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1.  Zinc-α-2-Glycoprotein: A Candidate Biomarker for Colon Cancer Diagnosis in Chinese Population 
Zinc-α-2-glycoprotein (AZGP1) is a 41-kDa secreted glycoprotein, which has been detected in several malignancies. The diagnostic value of AZGP1 in serum of prostate and breast cancer patients has been reported. Analyzing “The Cancer Genome Atlas” data, we found that in colon cancer AZGP1 gene expression was upregulated at transcriptional level. We hypothesized that AZGP1 could be used as a diagnostic marker of colon cancer. First, we confirmed AZGP1 expression was higher in a set of 28 tumor tissues than in normal colonic mucosa tissues by real-time quantitative PCR and western blot in a Chinese population. We verified that serum concentration of AZGP1 was higher in 120 colon cancer patients compared with 40 healthy controls by ELISA (p < 0.001). Then receiver-operating characteristic (ROC) curve analysis was used to evaluate the predictive diagnostic value of AZGP1 in serum. The area under the curve (AUC) of AZGP1 was 0.742 (p < 0.001, 95% confidence interval (CI) = 0.656–0.827) in between the AUC of carcinoembryonic antigen (CEA) and the AUC of CA19-9, suggesting that predictive diagnostic value of AZGP1 is between CEA and Carbohydrate 19-9 (CA19-9). The combination of AZGP1 with traditional serum biomarkers, CEA and CA19-9, could result in better diagnostic results. To further validate the diagnostic value of AZGP1, a tissue microarray containing 190 samples of primary colon cancer tissue paired with normal colonic tissue was analysed and the result showed that AZGP1 was significantly upregulated in 68.4% (130 of 190) of the primary cancer lesions. In contrast, there was a weakly positive staining in 29.5% (56 of 190) of the normal colonic tissue samples (p < 0.001). Leave-one-out cross-validation was performed on the serum data, and showed that the diagnostic value of AZGP1 had 63.3% sensitivity and 65.0% specificity. Combination of AZGP1, CEA and CA19-9 had improved diagnosis value accuracy with 74.2% sensitivity and 72.5% specificity. These results suggest that AZGP1 is a useful diagnostic biomarker in tissues and serum from a Chinese population.
doi:10.3390/ijms16010691
PMCID: PMC4307269  PMID: 25561225
AZGP1 protein; human; colon cancer; clinical marker; tissue array analysis
2.  Differential Regulation of TNF Receptor 1 and Receptor 2 in Adiponectin Expression following Myocardial Ischemia 
International journal of cardiology  2013;168(3):2201-2206.
Background
In vitro experiments demonstrate that adiponectin, a cardioprotective cytokine, is inhibited by tumor necrosis factor-alpha (TNFα). However, the role of TNFα in post-myocardial infarction (post-MI) adiponectin reduction remains unclear. More importantly, the TNF receptor type (TNFR1 or TNFR2) responsible for TNFα-mediated suppression of adiponectin production is unknown. The current study determined the role of TNFα in post-myocardial infarction (post-MI) adiponectin reduction, and identified the receptor type responsible for TNFα-mediated suppression of adiponectin production.
Methods and Results
Adult male wild type (WT) and three knockout variety (TNFα−/−, TNFR1−/−, and TNFR2−/−) mice were subjected to MI via coronary artery occlusion. Histological and biochemical analyses were performed 3 and 7 days post-MI. In WT mice, MI significantly increased plasma TNFα, reduced adipocyte adiponectin mRNA, and decreased plasma adiponectin levels. TNFα deletion had no significant effect upon basal adiponectin level, and partially restored adiponectin expression/production post-MI (P<0.01 vs. WT). Basal adiponectin levels were significantly increased in TNFR1−/− (P<0.05 vs. WT), and unchanged in TNFR2−/− mice. Importantly, suppressed adiponectin expression/production by MI or TNFα administration was markedly decreased by TNFR1 deletion (P<0.01 vs. WT), but exacerbated by TNFR2 deletion (P<0.05 vs. WT). Mechanistically, TNFR1 knockout significantly inhibited, whereas TNFR2 knockout further enhanced TNFα-induced mRNA and protein expression of ATF3, a transcriptional factor known to significantly inhibit adiponectin expression.
Conclusion
Our study demonstrates TNFα overproduction is responsible for reduced adiponectin expression/production following MI. Furthermore, we show TNFR1/TNFR2 exert opposite effects upon adiponectin expression/production via differential regulation of ATF3.
doi:10.1016/j.ijcard.2013.01.222
PMCID: PMC3676729  PMID: 23465561
Adiponectin; Diabetes; Tumor Necrosis Factor; Receptor
3.  Thioredoxin Reductase Was Nitrated in the Aging Heart After Myocardial Ischemia/Reperfusion 
Rejuvenation Research  2013;16(5):377-385.
