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1.  Concerted actions of the catechol O-methyltransferase and the cytosolic sulfotransferase SULT1A3 in the metabolism of catecholic drugs 
Biochemical pharmacology  2012;84(9):1186-1195.
Catecholic drugs had been reported to be metabolized through conjugation reactions, particularly methylation and sulfation. Whether and how these two Phase II conjugation reactions may occur in a concerted manner, however, remained unclear. The current study was designed to investigate the methylation and/or sulfation of five catecholic drugs. Analysis of the spent media of HepG2 cells metabolically labeled with [35S]sulfate in the presence of individual catecholic drugs revealed the presence of two [35S]sulfated metabolites for dopamine, epinephrine, isoproterenol, and isoetharine, but only one [35S]sulfated metabolite for apomorphine. Further analyses using tropolone, a catechol O-methyltransferase (COMT) inhibitor, indicated that one of the two [35S]sulfated metabolites of dopamine, epinephrine, isoproterenol, and isoetharine was a doubly conjugated (methylated and sulfated) product, since its level decreased proportionately with increasing concentrations of tropolone added to the labeling media. Moreover, while the inhibition of methylation resulted in a decrease of the total amount of [35S]sulfated metabolites, sulfation appeared to be capable of compensating the suppressed methylation in the metabolism of these four catecholic drugs. A two-stage enzymatic assay showed the sequential methylation and sulfation of dopamine, epinephrine, isoproterenol, and isoetharine mediated by, respectively, the COMT and the cytosolic sulfotransferase SULT1A3. Collectively, the results from the present study implied the concerted actions of the COMT and SULT1A3 in the metabolism of catecholic drugs.
PMCID: PMC3622478  PMID: 22917559
Methylation; Sulfation; COMTs; SULTs; Catecholic drugs
2.  Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases 
Journal of Biochemistry  2012;152(3):275-283.
Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [35S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.
PMCID: PMC3529569  PMID: 22763752
feed additive; ractopamine; salbutamol; sulfation; SULT
3.  Identification and Characterization of Zebrafish SULT1 ST9, SULT3 ST4, and SULT3 ST5 
By searching the GenBank database, we identified sequences encoding three new zebrafish cytosolic sulfotransferases (SULTs). These three new zebrafish SULTs, designated SULT1 ST9, SULT3 ST4, and SULT3 ST5, were cloned, expressed, purified, and characterized. SULT1 ST9 appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as β-naphthol, β-naphthylamine, caffeic acid and gallic acid. SULT3 ST4 showed strong activity toward endogenous compound such as dehydroepiandrosterone (DHEA), pregnenolone, and 17β-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, as well as xenobiotics including mestranol, β-naphthylamine, β-naphthol, and butylated hydroxyl anisole (BHA). pH-dependency and kinetic constants of these three enzymes were determined with DHEA, β-naphthol, and 17β-estradiol as substrates. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the expression of these three new zebrafish SULTs at different developmental stages during embryogenesis, through larval development, and on to maturity.
PMCID: PMC3521529  PMID: 22360938
Cytosolic sulfotransferase; SULT; 17β-estradiol; dehydroepiandrosterone; molecular cloning; developmental expression; zebrafish
4.  A comparative study of the sulfation of bile acids and a bile alcohol by the Zebra danio (Danio rerio) and human cytosolic sulfotransferases (SULTs) 
The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.
PMCID: PMC3515676  PMID: 21839837
Sulfotransferase; Sulfation; Zebra danio; Bile acid
5.  Identification, Characterization, and Ontogenic Study of a Catechol O-methyltransferase from Zebrafish 
To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5’- and 3’-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.
PMCID: PMC3515678  PMID: 21371608
Catechol O-methyltransferase; developmental expression; methylation; molecular cloning; zebrafish
6.  A Target-specific Approach for the Identification of Tyrosine-sulfated Hemostatic Proteins 
Analytical biochemistry  2009;390(1):88-90.
A simple methodology for the identification of hemostatic proteins that are subjected to post-translational tyrosine sulfation was developed. The procedure involved sequence analysis of members of the three hemostatic pathways using Sulfinator prediction algorithm, followed by [35S]sulfate-labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [35S]sulfate-labeled hemostatic proteins, and tyrosine O[35S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins, protein S, prekallikrein and plasminogen, were identified. Such a target-specific approach will allow for investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understating of the functional role of post-translational tyrosine sulfation.
