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1.  Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence 
The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylated in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induce cell cycle arrest and senescence, leading to impaired cell proliferation.
PMCID: PMC4296733  PMID: 17588532
recepteur d’origine Nantais (RON); epithelial-to-mesenchymal transition (EMT); cell proliferation; cell migration and invasion; senescence
2.  TrkC expression predicts favorable clinical outcome in invasive ductal carcinoma of breast independent of NT-3 expression 
Background: TrkC, a member of neurotrophin receptor family, functions not only as an oncogene, but also act as a tumor suppressor via a manner of dependence receptor in human malignant tumors. Little is known on the action of TrkC for the clinical prognosis and the progression of breast cancer according to the availability of its ligand NT-3. We sought to investigate the prognostic relevance of NT-3-TrkC axis in breast cancer and estimate its role during the process of breast cancer progression. Methods: 236 cases of invasive ductal carcinoma (IDC), 60 pure ductal carcinoma in situ (DCIS) and 30 normal breast tissue (NBT) between 2004 and 2005 were included in the study. Spearman’s rank correlation test was used to analyze the association of NT-3-TrkC expression and breast cancer progression. The Kaplan-Meier method and Cox proportional hazards model were performed to identify the relevant prognostic factors. Results: 50.4% IDC tumors displayed absent or low TrkC expression, while 49.6% was high TrkC expression. TrkC expression was negatively associated with lymph node metastasis (P = 0.029) and tumor proliferation (P = 0.015). Patients with lower TrkC expressing tumors had a higher risk of recurrence (odds ratio, 0.401; 95% confidence interval, 0.207-0.778; P = 0.007). The layered analysis indicated that patients with high TrkC expression tumors had a favor disease-free survival whether NT-3 and TrkC were co-expressed or solitarily expressed in the tumor (P = 0.000). NT-3 was demonstrated to be not a predictor of IDC patients’ prognosis. But NT-3 expression was inversely correlated with the progression of breast cancer (r = -0.341, P = 0.000), since more IDC tumors showed high NT-3 expression than DCIS tumors (51.7% vs. 25.9%), while no NBT showed high NT-3 expression, as well. Conclusion: The study indicates TrkC expression reduces tumor relapse independent of NT-3 availability in the IDC. Elevated NT-3 expression contributes to the progression of breast cancer.
PMCID: PMC4266714  PMID: 25520870
TrkC; NT-3; invasive ductal carcinoma (IDC); breast; prognosis; progression; dependence receptor (DR)
3.  Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs 
PLoS ONE  2014;9(10):e109725.
Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.
PMCID: PMC4201481  PMID: 25329998
4.  Knowledge and practices of Thai emergency physicians regarding the care of delirious elderly patients 
The Society for Academic Emergency Medicine (SAEM) Geriatric Emergency Medicine Task Force recommends assessment of delirium for all elderly emergency department (ED) patients. Little is known about emergency physicians' (EPs) opinions regarding care of delirious elderly patients. We sought to determine the knowledge and practice experience of members of the Thai Association for Emergency Medicine regarding the care of delirious elderly ED patients.
We surveyed all Thai emergency physicians from July to September 2013 using a brief online survey as this does not include any non-trained physician working in the private/provincial/community EDs, still a significant part of the ED workforce in Thailand.
We had a response rate of 50% (239/474) of which 95% (228/239) completed the survey. Respondents largely reported that <10% of their patients experience delirium. Eighty-five percent of the respondents recognized delirium as a problem that required active intervention, and 76% of the respondents thought it was underdiagnosed in the ED. Only 24% of the respondents reported routinely screening delirium in the ED and 16% reported using a specific screening tool for delirium assessment. Forty-two percent of the respondents reported treating delirium with a long acting benzodiazepine and 29% reported using haloperidol. Forty percent of respondents thought that oversedation was the most common complication associated with drug treatment of delirium.
Basic knowledge and perceptions surrounding the recognition, diagnosis, and treatment of delirium in elderly ED patients by Thai EPs vary. Most of the Thai EPs consider delirium in the ED an emergency condition, while far fewer screen for this condition. Future research and quality improvement should determine which single screening tool is appropriate for EPs in regular practice as well as how to standardize delirium management in the ED.
