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1.  Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor 
We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.
PMCID: PMC3961059  PMID: 24510772
High throughput flow cytometer; Fluorogen activating protein; G protein coupled receptor; Receptor trafficking; Live cell assay
2.  Dihydroartemisinin inhibits the mammalian target of rapamycin-mediated signaling pathways in tumor cells 
Carcinogenesis  2013;35(1):192-200.
Dihydroartemisinin (DHA), an antimalarial drug, has previously unrecognized anticancer activity, and is in clinical trials as a new anticancer agent for skin, lung, colon and breast cancer treatment. However, the anticancer mechanism is not well understood. Here, we show that DHA inhibited proliferation and induced apoptosis in rhabdomyosarcoma (Rh30 and RD) cells, and concurrently inhibited the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central controller for cell proliferation and survival, at concentrations (<3 μM) that are pharmacologically achievable. Of interest, in contrast to the effects of conventional mTOR inhibitors (rapalogs), DHA potently inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 but did not obviously affect mTORC2-mediated phosphorylation of Akt. The results suggest that DHA may represent a novel class of mTORC1 inhibitor and may execute its anticancer activity primarily by blocking mTORC1-mediated signaling pathways in the tumor cells.
PMCID: PMC3871936  PMID: 23929438
3.  Rumphellaoic Acid A, a Novel Sesquiterpenoid from the Formosan Gorgonian Coral Rumphella antipathies 
Marine Drugs  2014;12(12):5856-5863.
A novel sesquiterpenoid, rumphellaoic acid A (1), was isolated from the gorgonian coral Rumphella antipathies, and was found to possess a carbon skeleton that was obtained for the first time from a natural sources. The structure of 1 was elucidated by spectroscopic methods and this compound and was found to exert a moderate inhibitory effect on the release of elastase by human neutrophils.
PMCID: PMC4278205  PMID: 25486110
Rumphella antipathies; gorgonian; rumphellaoic acid; sesquiterpenoid; elastase
4.  Effect of Platelet-Rich Plasma and Bioactive Glass Powder for the Improvement of Rotator Cuff Tendon-to-Bone Healing in a Rabbit Model 
To test the hypothesis that a platelet-rich plasma (PRP) plus bioactive glass (BG) mixture could shorten the tendon-bone healing process in rotator cuff tendon repair, thirty mature male New Zealand white rabbits were randomly divided into three groups, Control, PRP, and PRP + BG. All groups underwent a surgical procedure to establish a rotator cuff tendon healing model. Mechanical examinations and histological assays were taken to verify the adhesion of the tendon-bone. Real-time PCR was adopted to analyze Bone Morphogenetic Protein-2 (BMP-2). The maximum load-to-failure value in mechanical examinations was significantly higher in the PRP + BG group than that in the control group after six weeks (Control 38.73 ± 8.58, PRP 54.49 ± 8.72, PRP + BG 79.15 ± 7.62, p < 0.001), but it was not significantly different at 12 weeks (PRP 74.27 ± 7.74, PRP + BG 82.57 ± 6.63, p = 0.145). In histological assays, H&E (hematoxylin-eosin) staining showed that the interface between the tendon-bone integration was much sturdier in the PRP + BG group compared to the other two groups at each time point, and more ordered arranged tendon fibers can be seen at 12 weeks. At six weeks, the mRNA expression levels of BMP-2 in the PRP + BG group were higher than those in the other groups (PRP + BG 0.65 ± 0.11, PRP 2.284 ± 0.07, Control 0.12 ± 0.05, p < 0.05). However, there was no significant difference in the mRNA expression levels of BMP-2 among the three groups at 12 weeks (p = 0.922, 0.067, 0.056). BMP-2 levels in PRP and PRP+BG groups were significantly lower at 12 weeks compared to six weeks (p = 0.006, <0.001).We found that the PRP + BG mixture could enhance tendon-bone healing in rotator cuff tendon repair.
PMCID: PMC4284689  PMID: 25464384
rotator cuff tendon; platelet-rich plasma; bioactive glass
5.  Staphylococcus epidermidis SrrAB Regulates Bacterial Growth and Biofilm Formation Differently under Oxic and Microaerobic Conditions 
Journal of Bacteriology  2014;197(3):459-476.