PMCID: PMC2695991  PMID: 19351526
Hemostasis; tyrosine sulfation; post-translational protein modification
7.  The Induction of Phagocytic activation by Mixtures of the Water Chlorination By-Products, Dichloroacetate- and Trichloroacetate in Mice after Subchronic Exposure 
In this study, groups of B6C3F1 male mice were treated with dichloroacetate (DCA), trichloroacetate (TCA), and mixtures of the compounds (Mix I, Mix II and Mix III) daily by gavage, for 13 weeks. The tested doses were 7.5, 15 and 30 mg DCA/kg/day and 12.5, 25 and 50 mg TCA/kg/day. The DCA: TCA ratios in Mix I, II and III were 7.5:12.5, 15:25 and 30:50 mg/kg/day, respectively. Peritoneal lavage cells were collected at the end of the treatment period and assayed for the biomarkers of phagocytic activation, including superoxide anion and tumor necrosis factor-alpha production, and myeloperoxidase activity. The mixtures produced non-linear effects on the biomarkers of phagocytic activation, with Mix I and II effects were found to be additive, but Mix III effects were found to be less than additive.
PMCID: PMC3646527  PMID: 23436740
Chloroacetates; Mixtures; Phagocytic activation
8.  Oleanolic Acid Suppresses Aerobic Glycolysis in Cancer Cells by Switching Pyruvate Kinase Type M Isoforms 
PLoS ONE  2014;9(3):e91606.
Warburg effect, one of the hallmarks for cancer cells, is characterized by metabolic switch from mitochondrial oxidative phosphorylation to aerobic glycolysis. In recent years, increased expression level of pyruvate kinase M2 (PKM2) has been found to be the culprit of enhanced aerobic glycolysis in cancer cells. However, there is no agent inhibiting aerobic glycolysis by targeting PKM2. In this study, we found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and consistently, abrogated Warburg effect in cancer cells. Suppression of aerobic glycolysis by OA is mediated by PKM2/PKM1 switch. Furthermore, mTOR signaling was found to be inactivated in OA-treated cancer cells, and mTOR inhibition is required for the effect of OA on PKM2/PKM1 switch. Decreased expression of c-Myc-dependent hnRNPA1 and hnRNPA1 was responsible for OA-induced switch between PKM isoforms. Collectively, we identified that OA is an antitumor compound that suppresses aerobic glycolysis in cancer cells and there is potential that PKM2 may be developed as an important target in aerobic glycolysis pathway for developing novel anticancer agents.
PMCID: PMC3953484  PMID: 24626155
9.  CS5931, a Novel Polypeptide in Ciona savignyi, Represses Angiogenesis via Inhibiting Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs) 
Marine Drugs  2014;12(3):1530-1544.
CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell) in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF) production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9) both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.
PMCID: PMC3967225  PMID: 24633253
CS5931; anti-angiogenesis; zebrafish; HUVECs; VEGF and MMPs
10.  Comparison of Spectral and Image Morphological Analysis for Egg Early Hatching Property Detection Based on Hyperspectral Imaging 
PLoS ONE  2014;9(2):e88659.
The use of non-destructive methods to detect egg hatching properties could increase efficiency in commercial hatcheries by saving space, reducing costs, and ensuring hatching quality. For this purpose, a hyperspectral imaging system was built to detect embryo development and vitality using spectral and morphological information of hatching eggs. A total of 150 green shell eggs were used, and hyperspectral images were collected for every egg on day 0, 1, 2, 3 and 4 of incubation. After imaging, two analysis methods were developed to extract egg hatching characteristic. Firstly, hyperspectral images of samples were evaluated using Principal Component Analysis (PCA) and only one optimal band with 822 nm was selected for extracting spectral characteristics of hatching egg. Secondly, an image segmentation algorithm was applied to isolate the image morphologic characteristics of hatching egg. To investigate the applicability of spectral and image morphological analysis for detecting egg early hatching properties, Learning Vector Quantization neural network (LVQNN) was employed. The experimental results demonstrated that model using image morphological characteristics could achieve better accuracy and generalization than using spectral characteristic parameters, and the discrimination accuracy for eggs with embryo development were 97% at day 3, 100% at day 4. In addition, the recognition results for eggs with weak embryo development reached 81% at day 3, and 92% at day 4. This study suggested that image morphological analysis was a novel application of hyperspectral imaging technology to detect egg early hatching properties.