PMCID: PMC4306074  PMID: 25635198
Delirium; Elderly; Emergency department; Emergency physician; Knowledge
5.  Expression of the SET protein in testes of mice at different developmental stages 
Asian Journal of Andrology  2014;16(5):689-693.
SET is a multifunctional protein involved in regulating many biological processes of the cell cycle. It is also a regulator of steroidogenesis in the ovary. However, the expression of SET protein in testis, and its function, still remains ambiguous. In this study, we observed the expression of SET in the testes of mice at different developmental stages, and have discussed its potential function in regulating spermatogenesis and androgen production. Forty-eight male mice at different developmental stages (1 week old as the infancy group; 4 weeks old as the prepubertal group; 12 weeks old as the adult group; over 12 months old as the ageing group) were used. Cellular location of SET protein in the testes was observed by immuno-histochemistry. Expression levels of Set mRNA and SET protein were analyzed by quantitative polymerase chain reaction and Western blotting. SET protein was expressed in spermatogonial cells and spermatocytes; the highest level was mainly in haploid and tetraploid cells of the prepubertal and adult groups, and Leydig cells of the adult and ageing groups. There was a low expression in Sertoli cells. Expression of Set mRNA in the prepubertal group was significantly higher than that in the adult group (P < 0.05), while expression of SET protein was at the highest level in the adult group (P < 0.05). SET protein is mainly expressed in spermatogonial cells and spermatocytes, and poorly expressed in Sertoli cells, suggesting that it is involved in spermatogenesis. Expression of SET protein in Leydig cells suggests a possible role in steroidogenesis.
PMCID: PMC4215651  PMID: 24923460
androgen production; Leydig cell; Sertoli cell; SET; spermatogenesis
6.  Laser-Machined Microcavities for Simultaneous Measurement of High-Temperature and High-Pressure 
Sensors (Basel, Switzerland)  2014;14(8):14330-14338.
Laser-machined microcavities for simultaneous measurement of high-temperature and high-pressure are demonstrated. These two cascaded microcavities are an air cavity and a composite cavity including a section of fiber and an air cavity. They are both placed into a pressure chamber inside a furnace to perform simultaneous pressure and high-temperature tests. The thermal and pressure coefficients of the short air cavity are ∼0.0779 nm/°C and ∼1.14 nm/MPa, respectively. The thermal and pressure coefficients of the composite cavity are ∼32.3 nm/°C and ∼24.4 nm/MPa, respectively. The sensor could be used to separate temperature and pressure due to their different thermal and pressure coefficients. The excellent feature of such a sensor head is that it can withstand high temperatures of up to 400 °C and achieve precise measurement of high-pressure under high temperature conditions.
PMCID: PMC4178988  PMID: 25106018
high pressure sensing; high temperature sensing; laser micromachining; micro-cavity; simultaneous measurement
7.  Association of caveolin-3 and cholecystokinin A receptor with cholesterol gallstone disease in mice 
AIM: To investigate the role of caveolin-3 (CAV3) and cholecystokinin A receptor (CCKAR) in cholesterol gallstone disease (CGD).
METHODS: To establish a mouse model of CGD, male C57BL/6 mice were fed with a lithogenic diet containing 1.0% cholic acid, 1.25% cholesterol and 15% fat; a similar control group was given a normal diet. The fresh liver weights and liver-to-body weight ratio were compared between the two groups after one month. Serum lipid profile and bile composition were determined with an autoanalyzer. The Cav3 and Cckar mRNA and CAV3 and CCKAR protein levels in the liver and gallbladder were determined via real-time polymerase chain reaction and Western blot, respectively.