Abstract
The age-related loss of anti-oxidant defense reduces recovery from myocardial ischemia/reperfusion injury (MI/R) in aged people. Our previous data showed that inactivation of thioredoxin (Trx) was involved in enhanced aging MI/R injury. Thioredoxin reductase (TrxR), the enzyme known to regulate Trx, is less efficient with age. The aim of the current study was to determine why TrxR activity was reduced and whether reduced TrxR activity contributed to enhanced aging MI/R injury. Both Trx and TrxR activity were decreased in the aging heart, and this difference was further amplified after MI/R. However, MI/R injury did not change TrxR expression between young and aging rats. Increased nitrogen oxide (NOx) but decreased nitric oxide (NO) bioavailability (decreased phosphorylated vasodilator-stimulated phosphoprotein) was observed in aging hearts. Peroxynitrite (ONOO−) was increased in aging hearts and was further amplified after MI/R. TrxR nitration in young and aging hearts was detected by immunoprecipitation (anti-nitrotyrosine) followed by immunoblotting (anti-TrxR). Compared with young hearts, TrxR nitration was increased in the aging hearts, and this was further intensified after MI/R. The ONOO− decomposition catalyst (FeTMPyp) reduced TrxR nitration and increased TrxR and Trx activity. More importantly, FeTMPyp attenuated the MI/R injury in aging hearts as evidenced by decreased caspase-3 and malondialdehyde (MDA) concentration and increased cardiac function. Increased ONOO− nitrated TrxR in the aging heart as a post-translational modification, which may be related to the enhanced MI/R injury of aging rats. Interventions that inhibit nitration and restore TrxR activity might be a therapy for attenuating enhanced MI/R injury in aging heart.
doi:10.1089/rej.2013.1437
PMCID: PMC3804317  PMID: 23802942
4.  C1q/TNF-Related Protein-9, a Novel Adipocyte-Derived Cytokine, Attenuates Adverse Remodeling in the Ischemic Mouse Heart via PKA Activation 
Circulation  2013;128(11 0 1):10.1161/CIRCULATIONAHA.112.000010.
Background
CTRP9 is a newly identified adiponectin paralog with established metabolic-regulatory properties. However, the role of CTRP9 in post-myocardial infarction (post-MI) remodeling remains completely unknown. This study determined whether C1q/TNF-related protein-9 (CTRP9) may regulate cardiac remodeling following acute myocardial infarction (AMI), and elucidated the underlying mechanisms.
Methods and Results
Male adult mice were subject to AMI by left anterior descending coronary artery (LAD) ligation or sham surgery, and treated with saline (vehicle) or globular CTRP9 via peritoneal implant osmotic-pumps for 6 weeks. H9C2 cardiac cell lines were utilized in vitro for determining underlying mechanisms. Adipocyte CTRP9 expression and plasma CTRP9 levels were both significantly reduced after AMI. Compared to vehicle, CTRP9 treatment improved animal survival rate (P<0.05), restored cardiac function (P<0.05), attenuated adverse remodeling (P<0.01), and ameliorated cardiomyocyte apoptosis and fibrosis following AMI (P<0.01). Among multiple anti-remodeling molecules determined, AMP-activated protein kinase (AMPK), protein kinase-A (PKA), and Akt were significantly activated in CTRP9-treated heart. Surprisingly, CTRP9 remains cardioprotective in cardiac-specific AMPK-DN mice. Additional in vitro experiments demonstrated that administration of either PKA inhibitor or PKA-specific siRNA virtually abolished CTRP9’s anti-apoptotic effect (P<0.05), whereas inhibition of Akt is less effective in blocking CTRP9 cardioprotection. Finally, CTRP9 phosphorylates BAD at its multiple anti-apoptotic sites, an effect blocked by PKA inhibitor.
Conclusions
We demonstrate that adipokine CTRP9 attenuates adverse cardiac remodeling following AMI, largely via a PKA-dependent pathway.
doi:10.1161/CIRCULATIONAHA.112.000010
PMCID: PMC3824619  PMID: 24030394
adipokine; remodeling; apoptosis; myocardial infarction
5.  Complex microstructures of ABC triblock copolymer thin films directed by polymer brushes based on self-consistent field theory 
Nanoscale Research Letters  2014;9(1):359.
