolled. The dosage of amoxicillin was considered to be appropriate when in accord with clinical practice guidelines, that is, 80–90 mg/kg/day. The outcome was defined according to the description of tympanic membrane on medical records. Multivariate logistic regression was used to analyze the relationship between antibiotic dosage and prognosis after adjusting for baseline factors. Results. The majority of prescriptions were under dosage (89.1%) but it was not noticeably associated with outcome (P = 0.41). The correlation between under dosage and poor prognosis was significant in children below 20 kg with bilateral acute otitis media (odds ratio 1.63; 95% CI 1.02–2.59, P = 0.04). Conclusion. Treating acute otitis media in children, high-dose amoxicillin with clavulanate as recommended in the clinical practice guideline was superior to conventional doses only in children under 20 kg with bilateral diseases.
doi:10.1155/2014/965096
PMCID: PMC3910352  PMID: 24523659
9.  Vertical blockade of the IGFR- PI3K/Akt/mTOR pathway for the treatment of hepatocellular carcinoma: the role of survivin 
Molecular Cancer  2014;13:2.
Background
To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC).
Methods
HCC cell lines (including Hep3B, Huh7, and PLC5) and HUVECs (human umbilical venous endothelial cells) were tested. The molecular targeting therapy agents tested included NVP-AEW541 (IGFR kinase inhibitor), MK2206 (Akt inhibitor), BEZ235 (PI3K/mTOR inhibitor), and RAD001 (mTOR inhibitor). Potential synergistic antitumor effects were tested by median dose-effect analysis in vitro and by xenograft HCC models. Apoptosis was analyzed by flow cytometry (sub-G1 fraction analysis) and Western blotting. The activities of pertinent signaling pathways and expression of apoptosis-related proteins were measured by Western blotting.
Results
Vertical blockade induced a more sustained inhibition of PI3K/Akt/mTOR signaling activities in all the HCC cells and HUVEC tested. Synergistic apoptosis-inducing effects, however, varied among different cell lines and drug combinations and were most prominent when NVP-AEW541 was combined with MK2206. Using an apoptosis array, we identified survivin as a potential downstream mediator. Over-expression of survivin in HCC cells abolished the anti-tumor synergy between NVP-AEW541 and MK2206, whereas knockdown of survivin improved the anti-tumor effects of all drug combinations tested. In vivo by xenograft studies confirmed the anti-tumor synergy between NVP-AEW541 and MK2206 and exhibited acceptable toxicity profiles.
Conclusions
Vertical blockade of the IGFR/PI3K/Akt/mTOR pathway has promising anti-tumor activity for HCC. Survivin expression may serve as a biomarker to predict treatment efficacy.
doi:10.1186/1476-4598-13-2
PMCID: PMC3882101  PMID: 24387108
Molecular targeted therapy; Insulin-like growth factor; NVP-AEW541; MK2206; BEZ235; RAD001
10.  Antiviral Natural Products and Herbal Medicines 
Viral infections play an important role in human diseases, and recent outbreaks in the advent of globalization and ease of travel have underscored their prevention as a critical issue in safeguarding public health. Despite the progress made in immunization and drug development, many viruses lack preventive vaccines and efficient antiviral therapies, which are often beset by the generation of viral escape mutants. Thus, identifying novel antiviral drugs is of critical importance and natural products are an excellent source for such discoveries. In this mini-review, we summarize the antiviral effects reported for several natural products and herbal medicines.