RESULTS: Establishment of the mouse CGD model was verified by the presence of cholesterol gallstones in mice fed the lithogenic diet. Compared with mice maintained on a normal diet, those fed the lithogenic diet had significantly higher mean liver-to-body weight ratio (0.067 ± 0.007 vs 0.039 ± 0.007, P < 0.01), serum total cholesterol (4.22 ± 0.46 mmol/L vs 2.21 ± 0.11 mmol/L, P < 0.001), bile total cholesterol (1.33 ± 0.33 mmol/L vs 0.21 ± 0.11 mmol/L, P < 0.001), and bile phospholipid concentrations (3.55 ± 1.40 mmol/L vs 1.55 ± 0.63 mmol/L, P = 0.04), but lower total bile acid concentrations (726.48 ± 51.83 μmol/L vs 839.83 ± 23.74 μmol/L, P = 0.007). The lithogenic diet was also associated with significantly lower CAV3 in the liver and lower CAV3 and CCKAR in the gallbladder compared with the control mice (all P < 0.05).
CONCLUSION: CAV3 and CCKAR may be involved in cholesterol gallstone disease.
PMCID: PMC4110583  PMID: 25071346
Cholesterol gallstone disease; Caveolin-3; Cholecystokinin A receptor; Lithogenic diet; Mechanism
8.  High gene delivery efficiency of alkylated low-molecular-weight polyethylenimine through gemini surfactant-like effect 
To our knowledge, the mechanism underlying the high transfection efficiency of alkylated low-molecular-weight polyethylenimine (PEI) is not yet well understood. In this work, we grafted branched PEI (molecular weight of 1,800 Da; bPEI1800) with lauryl chains (C12), and found that bPEI1800-C12 was structurally similar to gemini surfactant and could similarly assemble into micelle-like particles. Stability, cellular uptake, and lysosome escape ability of bPEI1800-C12/DNA polyplexes were all greatly enhanced after C12 grafting. bPEI1800-C12/DNA polyplexes exhibited significantly higher transfection efficiency than Lipofectamine™ 2000 in the presence of serum. Bioluminescence imaging showed that systemic injection of bPEI1800-C12/DNA polyplexes resulted in intensive luciferase expression in vivo and bioluminescence signals that could be detected even in the head. Altogether, the high transfection efficacy of bPEI1800-C12 was because bPEI1800-C12, being an analog of gemini surfactant, facilitated lysosome escape and induced the coil–globule transition of DNA to assemble into a highly organized micelle-like structure that showed high stability.
PMCID: PMC4122513  PMID: 25114526
self-organization; alkylation; luciferase; bioluminescence imaging
9.  Mutations of C-Reactive Protein (CRP) -286 SNP, APC and p53 in Colorectal Cancer: Implication for a CRP-Wnt Crosstalk 
PLoS ONE  2014;9(7):e102418.
C-reactive protein (CRP) is an established marker of inflammation with pattern-recognition receptor-like activities. Despite the close association of the serum level of CRP with the risk and prognosis of several types of cancer, it remains elusive whether CRP contributes directly to tumorigenesis or just represents a bystander marker. We have recently identified recurrent mutations at the SNP position -286 (rs3091244) in the promoter of CRP gene in several tumor types, instead suggesting that locally produced CRP is a potential driver of tumorigenesis. However, it is unknown whether the -286 site is the sole SNP position of CRP gene targeted for mutation and whether there is any association between CRP SNP mutations and other frequently mutated genes in tumors. Herein, we have examined the genotypes of three common CRP non-coding SNPs (rs7553007, rs1205, rs3093077) in tumor/normal sample pairs of 5 cancer types (n = 141). No recurrent somatic mutations are found at these SNP positions, indicating that the -286 SNP mutations are preferentially selected during the development of cancer. Further analysis reveals that the -286 SNP mutations of CRP tend to co-occur with mutated APC particularly in rectal cancer (p = 0.04; n = 67). By contrast, mutations of CRP and p53 or K-ras appear to be unrelated. There results thus underscore the functional importance of the -286 mutation of CRP in tumorigenesis and imply an interaction between CRP and Wnt signaling pathway.
PMCID: PMC4099363  PMID: 25025473
10.  Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons 
BioMed Research International  2014;2014:290182.
Tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine regulating neuroinflammation. Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.
PMCID: PMC4100394  PMID: 25093162
11.  Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis 
Cell  2013;153(5):1012-1024.
Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.