PMCID: PMC3923798  PMID: 24551130
11.  ASDCD: Antifungal Synergistic Drug Combination Database 
PLoS ONE  2014;9(1):e86499.
Finding effective drugs to treat fungal infections has important clinical significance based on high mortality rates, especially in an immunodeficient population. Traditional antifungal drugs with single targets have been reported to cause serious side effects and drug resistance. Nowadays, however, drug combinations, particularly with respect to synergistic interaction, have attracted the attention of researchers. In fact, synergistic drug combinations could simultaneously affect multiple subpopulations, targets, and diseases. Therefore, a strategy that employs synergistic antifungal drug combinations could eliminate the limitations noted above and offer the opportunity to explore this emerging bioactive chemical space. However, it is first necessary to build a powerful database in order to facilitate the analysis of drug combinations. To address this gap in our knowledge, we have built the first Antifungal Synergistic Drug Combination Database (ASDCD), including previously published synergistic antifungal drug combinations, chemical structures, targets, target-related signaling pathways, indications, and other pertinent data. Its current version includes 210 antifungal synergistic drug combinations and 1225 drug-target interactions, involving 105 individual drugs from more than 12,000 references. ASDCD is freely available at
PMCID: PMC3901703  PMID: 24475134
12.  Influenza Virus-Induced Lung Inflammation Was Modulated by Cigarette Smoke Exposure in Mice 
PLoS ONE  2014;9(1):e86166.
Although smokers have increased susceptibility and severity of seasonal influenza virus infection, there is no report about the risk of 2009 pandemic H1N1 (pdmH1N1) or avian H9N2 (H9N2/G1) virus infection in smokers. In our study, we used mouse model to investigate the effect of cigarette smoke on pdmH1N1 or H9N2 virus infection. Mice were exposed to cigarette smoke for 21 days and then infected with pdmH1N1 or H9N2 virus. Control mice were exposed to air in parallel. We found that cigarette smoke exposure alone significantly upregulated the lung inflammation. Such prior cigarette smoke exposure significantly reduced the disease severity of subsequent pdmH1N1 or H9N2 virus infection. For pdmH1N1 infection, cigarette smoke exposed mice had significantly lower mortality than the control mice, possibly due to the significantly decreased production of inflammatory cytokines and chemokines. Similarly, after H9N2 infection, cigarette smoke exposed mice displayed significantly less weight loss, which might be attributed to lower cytokines and chemokines production, less macrophages, neutrophils, CD4+ and CD8+ T cells infiltration and reduced lung damage compared to the control mice. To further investigate the underlying mechanism, we used nicotine to mimic the effect of cigarette smoke both in vitro and in vivo. Pre-treating the primary human macrophages with nicotine for 72 h significantly decreased their expression of cytokines and chemokines after pdmH1N1 or H9N2 infection. The mice subcutaneously and continuously treated with nicotine displayed significantly less weight loss and lower inflammatory response than the control mice upon pdmH1N1 or H9N2 infection. Moreover, α7 nicotinic acetylcholine receptor knockout mice had more body weight loss than wild-type mice after cigarette smoke exposure and H9N2 infection. Our study provided the first evidence that the pathogenicity of both pdmH1N1 and H9N2 viruses was alleviated in cigarette smoke exposed mice, which might partially be attributed to the immunosuppressive effect of nicotine.
PMCID: PMC3897646  PMID: 24465940
13.  The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis 
PLoS ONE  2014;9(1):e85626.
Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs.
PMCID: PMC3893223  PMID: 24454905
14.  Quantification of strain and charge co-mediated magnetoelectric coupling on ultra-thin Permalloy/PMN-PT interface 
Scientific Reports  2014;4:3688.
Strain and charge co-mediated magnetoelectric coupling are expected in ultra-thin ferromagnetic/ferroelectric multiferroic heterostructures, which could lead to significantly enhanced magnetoelectric coupling. It is however challenging to observe the combined strain charge mediated magnetoelectric coupling, and difficult in quantitatively distinguish these two magnetoelectric coupling mechanisms. We demonstrated in this work, the quantification of the coexistence of strain and surface charge mediated magnetoelectric coupling on ultra-thin Ni0.79Fe0.21/PMN-PT interface by using a Ni0.79Fe0.21/Cu/PMN-PT heterostructure with only strain-mediated magnetoelectric coupling as a control. The NiFe/PMN-PT heterostructure exhibited a high voltage induced effective magnetic field change of 375 Oe enhanced by the surface charge at the PMN-PT interface. Without the enhancement of the charge-mediated magnetoelectric effect by inserting a Cu layer at the PMN-PT interface, the electric field modification of effective magnetic field was 202 Oe. By distinguishing the magnetoelectric coupling mechanisms, a pure surface charge modification of magnetism shows a strong correlation to polarization of PMN-PT. A non-volatile effective magnetic field change of 104 Oe was observed at zero electric field originates from the different remnant polarization state of PMN-PT. The strain and charge co-mediated magnetoelectric coupling in ultra-thin magnetic/ferroelectric heterostructures could lead to power efficient and non-volatile magnetoelectric devices with enhanced magnetoelectric coupling.