The morphology and the phase diagram of ABC triblock copolymer thin film directed by polymer brushes are investigated by the self-consistent field theory in three dimensions. The polymer brushes coated on the substrate can be used as a good soft template to tailor the morphology of the block copolymer thin films compared with those on the hard substrates. The polymer brush is identical with the middle block B. By continuously changing the composition of the block copolymer, the phase diagrams are constructed for three cases with the fixed film thickness and the brush density: identical interaction parameters, frustrated and non-frustrated cases. Some ordered complex morphologies are observed: parallel lamellar phase with hexagonally packed pores at surfaces (LAM3 ll -HFs), perpendicular lamellar phase with cylinders at the interface (LAM⊥-CI), and perpendicular hexagonally packed cylinders phase with rings at the interface (C2⊥-RI). A desired direction (perpendicular or parallel to the coated surfaces) of lamellar phases or cylindrical phases can be obtained by varying the composition and the interactions between different blocks. The phase diagram of ABC triblock copolymer thin film wetted between the polymer brush-coated surfaces is very useful in designing the directed pattern of ABC triblock copolymer thin film.
doi:10.1186/1556-276X-9-359
PMCID: PMC4120731  PMID: 25114650
ABC triblock copolymer; Polymer brush; Morphology; Thin film; Self-consistent field theory; Phase diagram
6.  Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury 
European Heart Journal  2012;34(24):1834-1845.
Aims
Emerging evidence indicates that nuclear receptors play a critical regulatory role in cardiovascular physiology/pathology. Recently, farnesoid-X-receptor (FXR), a member of the metabolic nuclear receptor superfamily, has been demonstrated to be expressed in vascular cells, with important roles in vascular physiology/pathology. However, the potential cardiac function of FXR remains unclear. We investigated the cardiac expression and biological function of FXR.
Methods and results
Farnesoid-X-receptor was detected in both isolated neonatal rat cardiac myocytes and fibroblasts. Natural and synthetic FXR agonists upregulated cardiac FXR expression, stimulated myocyte apoptosis, and reduced myocyte viability dose- and time-dependently. Mechanistic studies demonstrated that FXR agonists disrupted mitochondria, characterized by mitochondrial permeability transition pores activation, mitochondrial potential dissipation, cytochrome c release, and both caspase-9 and -3 activation. Such mitochondrial apoptotic responses were abolished by siRNA-mediated silencing of endogenous FXR or pharmacological inhibition of mitochondrial death signalling. Furthermore, low levels of FXR were detected in the adult mouse heart, with significant (∼2.0-fold) upregulation after myocardial ischaemia/reperfusion (MI/R). Pharmacological inhibition or genetic ablation of FXR significantly reduced myocardial apoptosis by 29.0–53.4%, decreased infarct size by 23.4–49.7%, and improved cardiac function in ischaemic/reperfused myocardium.
Conclusion
These results demonstrate that nuclear receptor FXR acts as a novel functional receptor in cardiac tissue, regulates apoptosis in cardiomyocytes, and contributes to MI/R injury.
doi:10.1093/eurheartj/ehs011
PMCID: PMC3689100  PMID: 22307460
Nuclear receptors; Apoptosis; Myocytes
7.  Variations in the protein level of Omi/HtrA2 in the heart of aged rats may contribute to the increased susceptibility of cardiomyocytes to ischemia/reperfusion injury and cell death 
Age  2012;35(3):733-746.
Survival after acute myocardial infarction is decreased in elderly patients. The enhanced rates of apoptosis in the aging heart exacerbate myocardial ischemia/reperfusion (MI/R) injury. We have recently demonstrated that the X-linked inhibitor of apoptosis protein (XIAP), the most potent endogenous inhibitor of apoptosis, was decreased in aging rats’ hearts. XIAP was balanced by two mitochondria proteins, Omi/HtrA2 and Smac/DIABLO. However, the implicative role of XIAP, Omi/HtrA2, and Smac/DIABLO to aging-related MI/R injury has not been previously investigated. In our study, male aging rats (20–24 months) or young adult rats (4–6 months) were subjected to 30 min of myocardial ischemia followed by reperfusion. MI/R-induced cardiac injury was enhanced in aging rats, as evidenced by aggravated cardiac dysfunction, enlarged infarct size, and increased myocardial apoptosis (TUNEL and caspase-3 activity). Then, the XIAP, Omi/HtrA2, and Smac/DIABLO protein and mRNA expression was detected. XIAP protein and mRNA expression was decreased in both aging hearts and aging hearts subjected to MI/R. Meanwhile, myocardial XIAP protein expression was correlated to cardiac function after MI/R. However, Omi/HtrA2, but not Smac/DIABLO, expression was increased in aging hearts. Moreover, the translocation of Omi/HtrA2 from mitochondria to cytosol was increased in both aging hearts and aging hearts subjected to MI/R. Treatment with ucf-101 (a novel and specific Omi/HtrA2 inhibitor) attenuated XIAP degradation and caspase-3 activity and exerted cardioprotective effects. Taken together, these results demonstrated that increased expression and leakage of Omi/HtrA2 enhanced MI/R injury in aging hearts via degrading XIAP and promoting myocardial apoptosis.
doi:10.1007/s11357-012-9406-x
PMCID: PMC3636415  PMID: 22535253
Aging; Myocardial ischemia/reperfusion; Apoptosis; XIAP; Omi/HtrA2
8.  Specific Changes of Serum Proteins in Parkinson's Disease Patients 
PLoS ONE  2014;9(4):e95684.