PMCID: PMC3983474  PMID: 23706739
12.  The Inhibitory Effect of Binens Bipinnata L. Extract on U14 Tumour in Mice 
The objective of this paper was to study the in vitro and in vivo inhibitory effect of Bidens bipinnata L. extract on growth of cervical carcinoma U14 cells. MTT method was used to determine the inhibitory effect of Bidens bipinnata L. extract on U14 tumour cells, and the effects of Bidens bipinnata L. extract on inhibition rate of solid tumour and life prolongation rate of ascites tumour were observed through the establishment of two animal models of mouse cervical carcinoma U14 solid tumour and ascites tumour. In the in vitro MTT assay, the inhibition rate gradually increased with the increase of dose of Bidens bipinnata L. and the extension of time. Its inhibition rate was 70.44% at a concentration of 80µg/L. Solid tumour inhibition rates in the high- and low-dose groups and cisplatin group were 49.13%, 2.26% and 75.72% respectively; life prolongation rates in each ascites tumour group were 63.63%, 34.86% and 87.34% respectively. The Bidens bipinnata L. extract has a certain inhibitory effect on growth of mouse cervical carcinoma U14.
PMCID: PMC3794393  PMID: 24146503
Bidens bipinnata L. extract; U14; solid tumour; ascites tumour
13.  Predictors of recurrence in breast cancer subtypes with negative lymph node in a Chinese population 
To establish a series of objective parameters to predict the risk of relapse from axillary lymph node-negative (ANN) breast cancer, and evaluate the patterns of recurrence according to molecular subtypes, we collected information on 2126 consecutive breast cancer patients operated between 2002 and 2006. In this case-control study, 212 patients experiencing recurrence or breast cancer related death were defined as ‘poor group’. Another 212 patients were selected from the remaining cases with stratified sampling method to comprise the ‘good group’. Significant differences were found in vascular invasion, grade and molecular subtype between the two groups. Expression of ER and PR in the ‘poor group’ was lower (P < 0.05). However, positive rates of Ki67, p53 and VEGF in the ‘poor group’ were higher (P < 0.05). Multivariate analysis revealed that molecular subtype, expression of VEGF, tumor grade, and vascular invasion were closely correlated with bad outcome. Analysis of the ‘poor group’ demonstrated that ‘HER2 positive’ and ‘triple negative’ subtypes more commonly suffered from distant metastases and death. No metastasis was found in patients with pure invasive papillary carcinoma, invasive cribriform carcinoma or adenoid cystic carcinoma, whereas the diagnoses of invasive micropapillary carcinoma, invasive apocrine carcinoma, invasive papillary carcinoma mixed with invasive ductal carcinoma, or metaplastic carcinoma were correlated with distant metastasis and death. In conclusion, molecular subtype and expression of VEGF are useful markers for predicting prognosis of ANN breast cancer patients. ‘Luminal A-like’ subtype has better outcome than others. Moreover, molecular subtypes have different recurrence patterns.
PMCID: PMC4097226  PMID: 25031741
Breast cancer; molecular subtype; p53; VEGF; recurrence
14.  Effects of Time of Feeding on Psychostimulant Reward, Conditioned Place Preference, Metabolic Hormone Levels, and Nucleus Accumbens Biochemical Measures in Food-Restricted Rats 
Psychopharmacology  2013;227(2):307-320.
Chronic food restriction (FR) increases rewarding effects of abused drugs and persistence of a cocaine-conditioned place preference (CPP). When there is a single daily meal, circadian rhythms are correspondingly entrained, and pre- and postprandial periods are accompanied by different circulating levels of metabolic hormones that modulate brain dopamine function.
The present study assessed whether rewarding effects of d-amphetamine, cocaine, and persistence of cocaine CPP differ between FR subjects tested in the pre- and postprandial period.
Materials and methods
Rats were stereotaxically implanted with intracerebral microinjection cannulae and an electrode in lateral hypothalamus. Rewarding effects of d-amphetamine and cocaine were assessed using electrical self-stimulation in rats tested 1-4 or 18-21 hrs after the daily meal. Non-implanted subjects acquired a cocaine CPP while ad libitum fed, then were switched to FR and tested for CPP at these same times.