doi:10.4103/2225-4110.124335
PMCID: PMC4032839  PMID: 24872930
Antiviral; Drug development; Herbal medicines; Natural products
11.  Mesoporous silica nanoparticles synthesized from liquid crystal display manufacturing extracts as a potential candidate for a drug delivery carrier: evaluation of their safety and biocompatibility 
Mesoporous silica nanoparticles (MSNs) were synthesized as a promising drug delivery carrier due to the large surface area and porous characteristics. Our previous study successfully recycled wastes from the liquid crystal display (LCD) industry as the silica precursor. In this study, we substantiated the possibility of applying this material as a drug carrier. MSNs synthesized from the extraction of wastes from the manufacture of LCD panels were characterized as having an average diameter of 100 nm, a surface area of 788 m2/g, a uniform pore size distribution of 3.8 nm, and a pore volume of up to 1.04 cm3/g. Methotrexate and camptothecin were entrapped in MSNs at about 33.88% and 75.12%, respectively. The cell viability assay demonstrated that MSNs at 1 μg/mL had no significant influence on human lung fibroblast (WI-38) cells or ovarian cancer (ES-2) cells. A lactate dehydrogenase assay also indicated no inflammation occurred. Moreover, a hemolytic erythrocyte test indicated that the dose range of <100 μg/mL showed that 5% of erythrocytes were affected. After exposure to biofluids, the ordered structure was slightly degraded. The results revealed that MSNs synthesized from extraction of wastes from the manufacture of LCD panels had a good entrapment capacity for hydrophobic drugs and controllable safety conditions; they may be applied as a drug delivery carrier.
doi:10.2147/IJN.S50991
PMCID: PMC3798147  PMID: 24143088
mesoporous silica nanoparticles (MSNs); waste recycle; drug delivery carrier; safety; biocompatibility
12.  A Mitochondrial Phylogeny and Biogeographical Scenario for Asiatic Water Shrews of the Genus Chimarrogale: Implications for Taxonomy and Low-Latitude Migration Routes 
PLoS ONE  2013;8(10):e77156.
The six species and three subspecies in the genus Chimarrogale (Soricomorpha: Soricidae) are commonly referred to as Asiatic water shrews. The Chimarrogale are the most widely distributed group of Nectogaline shrews, extending throughout the Oriental region and Japan. Because of the limited numbers of specimens available for study, the phylogenetic relationships and biogeographical history of this genus have not been comprehensively discussed. We used mitochondrial cytochrome b gene sequences to estimate phylogenetic relationships and divergence times among four Chimarrogale species, including all three subspecies of Chimarrogale himalayica. We also conducted a species delimitation analysis and tested two alternative migration scenarios in Asia through species distribution modeling and a reconstruction of the ancestral distribution. Here, we present the first proposed hypothesis regarding the Asiatic water shrew phylogeny and reveal ten putative species within the four recognized species. Distinct phylogenetic statuses of Chimarrogale phaeura, Chimarrogale platycephala, and Chimarrogale styani were confirmed. Chimarrogale himalayica was strongly supported as paraphyletic. We suggest that three subspecies of Chimarrogale himalayica should be reconsidered as distinct species. However, these suggestions must be considered with caution because only a single locus of a mtDNA gene was used. Four additional putative species, possibly distributed in central southwestern China and Taiwan, are currently undescribed; therefore, comprehensive morphological analyses are warranted to test their taxonomic statuses. The estimated molecular divergence times indicated that rapid speciation occurred during the early Pliocene, and current distribution patterns may have been affected by global cooling during the Pliocene/Pleistocene boundary. Reconstruction of the ancestral distribution and species distribution modeling for Asiatic water shrews revealed a low-latitude migration route over which ancestral Chimarrogale migrated from Europe via Central Asia to their current distribution. Our results demonstrated that Asiatic water shrews could have evolved throughout the low-latitude migration route from Europe to East and Southeast Asia.
doi:10.1371/journal.pone.0077156
PMCID: PMC3790732  PMID: 24124605
13.  Autophagy 
Autophagy  2012;8(10):1477-1493.
Autophagy is a catabolic process that functions in recycling and degrading cellular proteins, and is also induced as an adaptive response to the increased metabolic demand upon nutrient starvation. However, the prosurvival role of autophagy in response to metabolic stress due to deprivation of glutamine, the most abundant nutrient for mammalian cells, is not well understood. Here, we demonstrated that when extracellular glutamine was withdrawn, autophagy provided cells with sub-mM concentrations of glutamine, which played a critical role in fostering cell metabolism. Moreover, we uncovered a previously unknown connection between metabolic responses to ATG5 deficiency and glutamine deprivation, and revealed that WT and atg5−/− MEFs utilized both common and distinct metabolic pathways over time during glutamine deprivation. Although the early response of WT MEFs to glutamine deficiency was similar in many respects to the baseline metabolism of atg5−/− MEFs, there was a concomitant decrease in the levels of essential amino acids and branched chain amino acid catabolites in WT MEFs after 6 h of glutamine withdrawal that distinguished them from the atg5−/− MEFs. Metabolomic profiling, oxygen consumption and pathway focused quantitative RT-PCR analyses revealed that autophagy and glutamine utilization were reciprocally regulated to couple metabolic and transcriptional reprogramming. These findings provide key insights into the critical prosurvival role of autophagy in maintaining mitochondrial oxidative phosphorylation and cell growth during metabolic stress caused by glutamine deprivation.
