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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.
doi:10.1016/j.bcp.2012.08.009
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.
doi:10.1093/jb/mvs073
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.
doi:10.1016/j.aquatox.2012.01.015
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.
doi:10.1016/j.jsbmb.2011.07.011
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.
doi:10.1016/j.aquatox.2010.12.016
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.
Summary
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.
doi:10.1016/j.ab.2009.04.002
PMCID: PMC2695991  PMID: 19351526
Hemostasis; tyrosine sulfation; post-translational protein modification
7.  Newly Emerging Mutations in the Matrix Genes of the Human Influenza A(H1N1)pdm09 and A(H3N2) Viruses Reduce the Detection Sensitivity of Real-Time Reverse Transcription-PCR 
New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.
doi:10.1128/JCM.02467-13
PMCID: PMC3911422  PMID: 24153120
8.  A Short Bowel (Small Intestine = 40 cm), No Ileocecal Valve, and Colonic Inertia Patient Works Well with Oral Intake Alone without Parenteral Nutrition 
Case Reports in Surgery  2014;2014:387307.
We present a 50-year-old male who suffered from ischemic bowel disease, having undergone massive resection of small intestine and ileocecal valve. He had to cope with 40 cm proximal jejunum and 70 cm distal colon remaining. In the postoperative period parenteral nutrition (PN) was used immediately for nutrition support and electrolyte imbalance correction. We gave him home PN as regular recommendation for the short bowel status after discharge from hospital. This patient has tolerated regular oral intake 2 months later and did not develop significant short bowel syndrome. There were several episodes of venous access infection which troubled this patient and admitted him for treatment during home PN. Therefore, we changed home PN to cyclic tapering pattern. The patient could maintain his nutrition and hydration with oral intake alone after tapering home PN 15 months later. He has survived more than one year without PN support and still maintained 80% ideal body weight with average albumin of 3.5 ± 0.2 mg/dL. Although patient was hospitalized every two months to supplement nutrients, however, this has greatly improved the quality of life.
doi:10.1155/2014/387307
PMCID: PMC4082853  PMID: 25018889
9.  miR-544a promotes the invasion of lung cancer cells by targeting cadherina 1 in vitro 
OncoTargets and therapy  2014;7:895-900.
Objective
To find out the effect of miR-544a on the invasion of lung cancer cells and to explore the underlying molecular mechanisms.
Methods
Micro-ribonucleic acid (miRNA) expression in two different invasive lung cancer cell lines 95C (low invasive ability) and 95D (high invasive ability) was analyzed by miRNA microarray and real-time quantitative polymerase chain reaction (PCR); miR-544a mimic was transfected to 95C, and its invasion ability was detected by transwell migration assay; we predicted the candidate miRNA target genes by TargetScan (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) software and verified the target genes by Western blot.
Results
The expression of miR-544a was significantly increased in 95D in miRNA microarray and quantitative PCR tests (P<0.05). After being transfected with miR-544a mimic, the invasion ability of 95C was enhanced (P<0.01). Moreover, transfection with miR-544a inhibitor decreased the invasion ability of 95D (P<0.01). miR-544a possibly combined with CDH1 (E-cadherin) predicted by the TargetScan analysis. 95C with miR-544a mimic reduced the expression of CDH1 and improved the expression of vimentin, while 95D with miR-544a inhibitor improved the expression of CDH1 and reduced the expression of vimentin.
Conclusion
miR-544a can promote the invasion of non-small cell lung cancer by downregulation of CDH1 and upregulation of vimentin.
doi:10.2147/OTT.S61695
PMCID: PMC4051809  PMID: 24940073
NSCLC; non-small cell lung cancer; E-cadherin; microRNA; EMT
10.  Examination with Next-Generation Sequencing Technology of the Bacterial Microbiota in Bronchoalveolar Lavage Samples after Traumatic Injury 
Surgical Infections  2013;14(3):275-282.
Abstract
Background
We examined the microbiota of bronchoalveolar lavage (BAL) samples with next-generation sequencing (NGS) technology to determine whether its results correlate with those of standard culture methods or affect patient outcome or both.