The aim of this study is to identify and validate protein change in the serum from PD patients. We used serum samples from 21 PD patients and 20 age-matched normal people as control to conduct a comparative proteomic study. We performed 2-DE and analyzed the differentially expressed protein spots by LC-MS/MS. In PD group 13 spots were shown to be differentially expressed compared to control group. They were identified as 6 proteins. Among these, 3 proteins were confirmed by Western blot analysis. It showed that the frequency of fibrinogen γ-chain (FGG) appeared 70% in PD, which could not be detected in control group. The protein of inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4) was found to exist two forms in serum. The full size (120 kDa) of the protein was increased and the fragmented ITI-H4 (35 kDa) was decreased in PD group. The ratio of full size ITI-H4 to fragmented ITI-H4 in PD patients was 3.85±0.29-fold higher than in control group. Furthermore, fragmented Apo A-IV (∼26 kDa) was mainly detected in control group, while it was rare to be found in PD group. Above findings might be useful for diagnosis of PD. When the expressions of FGG and 120 kDa ITI-H4 are increase, as well as ∼26 kDa Apo A-IV disappear would provide strong evidence for PD.
doi:10.1371/journal.pone.0095684
PMCID: PMC4000217  PMID: 24769800
9.  Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit 
Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.
doi:10.1111/jcmm.12187
PMCID: PMC3930416  PMID: 24286368
Cordycepin; AMPK; LKB1; Molecular docking
10.  Genome Sequences of an H5N1 Highly Pathogenic Avian Influenza Virus Isolated from Vaccinated Layers in China in 2012 
Genome Announcements  2013;1(6):e00936-13.
An H5N1 virus was isolated from vaccinated layers during an outbreak of highly pathogenic avian influenza (HPAI) in Ningxia, China, in 2012. Phylogenetic analysis revealed that the virus is a novel variant in clade 7.2, and the outbreak likely resulted from mutations in the viral hemagglutinin (HA) gene.
doi:10.1128/genomeA.00936-13
PMCID: PMC3837171  PMID: 24265490
11.  Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway 
PLoS ONE  2013;8(8):e70956.
Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.
doi:10.1371/journal.pone.0070956
PMCID: PMC3745454  PMID: 23976968
12.  C1q/TNF-Related Protein-3, a Newly Identified Adipokine, is a Novel Anti-Apoptotic, Pro-Angiogenic, and Cardioprotective Molecule in the Ischemic Mouse Heart 
Circulation  2012;125(25):3159-3169.
Background
Obesity/diabetes adversely affects post-ischemic heart remodeling via incompletely understood underlying mechanisms. C1q/TNF-related protein-3 (CTRP3) is a newly identified adipokine exerting beneficial metabolic regulation, similar to adiponectin. The current study determined whether CTRP3 may regulate post-ischemic cardiac remodeling and cardiac dysfunction, and, if so, sought to elucidate the involved underlying mechanisms.
Methods and Results
Male adult mice were subjected to myocardial infarction (MI) via left anterior descending (LAD) coronary artery occlusion. Both the effect of MI upon endogenous CTRP3 expression/production and the effect of exogenous CTRP3 (adenovirus or recombinant CTRP3) replenishment upon MI injury were investigated. MI significantly inhibited adipocyte CTRP3 expression and reduced plasma CTRP3 level, reaching nadir 3 days post-MI. CTRP3 replenishment improved survival rate (P<0.05), restored cardiac function, attenuated cardiomyocyte apoptosis, increased revascularization, and dramatically reduced interstitial fibrosis (P values all <0.01). CTRP3 replenishment had no significant effect upon cardiac AMP-activated protein kinase (AMPK) phosphorylation, but significantly increased Akt phosphorylation and expression of hypoxia inducing factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Surprisingly, treatment of human umbilical vascular endothelial cells (HUVECs) with CTRP3 did not directly affect NO production or tube formation. However, pre-conditioned medium from CTRP3-treated cardiomyocytes significantly enhanced HUVEC tube formation, an effect blocked by either pre-treatment of cardiomyocytes with a PI3K inhibitor, or pre-treatment of HUVECs with a VEGF inhibitor. Finally, pre-conditioned medium from CTRP3-knockdown 3T3 cells significantly enhanced hypoxia-induced cardiomyocyte injury.
Conclusions
CTRP3 is a novel anti-apoptotic, pro-angiogenic, and cardioprotective adipokine, whose expression is significantly inhibited following MI.
doi:10.1161/CIRCULATIONAHA.112.099937
PMCID: PMC3391311  PMID: 22653084
CTRP3; revascularization; myocardial ischemia; signal transduction
13.  Tumor Necrosis Factor-α and Lymphotoxin-α Mediate Myocardial Ischemic Injury via TNF Receptor 1, but Are Cardioprotective When Activating TNF Receptor 2 
PLoS ONE  2013;8(5):e60227.