PMCID: PMC3891213  PMID: 24418911
15.  Loss of long-term depression in the insular cortex after tail amputation in adult mice 
Molecular Pain  2014;10:1.
The insular cortex (IC) is an important forebrain structure involved in pain perception and taste memory formation. Using a 64-channel multi-electrode array system, we recently identified and characterized two major forms of synaptic plasticity in the adult mouse IC: long-term potentiation (LTP) and long-term depression (LTD). In this study, we investigate injury-related metaplastic changes in insular synaptic plasticity after distal tail amputation. We found that tail amputation in adult mice produced a selective loss of low frequency stimulation-induced LTD in the IC, without affecting (RS)-3,5-dihydroxyphenylglycine (DHPG)-evoked LTD. The impaired insular LTD could be pharmacologically rescued by priming the IC slices with a lower dose of DHPG application, a form of metaplasticity which involves activation of protein kinase C but not protein kinase A or calcium/calmodulin-dependent protein kinase II. These findings provide important insights into the synaptic mechanisms of cortical changes after peripheral amputation and suggest that restoration of insular LTD may represent a novel therapeutic strategy against the synaptic dysfunctions underlying the pathophysiology of phantom pain.
PMCID: PMC3912895  PMID: 24398034
16.  A Computational Framework to Infer Human Disease-Associated Long Noncoding RNAs 
PLoS ONE  2014;9(1):e84408.
As a major class of noncoding RNAs, long noncoding RNAs (lncRNAs) have been implicated in various critical biological processes. Accumulating researches have linked dysregulations and mutations of lncRNAs to a variety of human disorders and diseases. However, to date, only a few human lncRNAs have been associated with diseases. Therefore, it is very important to develop a computational method to globally predict potential associated diseases for human lncRNAs. In this paper, we developed a computational framework to accomplish this by combining human lncRNA expression profiles, gene expression profiles, and human disease-associated gene data. Applying this framework to available human long intergenic noncoding RNAs (lincRNAs) expression data, we showed that the framework has reliable accuracy. As a result, for non-tissue-specific lincRNAs, the AUC of our algorithm is 0.7645, and the prediction accuracy is about 89%. This study will be helpful for identifying novel lncRNAs for human diseases, which will help in understanding the roles of lncRNAs in human diseases and facilitate treatment. The corresponding codes for our method and the predicted results are all available at
PMCID: PMC3879311  PMID: 24392133
17.  Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  2013;51(7):2400-2402.
We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks.
PMCID: PMC3697666  PMID: 23616462
18.  Current Progress in Gene Delivery Technology Based on Chemical Methods and Nano-carriers 
Theranostics  2014;4(3):240-255.
Gene transfer methods are promising in the field of gene therapy. Current methods for gene transfer include three major groups: viral, physical and chemical methods. This review mainly summarizes development of several types of chemical methods for gene transfer in vitro and in vivo by means of nano-carriers like; calcium phosphates, lipids, and cationic polymers including chitosan, polyethylenimine, polyamidoamine dendrimers, and poly(lactide-co-glycolide). This review also briefly introduces applications of these chemical methods for gene delivery.
PMCID: PMC3915088  PMID: 24505233
Non-viral; Gene delivery; Vectors; Chemical Methods.
19.  Gene Features Selection for Three-Class Disease Classification via Multiple Orthogonal Partial Least Square Discriminant Analysis and S-Plot Using Microarray Data 
PLoS ONE  2013;8(12):e84253.
DNA microarray analysis is characterized by obtaining a large number of gene variables from a small number of observations. Cluster analysis is widely used to analyze DNA microarray data to make classification and diagnosis of disease. Because there are so many irrelevant and insignificant genes in a dataset, a feature selection approach must be employed in data analysis. The performance of cluster analysis of this high-throughput data depends on whether the feature selection approach chooses the most relevant genes associated with disease classes.