Methods
We collected BAL samples in the surgical intensive care unit (SICU) as part of the standard of care for intubated individuals who had a Clinical Pulmonary Infection Score (CPIS) ≥6 points. A portion of the BAL fluid was sequenced for the 16S region of ribosomal deoxyribonucleic acid (rDNA) with the Roche 454 FLX Titanium sequencer. Sequences were analyzed through a data-analysis pipeline to identify the appropriate taxonomic designation (∼species) of each 16s sequence. The bacterial microbiota of each BAL sample was compared with the bacteria identified in the sample through standard culture methods. Correlations between the taxonomic diversity of the microbiota and clinical outcome were examined through linear regression and Pearson correlation.
Results
Bronchoalveolar lavage samples from 12 individuals in the SICU who had a CPIS ≥6 points were examined through 454 pyrosequencing. The number of phylotypes (∼species) in the samples ranged from 15 to 129. The number of phyla in the BAL samples ranged from 3 to 14. There was little correlation between the bacteria identified by NGS and those identified with standard culture methods. The same predominant bacterial strain was identified by both culture and sequencing in only a single sample. The correlation between patient days on a ventilator and the number of species in BAL samples was significant (r=0.7435, p=0.0056; r2=0.5528).
Conclusions
Increasing diversity of the bacterial microbiota in BAL samples correlates with the duration of mechanical ventilation. Bacteria identified through standard culture methods were not well correlated with the findings of NGS.
doi:10.1089/sur.2012.095
PMCID: PMC3689187  PMID: 23647218
11.  (3Z)-3-[(Z)-2-(2-Oxoindolin-3-yl­idene)hydrazin-1-yl­idene]indolin-2-one 0.17-hydrate 
In the title compound, C16H10N4O2·0.17H2O, prepared by the one-step condensation reaction of isatin with hydrazine hydrate under microwave irradiation, the complete organic mol­ecule is generated by crystallographic inversion symmetry and therefore exists in an S-trans conformation. In the crystal, mol­ecules are linked by N—H⋯O hydrogen bonds, generating a three-dimensional framework with [001] channels, which are occupied by the disordered water mol­ecules.
doi:10.1107/S1600536814011805
PMCID: PMC4051016  PMID: 24940290
12.  Atorvastatin increases lipopolysaccharide-induced expression of tumour necrosis factor-α-induced protein 8-like 2 in RAW264.7 cells 
RAW264.7 cells are one of the major sources of productive inflammatory biomediators, including tumour necrosis factor-α (TNF-α) and interleukin (IL)-6. TNF-α-induced protein 8-like 2 (TIPE2) is an essential negative regulator of Toll-like and T-cell receptors, and the selective expression in the immune system prevents hyper-responsiveness and maintains immune homeostasis. The aim of the present study was to investigate whether atorvastatin upregulates the expression of TIPE2 and further regulates the inflammatory response and oxidation emergency response in RAW264.7 cells. RAW264.7 cells were incubated in Dulbecco’s modified Eagle’s medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. Following incubation, the medium was collected and the levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay. The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined. The results revealed that LPS increased the mRNA and protein expression levels of TIPE2, MIF and NF-κB, as well as the production of IL-6 and TNF-α, in a dose and time dependent manner in RAW264.7 cells. Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells. Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner. The observations indicated that atorvastatin upregulated LPS induced expression of TIPE2 and consequently inhibited MIF, NF-κB, NOS and COX-2 expression and the production of NO, PGE2, TNF-α and IL-6, increased HO-1 expression, and inhibited ROS activity in cultured RAW264.7 cells.
doi:10.3892/etm.2014.1722
PMCID: PMC4061217  PMID: 24944625
atorvastatin; RAW264.7 cells; tumour necrosis factor-α-induced protein 8-like 2; macrophage migration inhibitory factor; nuclear factor-κB
13.  High Frequency of Putative Ovarian Cancer Stem Cells With CD44/CK19 Coexpression Is Associated With Decreased Progression-Free Intervals In Patients With Recurrent Epithelial Ovarian Cancer 
Reproductive Sciences  2013;20(5):605-615.
Objective:
Epithelial ovarian cancer (EOC) cells with CD44 and CK19 coexpression may represent a subset of ovarian cancer stem cells (OCSCs). This study was conducted to evaluate the correlation of the frequency of putative OCSCs (CD44 + CK19 + OCSCs) with the clinicopathologic features and the prognostic value in patients with recurrent advanced stage EOC.