Objective
This study determines the roles of tumor necrosis factor-α (TNFα) and lymphotoxin-α (LTα) in post-myocardial infarction (post-MI) cardiac injury, and identifies the TNF receptor type responsible for TNFα- and LTα-mediated cardiac injury.
Methods and Results
Adult male wild type (WT), TNFα−/−, LTα−/−, TNFR1−/−, and TNFR2−/− mice were subjected to MI via coronary artery occlusion. Functional, histological, and biochemical analyses were performed 1 to 7 days post-MI. In WT mice, MI significantly increased both TNFα and LTα levels in plasma, but in distinct temporal manner. Plasma TNFα peaked 1 day after MI, and decreased toward baseline 3 days after MI. In contrast, plasma LTα became significantly increased 3 days post-MI, and remained elevated thereafter. TNFα deletion significantly improved cardiac function 3 days, but not 7 days, after MI. In contrast, LTα deletion had no effect upon cardiac dysfunction 3 days after MI, but improved cardiac function 7 days after MI. More importantly, knockout of TNFR1 and TNFR2 had opposite effects upon post-MI cardiac dysfunction, which was markedly attenuated by TNFR1 deletion (P<0.01 vs. WT), but exacerbated by TNFR2 deletion (P<0.05 vs. WT).
Conclusion
Our study demonstrates that TNFα and LTα overproduction contribute to early and late cardiac dysfunction after MI, respectively. We provide clear evidence that both TNFα and LTα mediate post-MI cardiac dysfunction via TNFR1 stimulation, whereas TNFR2 activation is cardioprotective against ischemic injury. Simultaneous inhibition of TNFα and LTα or specific TNFR1 function blockade may represent superior cardioprotective approaches over general TNF activity suppression.
doi:10.1371/journal.pone.0060227
PMCID: PMC3660398  PMID: 23704873
14.  Intraoperative Cryoprecipitate Transfusion and Its Association with the Incidence of Biliary Complications after Liver Transplantation-A Retrospective Cohort Study 
PLoS ONE  2013;8(5):e60727.
Background
Cryoprecipitate is largely used for acquired hypofibrinogenemia in the setting of massive hemorrhage in liver transplantation (LT). However, the influence of intraoperative cryoprecipitate transfusion on biliary complications (BC) after LT has not been studied in detail.
Study Design and Methods
In a series of 356 adult patients who received their first LT, the causes of BC were retrospectively studied by multivariate logistic regression analysis. The clinical relationship between intraoperative cryoprecipitate transfusion and BC occurrence was studied through a retrospective cohort study in patients. All patients received follow-ups for one year, and, during the follow-up period, the time of BC occurrence and liver biopsies were recorded.
Results
Intraoperative cryoprecipitate transfusion (RR = 3.46, 95% CI [1.72–6.97], P<0.001), cold ischemia time >8 h (RR = 4.24, 95% CI [2.28–7.92], P<0.01), and high-level Child-Pugh ( RR = 1.71, 95% CI [1.11–2.63], P = 0.014) are independent risk factors to predict BC after LT according to time-to-event analysis. One year BC-free survival probability of patients received intraoperative cryoprecipitate transfusions was significantly lower when compared to the group that received no cryoprecipitate(P<0.001). Moreover, BC patients in the cryoprecipitate transfusion group owned different liver pathological feature, pathological micro-thrombus formation and cholestasis were seen more often (41.4% vs 0%, 62.1% vs 12.5%, respectively) than no cryoprecipitate transfusion group.
Conclusion
These findings suggested that intraoperative cryoprecipitate transfusion was associated with BC after LT. The mechanism of BC occurrence might involve micro-thrombus formation and immune rejection.
doi:10.1371/journal.pone.0060727
PMCID: PMC3651089  PMID: 23675406
15.  Essential Role of Caveolin-3 in Adiponectin Signalsome Formation and Adiponectin Cardioprotection 
Objectives
Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection.
Methods and Results
Compared to wild type (WT), Cav-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery, P<0.05). Although the expression level of key APN signaling molecules were normal in Cav-3KO, the cardioprotective effects of APN observed in WT were either markedly reduced or completely lost in Cav-3KO. Molecular and cellular experiments revealed that AdipoR1 co-localizes with Cav-3, forming AdipoR1/Cav-3 complex via specific Cav-3 scaffolding domain binding motifs. AdipoR1/Cav-3 interaction is required for APN-initiated AMPK-dependent and AMPK-independent intracellular cardioprotective signalings. More importantly, APPL1 and adenylate cyclase (AC), two immediately downstream molecules respectively required for AMPK-dependent and AMPK-independent signaling, form a protein complex with AdipoR1 in a Cav-3 dependent fashion. Finally, pharmacological activation of both AMPK plus PKA significantly reduced myocardial infarct size and improved cardiac function in Cav-3KO animals.