Here we proposed a new method using multiple Orthogonal Partial Least Squares-Discriminant Analysis (mOPLS-DA) models and S-plots to select the most relevant genes to conduct three-class disease classification and prediction. We tested our method using Golub’s leukemia microarray data. For three classes with subtypes, we proposed hierarchical orthogonal partial least squares-discriminant analysis (OPLS-DA) models and S-plots to select features for two main classes and their subtypes. For three classes in parallel, we employed three OPLS-DA models and S-plots to choose marker genes for each class. The power of feature selection to classify and predict three-class disease was evaluated using cluster analysis. Further, the general performance of our method was tested using four public datasets and compared with those of four other feature selection methods. The results revealed that our method effectively selected the most relevant features for disease classification and prediction, and its performance was better than that of the other methods.
PMCID: PMC3875537  PMID: 24386356
20.  Pygopus Maintains Heart Function in Aging Drosophila Independently of Canonical Wnt Signaling 
Circulation. Cardiovascular genetics  2013;6(5):10.1161/CIRCGENETICS.113.000253.
Heart function declines with age, but the genetic factors underlying such deterioration are largely unknown. Wnt signaling is known to play a role in heart development, but it has not been shown to be important in adult heart function. We have investigated the nuclear adapter protein encoded by pygopus (pygo), which mediates canonical Wnt signaling, for roles in aging-related cardiac dysfunction.
Methods and Results
Using the Drosophila heart model, we show that cardiac-specific pygo knockdown in adult flies causes a significant (4- to 5-fold) increase in cardiac arrhythmias (P<0.001) that worsened with age and caused a significant decrease in contractility (−54%; P<0.001) with systolic dysfunction. Immunohistochemistry revealed structural abnormalities that worsened with age, and both functional and morphological alterations were ameliorated by pygo overexpression. Unexpectedly, knockdown of 2 other Wnt signaling components, β-cat/armadillo or TCF/pangolin, had relatively milder effects on cardiac function. Double-heterozygous combinations of mutants for pygo and canonical Wnt signaling components had no additional effect on heart function over pygo heterozygotes alone. However, double knockdown of pygo and Ca2+/calmodulin-dependent protein kinase II caused additional arrhythmia compared with pygo knockdown alone, suggesting that some of the effects of pygo are mediated by Ca2+ signaling. In the isoproterenol-induced hypertrophic mouse model, we show that Pygo1 protein levels are increased.
Our data indicate that Pygo plays a critical role in adult heart function that is Wnt signaling independent and is likely conserved in mammals.
PMCID: PMC3871875  PMID: 24046329
arrhythmias; cardiac; atrial fibrillation; cardiac defects; cardiomyopathies; hypertrophy; physiopathology; systolic time interval
22.  Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry 
Parasites & Vectors  2013;6:355.
Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the host’s immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry.
The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis.
The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes.
In this study, T. spiralis muscle larval surface proteins have been identified, which will provide useful information to elucidate the host-parasite interaction, identify the invasion-related proteins, early diagnostic antigens and the targets for a vaccine.
PMCID: PMC3866304  PMID: 24330777
T. spiralis; Muscle larvae; Surface proteins; Mass spectrometry
23.  MiR-19a is correlated with prognosis and apoptosis of laryngeal squamous cell carcinoma by regulating TIMP-2 expression 
MiRNAs are small, noncoding RNA molecules that act as posttranscriptional regulators of gene expression and function as important regulators in cancer-related processes. The miR-19a is overexpressed in various cancers and has been causally related to cellular proliferation and growth. To determine whether miR-19a plays a role in laryngeal squamous cell carcinoma (LSCC), we used quantitative real time PCR to detect miR-19a expression in LSCC tissues. We found that miR-19a is overexpressed in LSCC and correlated with neck nodal metastasis, poor differentiation and advanced stage. Statistical analysis suggests that higher level of miR-19a was associated with reduced overall survival. In vitro functional study showed that inhibition of miR-19a by antisense oligonucleotides (ASO) led to apoptosis and reduction of cell proliferation in LSCC cells. Furthermore, growth of LSCC xenograft tumors was significantly suppressed by repeated injection of ASO-miR-19a lentivirus. The TUNEL stain and transmission electron microscopy also detected increased apoptotic cells in ASO-miR-19a treated LSCC xenografts. In addition, both realtime PCR and western blot showed ASO-miR-19a can upregulate TIMP-2 expression and this suggests miR-19a is related with TIMP-2 pathway in LSCC cells. Taken together, these data suggest that miR-19a plays an oncogenic role in the progression of LSCC, and may serve as a biomarker or therapeutic target for patients with LSCC.