Methods:
A retrospective study was carried out on 33 patients with EOC and a uniformly treated tissue microarray was constructed. A multiplexed, immunofluorescence-based method of automated in situ quantitative measurement of protein analysis was used for evaluation of the frequency or density of CD44 + CK19 + OCSCs in EOC.
Results:
The mean follow-up time was 42.8 ± 27.1 months. High frequency of EOC cells with CD44+ or CD44+/CK19+ was associated with chemoresistance (P = .033 and P = .02, respectively). Using K-M analysis with log-rank test, a high frequency of putative OCSCs was associated with short disease-free interval (7.9 months vs 20.9 months, P = .019). In univariable analysis, the frequency of OCSCs, International Federation of Gynecology and Obstetrics stage and residual tumor volume were significant predictor variables and were entered into multivariable analysis (P = .019, .037, and .005, respectively). Although no independent significant predictor was found, the frequency of putative OCSCs was the most promising predictor variable compared with the other 2 variables (hazard ratio = 2.344, P = .052).
Conclusion:
Our findings suggest that high frequency of OCSCs (CD44+ and CK19+) in epithelial ovarian tumors correlates with short progression-free intervals.
doi:10.1177/1933719112461183
PMCID: PMC3635069  PMID: 23171677
ovarian cancer stem cells; CD44; CK19; AQUA
14.  Influenza A(H5N2) Virus Antibodies in Humans after Contact with Infected Poultry, Taiwan, 2012 
Emerging Infectious Diseases  2014;20(5):857-860.
Six persons in Taiwan who had contact with poultry infected with influenza A(H5N2) showed seroconversion for the virus by hemagglutinin inhibition or microneutralization testing. We developed an ELISA based on nonstructural protein 1 of the virus to differentiate natural infection from cross-reactivity after vaccination; 2 persons also showed seroconversion by this test.
doi:10.3201/eid2005.131393
PMCID: PMC4012807  PMID: 24750594
HPAI H5N2 virus; NS1 antibody; seroconversion; influenza; respiratory infections; poultry; antibodies; influenza A(H5N2) virus; viruses; humans; cross-reactivity; contact; Taiwan
15.  Pigment Epithelium-Derived Factor 34-mer Peptide Prevents Liver Fibrosis and Hepatic Stellate Cell Activation through Down-Regulation of the PDGF Receptor 
PLoS ONE  2014;9(4):e95443.
Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl4)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.
doi:10.1371/journal.pone.0095443
PMCID: PMC3998957  PMID: 24763086
16.  Correlation between serum IGF-1 and blood lead level in short stature children and adolescent with growth hormone deficiency 
This study aimed to investigate correlation between serum insulin-like growth factor-1 (IGF-1) and blood lead level in short stature children with growth hormone deficiency (GHD), and IGF-1 signal molecules were investigated in lead exposed rats. Our findings may provide evidence for clarifying pathogenesis of lead induced short stature in children. Methods: 880 short stature children were recruited from clinics and divided into GHD group and idiopathic short stature (ISS) group according to the GH peak in growth hormone stimulation test. The height, body weight, serum IGF-1 level and blood lead level were determined. A rat model of lead poisoning was used to establish and western blot assay was employed to detect the phosphorylation of signaling molecules (MAPK and PI3K/Akt) related to IGF-1 signaling pathway. Results: In GHD group, the height, body weight and serum IGF-1 level were significantly lower, but the blood lead level was significantly higher than those in ISS group (P<0.05). Western blot assay confirmed that the protein expression of phosphorylated ERK1/2, JNK, p38, Akt473 and Akt308 increased significantly (P<0.01) in lead exposure rats. Conclusion: Our study suggesting that reduction in IGF-1 in children with GHD is associated with blood lead level. Lead exposure may induce expression of phosphorylated MAPK and Akt signaling molecules. The activation of these molecules may influence binding of IGF-1 and tyrosine kinase receptor IGFIR to regulate cell growth via the MAPK and Akt signaling pathways, which then interfere with growth-promoting effect of IGF-1 in short children.