doi:10.4161/auto.21228
PMCID: PMC3679231  PMID: 22906967
ATG5; autophagy; glutamine; ATP; transcriptional reprogramming; altered metabolism
14.  Pitavastatin and Atorvastatin Double-Blind Randomized ComPArative Study among HiGh-Risk Patients, Including ThOse with Type 2 Diabetes Mellitus, in Taiwan (PAPAGO-T Study) 
PLoS ONE  2013;8(10):e76298.
Background
Evidence about the efficacy and safety of statin treatment in high-risk patients with hypercholesterolemia is available for some populations, but not for ethnic Chinese. To test the hypothesis that treatment with pitavastatin (2 mg/day) is not inferior to treatment with atorvastatin (10 mg/day) for reducing low-density lipoprotein cholesterol (LDL-C), a 12-week multicenter collaborative randomized parallel-group comparative study of high-risk ethnic Chinese patients with hypercholesterolemia was conducted in Taiwan. In addition, the effects on other lipid parameters, inflammatory markers, insulin-resistance-associated biomarkers and safety were evaluated.
Methods and Results
Between July 2011 and April 2012, 251 patients were screened, 225 (mean age: 58.7 ± 8.6; women 38.2% [86/225]) were randomized and treated with pitavastatin (n = 112) or atorvastatin (n = 113) for 12 weeks. Baseline characteristics in both groups were similar, but after 12 weeks of treatment, LDL-C levels were significantly lower: pitavastatin group = −35.0 ± 14.1% and atorvastatin group = −38.4 ± 12.8% (both: p < 0.001). For the subgroup with diabetes mellitus (DM) (n = 125), LDL-C levels (−37.1 ± 12.9% vs. −38.0 ± 13.1%, p = 0.62) were similarly lowered after either pitavastatin (n = 63) or atorvastatin (n = 62) treatment. Triglycerides, non-high density lipoprotein cholesterol, and apoprotein B were similarly and significantly lower in both treatment groups. In non-lipid profiles, HOMA-IR and insulin levels were higher to a similar degree in both statin groups. Hemoglobin A1C was significantly (p = 0.001) higher in the atorvastatin group but not in the pitavastatin group. Both statins were well tolerated, and both groups had a similar low incidence of treatment-emergent adverse events.
Conclusion
Both pitavastatin (2 mg/day) and atorvastatin (10 mg/day) were well tolerated, lowered LDL-C, and improved the lipid profile to a comparable degree in high-risk Taiwanese patients with hypercholesterolemia.
Trial Registration
ClinicalTrials.gov NCT01386853 http://clinicaltrials.gov/ct2/show/NCT01386853?term=NCT01386853&rank=1
doi:10.1371/journal.pone.0076298
PMCID: PMC3788128  PMID: 24098467
15.  The Anticancer Properties and Apoptosis-inducing Mechanisms of Cinnamaldehyde and the Herbal Prescription Huang-Lian-Jie-Du-Tang (黃連解毒湯 Huáng Lián Jiě Dú Tang) in Human Hepatoma Cells 
Hepatocellular carcinoma (HCC) has long been one of the most important causes of cancer mortality in the world. Many natural products and traditional herbal medicines have been used to treat HCC in Asian countries such as Japan, Korea, Taiwan, and China. The present review aims to describe the anticancer properties and apoptotic mechanisms of cinnamaldehyde, the bioactive ingredient isolated from cinnamon trees, and the herbal prescription Huang-Lian-Jie-Du-Tang (黃連解毒湯 Huáng Lián Jiě Dú Tang; HLJDT) against human hepatoma cells in vitro and in vivo. Implication of their treatment for the development of targeted therapy against HCC is discussed.