PMCID: PMC3885460  PMID: 24427326
miR-19a; LSCC; TIMP-2; apoptosis
24.  Therapeutic effects of sesame oil on monosodium urate crystal-induced acute inflammatory response in rats 
SpringerPlus  2013;2:659.
Sesame oil has been used in traditional Taiwanese medicine to relieve the inflammatory pain in people with joint inflammation, toothache, scrapes, and cuts. However, scientific evidence related to the effectiveness or action mechanism of sesame oil on relief of pain and inflammation has not been examined experimentally. Here, we investigated the therapeutic effect of sesame oil on monosodium urate monohydrate (MSU) crystal-induced acute inflammatory response in rats. Air pouch, a pseudosynovial cavity, was established by injecting 24 mL of filtered sterile air subcutaneously in the backs of the rats. At day 0, inflammation in air pouch was induced by injecting MSU crystal (5 mg/rat, suspended in sterilized phosphate buffered saline, pH 7.4), while sesame oil (0, 1, 2, or 4 mL/kg, orally) was given 6 h after MSU crystal injection. Parameters in lavage and skin tissue from the air pouches were assessed 6 h after sesame oil was given. Sesame oil decreased MSU crystal-induced total cell counts, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 levels in lavage and pouch tissue. Sesame oil significantly decreased leukocyte and neutrophil counts in lavage compared with MSU crystal alone group. Sesame oil decreased activated mast cell counts in skin tissue in MSU crystal-treated rats. Sesame oil significantly decreased nuclear factor (NF)-κB activity and IL-4 level in isolated mast cells from rats treated with MSU crystal. Furthermore, sesame oil decreased lavage complement proteins C3a and C5a levels in MSU crystal-treated rats. In conclusion, sesame oil shows a potent therapeutic effect against MSU crystal-induced acute inflammatory response in rats.
PMCID: PMC3866373  PMID: 24353977
Gout; Hyperuricemia; Monosodium urate monohydrate; Mast cell; Inflammation; Arthritis
25.  Elevaed Levels of Serum Glial FibrillaryAcidic Protein Breakdown Products in Mild and Moderate Traumatic Brain Injury are Associated With Intracranial Lesions and Neurosurgical Intervention 
Annals of emergency medicine  2011;59(6):10.1016/j.annemergmed.2011.08.021.
This study examined whether serum levels of GFAP breakdown products (GFAP-BDP) were elevated in mild and moderate TBI compared to controls and if they were associated with traumatic intracranial lesions on CT scan (+CT) and having a neurosurgical intervention (NSI).
This prospective cohort study enrolled adult patients presenting to three Level 1 Trauma Centers following blunt head trauma with loss of consciousness, amnesia, or disorientation and a GCS 9–15. Control groups included normal uninjured controls and trauma controls presenting to the ED with orthopedic injuries or an MVC without TBI. Blood samples were obtained in all patients within 4 hours of injury and measured by ELISA for GFAP-BDP (ng/ml).
Of the 307 patients enrolled, 108 were TBI patients (97 with GCS 13–15, and 11 with GCS 9–12) and 199 were controls (176 normal controls and 16 MVC controls and 7 orthopedic controls). ROC curves demonstrated that early GFAP-BDP levels were able to distinguish TBI from uninjured controls with an AUC of 0.90 (95%CI 0.86–0.94) and differentiated TBI with a GCS 15 with an AUC 0.88 (95%CI 0.82–0.93). Thirty two TBI patients (30%) had lesions on CT. The AUC for discriminating those patients with CT lesions versus those without CT lesions was 0.79 (95%CI 0.69–0.89). Moreover, the ROC curve for distinguishing NSI from no NSI yielded an AUC of 0.87 (95%CI 0.77–0.96).
GFAP-BDP is detectable in serum within an hour of injury and is associated with measures of injury severity including the GCS score, CT lesions and neurosurgical intervention. Further study is required to validate these findings before clinical application.
PMCID: PMC3830977  PMID: 22071014
Traumatic Brain Injury; head injury; trauma; human; biomarkers; proteomics; diagnostic; Serum; Computed Tomography; neurosurgical intervention; sensitivity; specificity

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