PMCID: PMC4057833  PMID: 24955154
Growth hormone deficiency; insulin-like growth factor I; lead exposure animal model; short stature; signaling pathway
17.  Zinc Regulates the Acute Phase Response and Serum Amyloid A Production in Response to Sepsis through JAK-STAT3 Signaling 
PLoS ONE  2014;9(4):e94934.
Sepsis rapidly activates the host inflammatory response and acute phase response. Severe sepsis, complicated by multiple organ failure, is associated with overwhelming inflammation and high mortality. We previously observed that zinc (Zn) deficiency significantly increases mortality in a mouse model of polymicrobial sepsis due to over-activation of the inflammatory response. In order to identify potential mechanisms that account for Zn-responsive effects, we generated whole exome expression profiles from the lung tissue of septic mice that were maintained on Zn modified diets. Based on systems analysis, we observed that Zn deficiency enhances the acute phase response and particularly the JAK-STAT3 pathway, resulting in increased serum amyloid A production. In vitro studies of primary hepatocytes and HepG2 cells substantiated that Zn-deficiency augments serum amyloid A production through up-regulation of the JAK-STAT3 and NF-κB pathways. In contrast, Zn inhibited STAT3 activation through the up-regulation of SHP1 activity. Collectively, these findings demonstrate that Zn deficiency enhances the acute phase response through up-regulation of the JAK-STAT3 pathway, thereby perpetuating increased inflammation that may lead to increased morbidity and mortality in response to sepsis.
doi:10.1371/journal.pone.0094934
PMCID: PMC3986341  PMID: 24732911
18.  Modeling and Simulation of Complex Network Attributes on Coordinating Large Multiagent System 
The Scientific World Journal  2014;2014:412479.
With the expansion of distributed multiagent systems, traditional coordination strategy becomes a severe bottleneck when the system scales up to hundreds of agents. The key challenge is that in typical large multiagent systems, sparsely distributed agents can only communicate directly with very few others and the network is typically modeled as an adaptive complex network. In this paper, we present simulation testbed CoordSim built to model the coordination of network centric multiagent systems. Based on the token-based strategy, the coordination can be built as a communication decision problem that agents make decisions to target communications and pass them over to the capable agents who will potentially benefit the team most. We have theoretically analyzed that the characters of complex network make a significant difference with both random and intelligent coordination strategies, which may contribute to future multiagent algorithm design.
doi:10.1155/2014/412479
PMCID: PMC3998005  PMID: 24955399
19.  Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling 
Molecular Medicine Reports  2014;9(6):2111-2116.
Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration.
doi:10.3892/mmr.2014.2109
PMCID: PMC4055482  PMID: 24699800
neonatal mouse; heart; regeneration; digital gene expression profile
20.  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.
doi:10.1002/jbt.21476
PMCID: PMC3646527  PMID: 23436740
Chloroacetates; Mixtures; Phagocytic activation
21.  Effect of salidroside on lung injury by upregulating peroxisome proliferator-activated receptor γ expression in septic rats 
Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. Thus, the aim of the present study was to investigate the beneficial effects of salidroside on ameliorating cecal ligation and puncture (CLP)-induced lung inflammation. Rats underwent CLP surgery to induce ALI and 800 mg/kg salidroside (i.v.) was administered 24 h after the CLP challenge. Subsequently, biochemical changes in the blood and lung tissues, as well as morphological and histological alterations in the lungs, that were associated with inflammation and injury were analysed. CLP was shown to significantly increase the serum levels of plasma tumour necrosis factor-α and interleukin-6, -1β and-10. In addition, CLP increased pulmonary oedema, thickened the alveolar septa and caused inflammation in the lung cells. These changes were ameliorated by the administration of 800 mg/kg salidroside (i.v.) 24 h after the CLP challenge. This post-treatment drug administration also significantly attenuated the lipopolysaccharide-induced activation of nuclear factor-κβ and increased the release of peroxisome proliferator-activated receptor γ in the lung tissue. Therefore, salidroside administered following the induction of ALI by CLP significantly prevented and reversed lung tissue injuries. The positive post-treatment effects of salidroside administration indicated that salidroside may be a potential candidate for the management of lung inflammation in CLP-induced endotoxemia and septic shock.