SrrAB expression in Staphylococcus epidermidis strain 1457 (SE1457) was upregulated during a shift from oxic to microaerobic conditions. An srrA deletion (ΔsrrA) mutant was constructed for studying the regulatory function of SrrAB. The deletion resulted in retarded growth and abolished biofilm formation both in vitro and in vivo and under both oxic and microaerobic conditions. Associated with the reduced biofilm formation, the ΔsrrA mutant produced much less polysaccharide intercellular adhesion (PIA) and showed decreased initial adherence capacity. Microarray analysis showed that the srrA mutation affected transcription of 230 genes under microaerobic conditions, and 51 genes under oxic conditions. Quantitative real-time PCR confirmed this observation and showed downregulation of genes involved in maintaining the electron transport chain by supporting cytochrome and quinol-oxidase assembly (e.g., qoxB and ctaA) and in anaerobic metabolism (e.g., pflBA and nrdD). In the ΔsrrA mutant, the expression of the biofilm formation-related gene icaR was upregulated under oxic conditions and downregulated under microaerobic conditions, whereas icaA was downregulated under both conditions. An electrophoretic mobility shift assay further revealed that phosphorylated SrrA bound to the promoter regions of icaR, icaA, qoxB, and pflBA, as well as its own promoter region. These findings demonstrate that in S. epidermidis SrrAB is an autoregulator and regulates biofilm formation in an ica-dependent manner. Under oxic conditions, SrrAB modulates electron transport chain activity by positively regulating qoxBACD transcription. Under microaerobic conditions, it regulates fermentation processes and DNA synthesis by modulating the expression of both the pfl operon and nrdDG.
PMCID: PMC4285975  PMID: 25404696
6.  An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates 
PLoS ONE  2014;9(10):e111518.
The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.
PMCID: PMC4216080  PMID: 25360558
7.  Ursolic Acid Inhibits Superoxide Production in Activated Neutrophils and Attenuates Trauma-Hemorrhage Shock-Induced Organ Injury in Rats 
PLoS ONE  2014;9(10):e111365.
Neutrophil activation is associated with the development of organ injury after trauma–hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma–hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma–hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma–hemorrhagic shock-induced organ injury in rats.
PMCID: PMC4216084  PMID: 25360589
8.  Genome-Wide Association Studies Using Haplotypes and Individual SNPs in Simmental Cattle 
PLoS ONE  2014;9(10):e109330.
Recent advances in high-throughput genotyping technologies have provided the opportunity to map genes using associations between complex traits and markers. Genome-wide association studies (GWAS) based on either a single marker or haplotype have identified genetic variants and underlying genetic mechanisms of quantitative traits. Prompted by the achievements of studies examining economic traits in cattle and to verify the consistency of these two methods using real data, the current study was conducted to construct the haplotype structure in the bovine genome and to detect relevant genes genuinely affecting a carcass trait and a meat quality trait. Using the Illumina BovineHD BeadChip, 942 young bulls with genotyping data were introduced as a reference population to identify the genes in the beef cattle genome significantly associated with foreshank weight and triglyceride levels. In total, 92,553 haplotype blocks were detected in the genome. The regions of high linkage disequilibrium extended up to approximately 200 kb, and the size of haplotype blocks ranged from 22 bp to 199,266 bp. Additionally, the individual SNP analysis and the haplotype-based analysis detected similar regions and common SNPs for these two representative traits. A total of 12 and 7 SNPs in the bovine genome were significantly associated with foreshank weight and triglyceride levels, respectively. By comparison, 4 and 5 haplotype blocks containing the majority of significant SNPs were strongly associated with foreshank weight and triglyceride levels, respectively. In addition, 36 SNPs with high linkage disequilibrium were detected in the GNAQ gene, a potential hotspot that may play a crucial role for regulating carcass trait components.
PMCID: PMC4203724  PMID: 25330174
9.  Dual Effects of Alpha-Arbutin on Monophenolase and Diphenolase Activities of Mushroom Tyrosinase 
PLoS ONE  2014;9(10):e109398.
The effects of α-arbutin on the monophenolase and diphenolase activities of mushroom tyrosinase were investigated. The results showed that α-arbutin inhibited monophenolase activity but it activated diphenolase activity. For monophenolase activity, IC50 value was 4.5 mmol·L−1 and 4.18 mmol·L−1 of α-arbutin could extend the lag time from 40.5 s to 167.3 s. Alpha- arbutin is proposed to be regarded as a triphenolic substrate by the enzyme during catalyzation, leading to the suicide inactivation of the active site of tyrosinase. For diphenolase activity, α-arbutin acted as an activator and its activation mechanism was mixed type activation. To reveal such activation, it should be mainly refered to the conformational changes in tyrosinase caused by the interaction of α-arbutin with residues located at the entrance to the active site, and the decrease of the effect of suicide inactivation.
PMCID: PMC4193772  PMID: 25303458
10.  Urethral Dysfunction in Female Mice with Estrogen Receptor β Deficiency 
PLoS ONE  2014;9(10):e109058.