Conclusion
Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.
doi:10.1161/ATVBAHA.111.242164
PMCID: PMC3312581  PMID: 22328772
Diabetes; Myocardial Ischemia/Reperfusion; Adipocytokine; Signaling Mechanism
16.  Activation of the Nuclear Factor E2-Related Factor 2/Antioxidant Response Element Pathway Is Neuroprotective after Spinal Cord Injury 
Journal of Neurotrauma  2012;29(5):936-945.
Abstract
The activation of oxidative damage, neuroinflammation, and mitochondrial dysfunction has been implicated in secondary pathomechanisms following spinal cord injury (SCI). These pathophysiological processes lead to cell death and are tightly regulated by nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) signaling. Here, we investigated whether activation of Nrf2/ARE is neuroprotective following SCI. Female Fischer rats were subjected to mild thoracic SCI (T8) using the New York University injury device. As early as 30 min after SCI, levels of Nrf2 transcription factor were increased in both nuclear and cytoplasmic fractions of neurons and astrocytes at the lesion site and remained elevated for 3 days. Treatment of injured rats with sulforaphane, an activator of Nrf2/ARE signaling, significantly increased levels of Nrf2 and glutamate-cysteine ligase (GCL), a rate-limiting enzyme for synthesis of glutathione, and decreased levels of inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) thus leading to a reduction in contusion volume and improvement in coordination. These results show that activation of the Nrf2/ARE pathway following SCI is neuroprotective and that sulforaphane is a viable compound for neurotherapeutic intervention in blocking pathomechanisms following SCI.
doi:10.1089/neu.2011.1922
PMCID: PMC3303102  PMID: 21806470
antioxidant response element; inflammation; neuroprotection; nuclear factor E2-related factor 2; oxidative stress; SCI
17.  IOL Tilt and Decentration Estimation from 3 Dimensional Reconstruction of OCT Image 
PLoS ONE  2013;8(3):e59109.
Purpose
To evaluate intraocular lens (IOL) tilt and decentration by anterior segment optical coherence tomography (AS-OCT) using 3-dimensional (3D) reconstruction method.
Design
Prospective observational case series.
Participants
Thirty-nine patients (39 eyes) were included.
Methods
The IOL positions of all eyes were examined by AS-OCT. Images were obtained in 4 axes (0–180 degrees, 45–225 degrees, 90–270 degrees, and 135–315 degrees) using the quadrant-scan model. The cross-sectional images were analyzed with MATLAB software.
Main Outcome Measures
The angle (θ) between the reference pupillary plane and the IOL plane, the distances between the center points of the pupil circle and the IOL on the x-axis (dx) and y-axis (dy) and the spatial distance (ds) were calculated after 3D-reconstruction.
Results
The mean angle (θ) between the pupillary plane and the IOL plane was 2.94±0.99 degrees. The mean IOL decentration of dx and dy was 0.32±0.26 mm and 0.40±0.27 mm, respectively. The ds of the IOL decentration was 0.56±0.31 mm. There was no significant correlation between the ocular residual astigmatism (ORA) and the tilted angle or the decentration distance. There was a significant correlation between the ORA and total astigmatism (r = 0.742, P<0.001). There was no significant correlation between the postoperative best corrected visual acuity (BCVA) and the ORA (r = 0.156; P = 0.344), total astigmatism (r = 0.012; P = 0.942), tilted angle (θ; r = 0.172; P = 0.295) or decentration distance (dx: r = 0.191, P = 0.244; dy: r = 0.253, P = 0.121; ds: r = 0.298, P = 0.065).
Conclusions
AS-OCT can be used as an alternative for the analysis of IOL tilt and decentration using 3D-reconstruction.
doi:10.1371/journal.pone.0059109
PMCID: PMC3598664  PMID: 23554982
18.  ASTROGLIOSIS INVOLVES ACTIVATION OF RIG-LIKE SIGNALING IN THE INNATE IMMUNE RESPONSE AFTER SPINAL CORD INJURY 
Glia  2011;60(3):414-421.
Spinal cord injury (SCI) induces a glial response in which astrocytes become activated and produce inflammatory mediators. The molecular basis for regulation of glial-innate immune responses remains poorly understood. Here, we examined the activation of retinoic acid inducible gene (RIG)-like receptors (RLRs) and their involvement in regulating inflammation following SCI. We show that astrocytes express two intracellular RLRs: RIG-I and melanoma differentiation-associated gene 5 (MDA5). SCI and stretch injury of cultured astrocytes stimulated RLR signaling as determined by phosphorylation of IRF3 leading to production of type I interferons (IFNs). RLR signaling stimulation with synthetic RNA resulted in RLR activation, phosphorylation of interferon regulatory factor 3 (IRF3), and increased expression of glial fibrillary acidic protein and vimentin, two hallmarks of reactive astrocytes. Moreover, mitochondrial E3 ubiquitin protein ligase 1 (MUL1), an RLR inhibitor, decreased production of glial fibrillary acidic protein (GFAP) and vimentin following RIG-I signaling stimulation. Our findings identify a role for RLR signaling and type I IFN in regulating astrocyte innate immune responses after SCI.