doi:10.4103/2225-4110.119732
PMCID: PMC3924998  PMID: 24716182
Anticancer; Apoptosis; Cinnamaldehyde; Hepatoma; Huang-Lian-Jie-Du-Tang
16.  Stress Responsive Proteins Are Actively Regulated during Rice (Oryza sativa) Embryogenesis as Indicated by Quantitative Proteomics Analysis 
PLoS ONE  2013;8(9):e74229.
Embryogenesis is the initial step in a plant’s life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation.
doi:10.1371/journal.pone.0074229
PMCID: PMC3776822  PMID: 24058531
17.  Quantitative Evaluation of the Mitochondrial Proteomes of Drosophila melanogaster Adapted to Extreme Oxygen Conditions 
PLoS ONE  2013;8(9):e74011.
Mitochondria are the primary organelles that consume oxygen and provide energy for cellular activities. To investigate the mitochondrial mechanisms underlying adaptation to extreme oxygen conditions, we generated Drosophila strains that could survive in low- or high-oxygen environments (LOF or HOF, respectively), examined their mitochondria at the ultrastructural level via transmission electron microscopy, studied the activity of their respiratory chain complexes, and quantitatively analyzed the protein abundance responses of the mitochondrial proteomes using Isobaric tag for relative and absolute quantitation (iTRAQ). A total of 718 proteins were identified with high confidence, and 55 and 75 mitochondrial proteins displayed significant differences in abundance in LOF and HOF, respectively, compared with the control flies. Importantly, these differentially expressed mitochondrial proteins are primarily involved in respiration, calcium regulation, the oxidative response, and mitochondrial protein translation. A correlation analysis of the changes in the levels of the mRNAs corresponding to differentially regulated mitochondrial proteins revealed two sets of proteins with different modes of regulation (transcriptional vs. post-transcriptional) in both LOF and HOF. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to extreme oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.
doi:10.1371/journal.pone.0074011
PMCID: PMC3771901  PMID: 24069262
18.  Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry 
BMC Microbiology  2013;13:187.
Background
We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry.
Results
Extensive analysis of the tannins’ mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses.
Conclusions
CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified.
doi:10.1186/1471-2180-13-187
PMCID: PMC3750913  PMID: 23924316
Chebulagic acid; Punicalagin; Tannin; Broad-spectrum antivirals; Viral entry; Glycosaminoglycans
19.  Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway 
PLoS ONE  2013;8(6):e66589.
Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.
doi:10.1371/journal.pone.0066589
PMCID: PMC3688529  PMID: 23818948
20.  Human Platelet Antigen Alleles in 998 Taiwanese Blood Donors Determined by Sequence-Specific Primer Polymerase Chain Reaction 
BioMed Research International  2013;2013:973789.
Polymorphism of human platelet antigens (HPAs) leads to alloimmunizations and immune-mediated platelet disorders including fetal-neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population are important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific primer polymerase chain reaction. The HPA allele frequency was 99.55, and 0.45% for HPA-1a and -1b; 96.49, and 3.51% for HPA-2a and -2b; 55.81, and 44.19% for HPA-3a and -3b; 99.75, and 0.25% for HPA-4a and -4b; 98.50, and 1.50% for HPA-5a and -5b; 97.75 and 2.25% for HPA-6a and -6b; 53.71 and 46.29% for HPA-15a and -15b. HPA-15b and HPA-3a, may be considered the most important, followed by HPA-2, -6, -1, -5, and -4 systems, as a cause of FNAIT, PTP, and PTR based on allele frequency. HPA-4b and HPA-5b role cannot be excluded based on their immunogenicity. A larger-scale study will now be conducted to confirm these hypotheses and to establish an apheresis donor database for the procurement of HPA-matched apheresis platelets for patients with PTR.
doi:10.1155/2013/973789
PMCID: PMC3705808  PMID: 23865077
21.  Oxaliplatin-based Chemotherapy Might Provide Longer Progression-Free Survival in KRAS Mutant Metastatic Colorectal Cancer1 
Translational Oncology  2013;6(3):363-369.