Rewarding effects of intra-nucleus accumbens (NAc) d-amphetamine, intraventricular cocaine, and persistence of cocaine CPP did not differ between rats tested 18-21 hrs food deprived, when ghrelin and insulin levels were at peak and nadir, respectively, and those tested 1-4 hrs after feeding. Rats that expressed a persistent CPP had elevated levels of p-ERK1, GluA1, and p-Ser845-GluA1 in NAc core, and the latter correlated with CPP expression.
Psychostimulant reward and persistence of CPP in FR rats are unaffected by time of testing relative to the daily meal. Further, NAc biochemical responses previously associated with enhanced drug responsiveness in FR rats are associated with persistent CPP expression.
PMCID: PMC3637844  PMID: 23354537
food restriction; nucleus accumbens; reward; self-stimulation; d-amphetamine; cocaine; conditioned place preference; ghrelin; insulin; ERK 1/2; GluA1
15.  Spin-Labeled Analogs of CMP-NeuAc as NMR Probes of the α-2,6-Sialyltransferase ST6Gal I 
Chemistry & biology  2007;14(4):409-418.
Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
PMCID: PMC3968682  PMID: 17462576
16.  Plasma microRNA-320, microRNA-let-7e and microRNA-21 as novel potential biomarkers for the detection of retinoblastoma 
Biomedical Reports  2014;2(3):424-428.
Retinoblastoma (RB) is a childhood malignancy caused by inactivation of the RB gene, with neuron-specific enolase (NSE) levels considered as its diagnostic marker. MicroRNAs (miRNAs) have been proven to play a significant role in multiple physiological and pathological processes and several miRNAs were identified as tumor biomarkers in recent studies. In the present study, 65 plasma samples were collected from RB patients and 65 samples from healthy individuals to serve as controls. The miRNA levels were measured via quantitative reverse transcription-polymerase chain reaction and their association with RB was assessed by statistical data analysis and receiver operating characteristic curves. Plasma miRNA (miR)-320, miR-let-7e and miR-21 levels were downregulated in the patient samples, the areas under the curves (AUCs) were 0.548–0.660, whereas the AUCs of combined classifiers were ≥0.990. The plasma miRNA levels, particularly of miR-320, were found to be of value in RB diagnosis and may be considered as novel diagnostic biomarkers.
PMCID: PMC3990201  PMID: 24748987
retinoblastoma; microRNA; plasma; biomarker
17.  MicroRNA-7a/b Protects against Cardiac Myocyte Injury in Ischemia/Reperfusion by Targeting Poly(ADP-Ribose) Polymerase 
PLoS ONE  2014;9(3):e90096.
MicroRNA-7 (miR-7) is highly connected to cancerous cell proliferation and metastasis. It is also involved in myocardial ischemia-reperfusion (I/R) injury and is upregulated in cardiomyocyte under simulated I/R (SI/R). We aimed to investigate the role of miR-7 during myocardial I/R injury in vitro and in vivo and a possible gene target.
Methods and Results
Real-time PCR revealed that miR-7a/b expression was upregulated in H9c2 cells after SI/R. Flow cytometry showed SI/R-induced cell apoptosis was decreased with miR-7a/b mimic transfection but increased with miR-7a/b inhibitor in H9c2 cells. In a rat cardiac I/R injury model, infarct size determination and TUNEL assay revealed that miR-7a/b mimic decreased but miR-7a/b inhibitor increased cardiac infarct size and cardiomyocyte apoptosis as compared with controls. We previously identified an important gene connected with cell apoptosis -- poly(ADP-ribose) polymerase (PARP) -- as a candidate target for miR-7a/b and verified the target by luciferase reporter activity assay and western blot analysis.
miR-7a/b is sensitive to I/R injury and protects myocardial cells against I/R-induced apoptosis by negatively regulating PARP expression in vivo and in vitro. miR-7a/b may provide a new therapeutic approach for treatment of myocardial I/R injury. Poly(ADP-ribose) polymerase.