doi:10.1002/glia.22275
PMCID: PMC3265608  PMID: 22161971
Innate immunity; Neuroinflammation; Spinal Cord Injury; Astrocytes
19.  C1q/TNF-Related Proteins (CTRPs), A Family of Novel Adipokines, Induce Vascular Relaxation through the Adiponectin Receptor-1/AMPK/eNOS/Nitric Oxide Signaling Pathway 
Objective
Reduced plasma adiponectin (APN) in diabetic patients is associated with endothelial dysfunction. However, APN knockout animals manifest modest systemic dysfunction unless metabolically challenged. The protein family CTRPs (C1q/TNF-related proteins) has recently been identified as APN paralogs and some CTRP members share APN’s metabolic regulatory function. However, the vasoactive properties of CTRPs remain completely unknown.
Methods and Results
The vasoactivity of currently identified murine CTRP members was assessed in aortic vascular rings and underlying molecular mechanisms was elucidated in HUVECs. Of eight CTRPs, CTRPs 3, 5, and 9 caused significant vasorelaxation. The vasoactive potency of CTRP9 exceeded that of APN (3-fold), and is endothelium-dependent and nitric oxide (NO) mediated. Mechanistically, CTRP9 increased AMPK/Akt/eNOS phosphorylation and increased NO production. AMPK knockdown completely blocked CTRP9-induced Akt/eNOS phosphorylation and NO production. Akt knockdown had no significant effect upon CTRP9-induced AMPK phosphorylation, but blocked eNOS phosphorylation and NO production. Adiponectin receptor 1 (AdipoR1), but not receptor 2, knockdown blocked CTRP9-induced AMPK/Akt/eNOS phosphorylation and NO production. Finally, pre-incubating vascular rings with an AMPK-inhibitor abolished CTRP9-induced vasorelaxive effects.
Conclusion
We have provided the first evidence that CTRP9 is a novel vasorelaxive adipocytokine which may exert vasculoprotective effects via the AdipoR1/AMPK/eNOS dependent/NO mediated signaling pathway.
doi:10.1161/ATVBAHA.111.231050
PMCID: PMC3197867  PMID: 21836066
Endothelial Function; Nitric Oxide; Diabetes; Signal Transduction
20.  Reduced Cardioprotective Action of Adiponectin in High-Fat Diet–Induced Type II Diabetic Mice and Its Underlying Mechanisms 
Antioxidants & Redox Signaling  2011;15(7):1779-1788.
Abstract
Diabetes exacerbates ischemic heart disease morbidity and mortality via incompletely understood mechanisms. Although adiponectin (APN) reduces myocardial ischemia/reperfusion (MI/R) injury in nondiabetic animals, whether APN's cardioprotective actions are altered in diabetes, a pathologic condition with endogenously reduced APN, has never been investigated. High-fat diet (HD)–induced diabetic mice and normal diet (ND) controls were subjected to MI via coronary artery ligation, and given vehicle or APN globular domain (gAPN, 2 μg/g) 10 min before reperfusion. Compared to ND mice (where gAPN exerted pronounced cardioprotection), HD mice manifested greater MI/R injury, and a tripled gAPN dose was requisite to achieve cardioprotective extent seen in ND mice (i.e., infarct size, apoptosis, and cardiac function). APN reduces MI/R injury via AMP-activated protein kinase (AMPK)–dependent metabolic regulation and AMPK-independent antioxidative/antinitrative pathways. Compared to ND, HD mice manifested significantly blunted gAPN-induced AMPK activation, basally and after MI/R (p < 0.05). Although both low- and high-dose gAPN equally attenuated MI/R-induced oxidative stress (i.e., NADPH oxidase expression and superoxide production) and nitrative stress (i.e., inducible nitric oxide synthase expression, nitric oxide production, and peroxynitrite formation) in ND mice, only high-dose gAPN efficaciously did so in HD mice. We demonstrate for the first time that HD-induced diabetes diminished both AMPK-dependent and AMPK-independent APN cardioprotection, suggesting an unreported diabetic heart APN resistance. Antioxid. Redox Signal. 15, 1779–1788.
doi:10.1089/ars.2010.3722
PMCID: PMC3159116  PMID: 21091073
21.  The Absence of Nrf2 Enhances NF-κB-Dependent Inflammation following Scratch Injury in Mouse Primary Cultured Astrocytes 
Mediators of Inflammation  2012;2012:217580.