The identification of better regimens in currently available chemotherapeutic agents is crucial for treating patients with KRAS mutant metastatic colorectal cancer (mCRC). Records of mCRC patients who received first-line oxaliplatin-based or irinotecan-based regimens were reviewed retrospectively. Clinicopathologic features and treatment outcome of patients with first-line progression-free survival (PFS) and overall survival (OS) in association with KRAS mutation status were analyzed using the Cox proportional hazard model. Between 2007 and 2010, a total of 118 mCRC patients were enrolled. Among them, 67 were males and 51 were females. In patients who received first-line oxaliplatin-based regimens, the PFS was significantly longer in KRAS mutant patients (N = 32) than that in KRAS wild-type patients (N = 51). The median PFS was 8.5 months in KRAS mutant versus 5.8 months in KRAS wild-type patients (P = .008). In contrast, in patients who received first-line irinotecan-based regimens, the PFS was shorter in KRAS mutant patients (N = 15) than that in KRAS wild-type patients (N = 20). Median PFS was 3.9 months in KRAS mutant versus 6.0 months in KRAS wild-type patients (P = .23). Median OS between KRAS mutant and wild-type patients was not significantly different in both oxaliplatin-based and irinotecan-based regimens. In multivariate analyses, KRAS mutation remains an independent predictive factor for longer PFS in first-line oxaliplatin-based regimens. In conclusion, oxaliplatin-based chemotherapy in KRAS mutant mCRC might result in longer PFS than in KRAS wild-type mCRC.
PMCID: PMC3660806  PMID: 23730417
22.  KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells 
PLoS ONE  2012;7(11):e50701.
Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.
doi:10.1371/journal.pone.0050701
PMCID: PMC3508995  PMID: 23209813
23.  Correction: An Improvement of Shotgun Proteomics Analysis by Adding Next-Generation Sequencing Transcriptome Data in Orange 
PLoS ONE  2012;7(10):10.1371/annotation/920bd689-3af7-418f-8149-43e683e18852.
doi:10.1371/annotation/920bd689-3af7-418f-8149-43e683e18852
PMCID: PMC3476949
24.  An Improvement of Shotgun Proteomics Analysis by Adding Next-Generation Sequencing Transcriptome Data in Orange 
PLoS ONE  2012;7(6):e39494.
Background
Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet.
Methodology/Principal Findings
In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data.
Conclusions/Significance
We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification.
doi:10.1371/journal.pone.0039494
PMCID: PMC3387166  PMID: 22768084
25.  Renal Protective Effect of Xiao-Chai-Hu-Tang on Diabetic Nephropathy of Type 1-Diabetic Mice 
Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese medicine formula consisting of seven medicinal plants, is used in the treatment of various diseases. We show here that XCHT could protect type-1 diabetic mice against diabetic nephropathy, using streptozotocin (STZ)-induced diabetic mice and high-glucose (HG)-exposed rat mesangial cell (RMC) as models. Following 4 weeks of oral administration with XCHT, renal functions and renal hypertrophy significantly improved in the STZ-diabetic mice, while serum glucose was only moderately reduced compared to vehicle treatment. Treatment with XCHT in the STZ-diabetic mice and HG-exposed RMC resulted in a decrease in expression levels of TGF-β1, fibronectin, and collagen IV, with concomitant increase in BMP-7 expression. Data from DPPH assay, DHE stain, and CM-H2DCFDA analysis indicated that XCHT could scavenge free radicals and inhibit high-glucose-induced ROS in RMCs. Taken together, these results suggest that treatment with XCHT can improve renal functions in STZ-diabetic mice, an effect that is potentially mediated through decreasing oxidative stress and production of TGF-β1, fibronectin, and collagen IV in the kidney during development of diabetic nephropathy. XCHT, therefore merits further investigation for application to improve renal functions in diabetic disorders.
doi:10.1155/2012/984024
PMCID: PMC3310293  PMID: 22474533

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