PMCID: PMC3940823  PMID: 24594984
18.  Effects of Upregulation of Hsp27 Expression on Oocyte Development and Maturation Derived from Polycystic Ovary Syndrome 
PLoS ONE  2013;8(12):e83402.
Heat shock protein 27 (Hsp27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS) however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.
PMCID: PMC3877038  PMID: 24391762
19.  Promiscuous Interactions of gp78 E3 ligase CUE domain with polyubiquitin chains 
Structure (London, England : 1993)  2012;20(12):2138-2150.
Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin and a dynamic, promiscuous interaction with diubiquitin chains. This leads to a model where the CUE domain functions to both facilitate substrate binding and enables switching between adjacent ubiquitin molecules of a growing chain to facilitate processivity in ubiquitination.
PMCID: PMC3518592  PMID: 23123110
20.  Prolonged Emergency Department Length of Stay is not Associated with Worse Outcomes in Patients with Intracerebral Hemorrhage 
Neurocritical care  2012;17(3):334-342.
Prolonged emergency department length of stay (EDLOS) has been associated with worse patient outcomes, longer inpatient stays, and failure to meet quality measures in several acute medical conditions, but these findings have not been consistently reproduced. We performed this study to explore the hypothesis that longer EDLOS would be associated with worse outcomes in a large cohort of patients presenting with spontaneous intracerebral hemorrhage (ICH).
We performed a secondary analysis of a prospective cohort of consecutive patients with spontaneous ICH who presented to a single academic referral center from February 2005 to October 2009. The primary exposure variable was EDLOS, and our primary outcome was neurologic status at hospital discharge, measured with a modified Rankin scale (mRS). Secondary outcomes were ICU length of stay, total hospital length of stay, and total hospital costs.
Our cohort included 616 visits of which 42 were excluded, leaving 574 patient encounters for analysis. Median age was 75 years (IQR 63–82), median EDLOS 5.1 h (IQR 3.7–7.1) and median discharge mRS 4 (IQR 3–6). Thirty percent of the subjects died in-hospital. Multivariable proportional odds logistic regression, controlling for age, initial Glasgow Coma Scale, initial hematoma volume, ED occupancy at registration, and the need for intubation or surgical intervention, demonstrated no association between EDLOS and outcome. Furthermore, multivariable analysis revealed no association of increased EDLOS with ICU or hospital length of stay or hospital costs.
We found no effect of EDLOS on neurologic outcome or resource utilization for patients presenting with spontaneous ICH.
PMCID: PMC3684176  PMID: 21912953
Emergency medicine; Emergency department crowding; Emergency department length of stay; Intracerebral hemorrhage
21.  Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System 
The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II) on collagen synthesis in hypoxic human lung fibroblast (HLF) cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR) after hypoxic treatment. Additionally, the collagen type I (Col-I), AT1R and nuclear factor κappaB (NF-κB) protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA). We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST), an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC), a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.
PMCID: PMC3876092  PMID: 24336063
angiotensin II; hypoxic HLF-1; collagen synthesis; nuclear factor κappaB
22.  Modeling and Optimization of the Multiobjective Stochastic Joint Replenishment and Delivery Problem under Supply Chain Environment 
The Scientific World Journal  2013;2013:916057.
As a practical inventory and transportation problem, it is important to synthesize several objectives for the joint replenishment and delivery (JRD) decision. In this paper, a new multiobjective stochastic JRD (MSJRD) of the one-warehouse and n-retailer systems considering the balance of service level and total cost simultaneously is proposed. The goal of this problem is to decide the reasonable replenishment interval, safety stock factor, and traveling routing. Secondly, two approaches are designed to handle this complex multi-objective optimization problem. Linear programming (LP) approach converts the multi-objective to single objective, while a multi-objective evolution algorithm (MOEA) solves a multi-objective problem directly. Thirdly, three intelligent optimization algorithms, differential evolution algorithm (DE), hybrid DE (HDE), and genetic algorithm (GA), are utilized in LP-based and MOEA-based approaches. Results of the MSJRD with LP-based and MOEA-based approaches are compared by a contrastive numerical example. To analyses the nondominated solution of MOEA, a metric is also used to measure the distribution of the last generation solution. Results show that HDE outperforms DE and GA whenever LP or MOEA is adopted.