It has been proved that Nrf2 depletion enhances inflammatory process through activation of NF-κB in the brain after TBI, but little is known about the relationship between Nrf2 and NF-κB in astrocytes after TBI. Hence, we used primary cultured astrocytes from either Nrf2 wildtype or knockout mice to study the influence of Nrf2 on the activation of NF-κB and expression of proinflammatory cytokines in a model of TBI in vitro. Primary cultured astrocytes were scratched to mimic the traumatic injury in vitro. Then the DNA-binding activity of NF-κB was evaluated by EMSA. The mRNA and protein levels of TNF-α, IL-1β, IL-6, and MMP9 were also evaluated. Gelatin zymography was performed to detect the activity of MMP9. The activity of NF-κB and expression of proinflammatory cytokines mentioned above were upregulated at 24 h after scratch. The expression and activity of MMP9 were also elevated. And such tendency was much more prominent in Nrf2 KO astrocytes than that in WT astrocytes. These results suggest that the absence of Nrf2 may induce more aggressive inflammation through activation of NF-κB and downstream proinflammatory cytokines in astrocytes.
doi:10.1155/2012/217580
PMCID: PMC3317373  PMID: 22529521
22.  Level of G Protein-Coupled Receptor Kinase-2 Determines Myocardial Ischemia-Reperfusion Injury via Pro- and Anti-Apoptotic Mechanisms 
Circulation research  2010;107(9):1140-1149.
Rationale
Activation of pro-survival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia-reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs) and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.
Objective
A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been done with GRK2 knockout mice as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model.
Methods and Results
We demonstrate, using cardiac-specific GRK2 and βARKct expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2 overexpressing mice (45.0±2.8% vs. 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, p<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via βARKct and these anti-apoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of β2ARs abolished βARKct-mediated cardioprotection suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival.
Conclusion
The novel effect of reducing acute ischemic myocardial injury via increased Akt activity adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct to not only chronic HF but also potentially in acute ischemic injury conditions.
doi:10.1161/CIRCRESAHA.110.221010
PMCID: PMC2966514  PMID: 20814022
acute myocardial ischemia; ischemia/reperfusion injury; cardioprotection; G protein-coupled receptor kinase-2; βARKct; Myocyte apoptosis
23.  Reduced Vascular Responsiveness to Adiponectin in Hyperlipidemic Rats -- Mechanisms and Significance 
Deficiency of adiponectin (APN), an adipocyte-derived vascular protective molecule, contributes to diabetic vascular injury. The current study determined whether obesity/hyperlipidemia may alter the vascular response to APN, and investigated the involved mechanisms and pathologic significance. Adult male Sprague-Dawley rats were fed a regular or high-fat diet (HF) for 4–16 weeks. Circulating APN levels, aortic pAMPK/AMPK, peNOS/eNOS, and APN receptor expression levels were determined. Compared to time-matched animals fed control diet, plasma APN levels in HF-diet animals were significantly increased at 8 weeks, and rapidly declined thereafter. Despite unchanged or elevated circulating APN levels, phosphorylated AMPK and eNOS in vascular tissue were significantly reduced at all observed time points. Recombinant full length APN (rAPN) induced AMPK/eNOS phosphorylation and vasodilatation were significantly reduced in 16-week obese/hyperlipidemic aortic segments. Vascular APN receptor 1 (AdipoR1) and receptor 2 (AdipoR2) expression were significantly reduced 16 weeks after HF-diet. Pre-incubation of rAPN with obese/hyperlipidemic plasma, but not with normal plasma, significantly reduced its AMPK and eNOS activation effect, and blunted its protective effect against TNFα-induced HUVEC apoptosis. This study demonstrated for the first time that obesity/hyperlipidemia reduces vascular responsiveness to APN. Modification/inactivation of APN by unidentified factors present in obese/hyperlipidemic plasma, decreased vascular AdipoR1/R2 expression, and reduced circulating APN levels contribute to reduced vascular responsiveness to APN at different stages of the obese condition. Reduced APN bioactivity allows unmitigated TNFα pro-apoptotic and pro-inflammatory actions, contributing to vascular injury in obesity/hyperlipidemia.
doi:10.1016/j.yjmcc.2010.03.002
PMCID: PMC2904862  PMID: 20303976
Vascular injury; Cytokines; Obesity; Dyslipidemia; Adiponectin
24.  Eugenol Reduces the Expression of Virulence-Related Exoproteins in Staphylococcus aureus▿  
Applied and Environmental Microbiology  2010;76(17):5846-5851.
Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both Gram-positive and Gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 μg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of α-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 μg/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.
doi:10.1128/AEM.00704-10
PMCID: PMC2935054  PMID: 20639367
25.  Subinhibitory Concentrations of Perilla Oil Affect the Expression of Secreted Virulence Factor Genes in Staphylococcus aureus 
PLoS ONE  2011;6(1):e16160.
Background
The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.
Methodology/Principal Findings
A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).
Conclusions/Significance
The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.
doi:10.1371/journal.pone.0016160
PMCID: PMC3023776  PMID: 21283822

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