doi:10.3892/etm.2014.1629
PMCID: PMC4043580  PMID: 24926325
sepsis; peroxisome proliferator-activated receptor γ; salidroside; rat; cecal ligation and puncture; lung injury
22.  A novel animal model of osteonecrosis of the femoral head induced using a magnetic resonance imaging-guided argon-helium cryotherapy system 
The aim of the present study was to establish a novel animal model of osteonecrosis of the femoral head (ONFH) using a magnetic resonance imaging (MRI)-guided argon-helium cryotherapy system. A total of 48 rabbits were used to generate the ONFH models. In group I, the left femoral head of the rabbits received two cycles of argon-helium freezing-thawing under MRI guidance, while in group II, the right femoral head of each rabbit received only one cycle of argon-helium freezing-thawing. X-ray and histological examinations were performed. The percentages of lacunae in the femoral heads of group I at weeks 4, 8 and 12 following surgery (49.75±3.17, 62.06±4.12 and 48.25±2.76%, respectively) were higher than those in group II (39.13±4.48, 50.69±3.84 and 37.50±3.86%, respectively). In addition, the percentage of empty lacunae in group I was 62.06% at week 8 following surgery. Therefore, an animal model of ONFH was successfully established using an argon-helium cryotherapy system. The percentage of empty lacunae in group I was higher than that in group II at weeks 4, 8 and 12 after surgery.
doi:10.3892/etm.2014.1625
PMCID: PMC4043595  PMID: 24926337
osteonecrosis; femoral head; magnetic resonance imaging; cryotherapy; animal model
23.  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.
doi:10.1371/journal.pone.0091606
PMCID: PMC3953484  PMID: 24626155
24.  Effect of melilotus extract on lung injury by upregulating the expression of cannabinoid CB2 receptors in septic rats 
Background
M. Suaveolens Ledeb has long been used in China to treat inflammatory infectious diseases. Melilotus is extracted from Melilotus Suaveolens Ledeb and its therapeutic potential is associated with its anti-inflammatory activity. However, the precise mechanisms underlying its effects are unknown. This study was conducted to evaluate the protective effects of melilotus extract in a rat cecal ligation and puncture (CLP)-induced animal model of acute lung injury (ALI).
Methods
A sepsis model was induced by CLP-like lung inflammation. Two hours prior to CLP administration, the treatment group was administered melilotus extract via oral injection. RT-PCR and Western blotting were used to test the expression of cannabinoid receptor (CB)2, NF-κβ and IκB from single peripheral blood mononuclear cells and lung tissues respectively. Enzyme linked immune sorbent assay was used to detect serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and IL-12. The numbers of neutrophils, lymphocytes, macrophages and total cells in the bronchoalveolar lavage (BAL) fluid were counted. For histologic analysis, hematoxylin and eosin (H&E) stains were evaluated.
Results
After inducing ALI by CLP for 24 hours, melilotus extract up-regulated peripheral blood mononuclear cell CB2 expression, blocked the activity of NF-κβ65, and the number of neutrophils, lymphocytes and total cells were significantly lower in the melilotus extract group than the control group. In addition, TNF-α and IL-6 levels were significantly decreased in the melilotus extract group. Histological results demonstrated the attenuation effect of melilotus extract on CLP-induced lung inflammation. CB2 was negatively correlated to NF-κβ mRNA and proteins, respectively (r = -0.377, P < 0.05; r = -0.441, P < 0.05).
Conclusion
The results of this study indicated melilotus extract significantly reduced CLP-induced lung inflammation by up-regulating CB2 expression. The remarkable protective effects of melilotus extract suggest its therapeutic potential in CLP induced-acute lung injury treatment.
doi:10.1186/1472-6882-14-94
PMCID: PMC3995869  PMID: 24612782
Sepsis; Melilotus extract; Cannabinoid receptors; Cecal ligation and puncture; Rats
25.  A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development 
Journal of Experimental Botany  2014;65(8):2023-2037.
Summary
The lily anther-specific gene, LLA1271, encodes an adhesin-like protein found for the first time in higher plants. The protein is deposited on the surface of microspores, moderately affecting exine formation.
The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.
doi:10.1093/jxb/eru051
PMCID: PMC3991738  PMID: 24591055
Adhesin; anther; exine; hormone; lily (Lilium longiflorum Thunb.); microspore; tapetum.

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