PMCID: PMC3835819  PMID: 24302880
23.  Glyceryl Trinitrate for Prevention of Post-ERCP Pancreatitis and Improve the Rate of Cannulation: A Meta-Analysis of Prospective, Randomized, Controlled Trials 
PLoS ONE  2013;8(10):e75645.
Acute pancreatitis is the most common complication of diagnostic and therapeutic endoscopic retrograde cholangiopancreatography (ERCP). Several clinical trials used glyceryl trinitrate (GTN) to prevent the incidence of post-ERCP pancreatitis (PEP). However, the results were still controversial.
To conduct a meta-analysis of published, full-length, randomized controlled trials evaluating the effect of prophylactic GTN on the prevention of PEP, improve the rate of cannulation and the prevention of hyperamylasemia.
Literature searches were conducted using PubMed, EMBASE, The Cochrane Library and Web of Knowledge databases, using keywords "post-ERCP" and "pancreatitis" and limited in randomized controlled trials.
Twelve RCTs involving 2649 patients were included. Eleven RCTs compared GTN with placebo for PEP prevention. Meta-analysis showed the overall incidence of PEP was significantly reduced by GTN treatment (RR 0.67; 95% CI, 0.52-0.87). Nevertheless, GTN administration did not decrease the incidence of moderate to severe PEP (RR 0.70; 95% CI, 0.42-1.15). Subgroup analyses revealed that GTN administered by sublingual was more effective than transdermal and topical in reducing the incidence of PEP. Besides, the prophylactic effect of GTN was far more obvious in the group of high PEP incidence than in the group of low PEP incidence. Additionally, the incidence of hyperamylasemia was significantly reduced by GTN treatment (RR 0.69; 95% CI, 0.54-0.90). No differences of the successful cannulation rate of bile ducts (RR 1.03; 95% CI, 0.99-1.06) attributable to GTN were observed.
Prophylactic use of GTN reduced the overall incidence of PEP and hyperamylasemia. However, GTN was not helpful for the severity of PEP and the rate of cannulation.
PMCID: PMC3787965  PMID: 24098392
24.  Leptin Induces IL-6 Expression through OBRl Receptor Signaling Pathway in Human Synovial Fibroblasts 
PLoS ONE  2013;8(9):e75551.
Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Leptin is abundantly expressed in osteoarthritis (OA) cartilage and synovium. However, the relationship between leptin and interleukin-6 (IL-6) in OA synovial fibroblasts (OASFs) remains obscure.
Methodology/Principal Findings
Stimulation of OASFs with leptin induced IL-6 expression in a concentration- and time-dependent manner. OASFs expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. However, OBRl, but not OBRs, antisense oligonucleotide (AS-ODN) abolished the leptin-mediated increase of IL-6 expression. Transfection with insulin receptor substrate (IRS)-1 siRNA decreased leptin-induced IL-6 production. In addition, pretreatment of cells with PI3K, Akt, or AP-1 inhibitor also inhibited the potentiating action of leptin. Leptin-induced AP-1 activation was inhibited by OBRl, IRS-1, PI3K, or Akt inhibitors and siRNAs.
Our results showed that leptin activates the OBRl receptor, which in turn activates IRS-1, PI3K, Akt, and AP-1 pathway, leading to up-regulation of IL-6 expression.
PMCID: PMC3785513  PMID: 24086566
25.  Fluopsin C induces oncosis of human breast adenocarcinoma cells 
Acta Pharmacologica Sinica  2013;34(8):1093-1100.
Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro.
Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (Δψm). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining.
Fluopsin C (0.5-8 μmol/L) reduced the cell viability in dose- and time-dependent manners. Its IC50 values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 μmol/L, respectively. Fluopsin C (2 μmol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and Δψm. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC50 values at 24 h of 2.7 and 2.4 μmol/L, respectively.
Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored.
PMCID: PMC4003024  PMID: 23708552
fluopsin C; Pseudomonas jinanesis; oncosis; human breast adenocarcinoma; membrane blebs

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