Estrogen has various regulatory functions in the growth, development, and differentiation of the female urogenital system. This study investigated the roles of ERβ in stress urinary incontinence (SUI). Wild-type (ERβ+/+) and knockout (ERβ−/−) female mice were generated (aged 6–8 weeks, n = 6) and urethral function and protein expression were measured. Leak point pressures (LPP) and maximum urethral closure pressure (MUCP) were assessed in mice under urethane anesthesia. After the measurements, the urethras were removed for proteomic analysis using label-free quantitative proteomics by nano-liquid chromatography–mass spectrometry (LC-MS/MS) analysis. The interaction between these proteins was further analysed using MetaCore. Lastly, Western blot was used to confirm the candidate proteins. Compared with the ERβ+/+ group, the LPP and MUCP values of the ERβ−/− group were significantly decreased. Additionally, we identified 85 differentially expressed proteins in the urethra of ERβ−/− female mice; 57 proteins were up-regulated and 28 were down-regulated. The majority of the ERβ knockout-modified proteins were involved in cell-matrix adhesion, metabolism, immune response, signal transduction, nuclear receptor translational regelation, and muscle contraction and development. Western blot confirmed the up-regulation of myosin and collagen in urethra. By contrast, elastin was down-regulated in the ERβ−/− mice. This study is the first study to estimate protein expression changes in urethras from ERβ−/− female mice. These changes could be related to the molecular mechanism of ERβ in SUI.
PMCID: PMC4183540  PMID: 25275480
11.  Strain-enhanced tunneling magnetoresistance in MgO magnetic tunnel junctions 
Scientific Reports  2014;4:6505.
While the effects of lattice mismatch-induced strain, mechanical strain, as well as the intrinsic strain of thin films are sometimes detrimental, resulting in mechanical deformation and failure, strain can also be usefully harnessed for applications such as data storage, transistors, solar cells, and strain gauges, among other things. Here, we demonstrate that quantum transport across magnetic tunnel junctions (MTJs) can be significantly affected by the introduction of controllable mechanical strain, achieving an enhancement factor of ~2 in the experimental tunneling magnetoresistance (TMR) ratio. We further correlate this strain-enhanced TMR with coherent spin tunneling through the MgO barrier. Moreover, the strain-enhanced TMR is analyzed using non-equilibrium Green's function (NEGF) quantum transport calculations. Our results help elucidate the TMR mechanism at the atomic level and can provide a new way to enhance, as well as tune, the quantum properties in nanoscale materials and devices.
PMCID: PMC4179128  PMID: 25266219
12.  Generalized Linear Model for Mapping Discrete Trait Loci Implemented with LASSO Algorithm 
PLoS ONE  2014;9(9):e106985.
Generalized estimating equation (GEE) algorithm under a heterogeneous residual variance model is an extension of the iteratively reweighted least squares (IRLS) method for continuous traits to discrete traits. In contrast to mixture model-based expectation–maximization (EM) algorithm, the GEE algorithm can well detect quantitative trait locus (QTL), especially large effect QTLs located in large marker intervals in the manner of high computing speed. Based on a single QTL model, however, the GEE algorithm has very limited statistical power to detect multiple QTLs because of ignoring other linked QTLs. In this study, the fast least absolute shrinkage and selection operator (LASSO) is derived for generalized linear model (GLM) with all possible link functions. Under a heterogeneous residual variance model, the LASSO for GLM is used to iteratively estimate the non-zero genetic effects of those loci over entire genome. The iteratively reweighted LASSO is therefore extended to mapping QTL for discrete traits, such as ordinal, binary, and Poisson traits. The simulated and real data analyses are conducted to demonstrate the efficiency of the proposed method to simultaneously identify multiple QTLs for binary and Poisson traits as examples.
PMCID: PMC4161361  PMID: 25210765
13.  Biological activities of fusarochromanone: a potent anti-cancer agent 
BMC Research Notes  2014;7(1):601.
Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity.
Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 μM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells.
Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101’s molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.
PMCID: PMC4168212  PMID: 25187308
Pro-apoptotic; Anti-cancer; Anti-angiogenic; Small bioactive molecule; Fusarochromanone
14.  Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation 
Nature neuroscience  2014;17(3):471-480.
Rapid and reversible methods for altering the level of endogenous proteins are critically important for studying biological systems and developing therapeutics. Here, we describe a membrane permeable targeting peptide-based method that rapidly and reversibly knocks down endogenous proteins through chaperone-mediated autophagy in vitro and in vivo. We demonstrated the specificity, efficacy and generalizability of the method by showing efficient knockdown of various proteins including death associated protein kinase 1 (160kDa), scaffolding protein PSD-95 (95kDa) and α-synuclein (18kDa) with their respective targeting peptides in a dose-, time- and lysosomal activity-dependent manner in neuronal cultures. More significantly, we showed that when given systemically, the peptide system efficiently knocked down the targeted protein in the brain of intact rats. Our study provides a robust and convenient research tool to manipulate endogenous protein levels, and may also lead to the development of protein knockdown-based novel therapeutics for treating various human diseases.
PMCID: PMC3937121  PMID: 24464042 CAMSID: cams3904
15.  Rumphellols A and B, New Caryophyllene Sesquiterpenoids from a Formosan Gorgonian Coral, Rumphella antipathies 
Two new marine-derived caryophyllene-type sesquiterpenoids, rumphellols A and B (1 and 2), were obtained from the gorgonian coral, Rumphella antipathies, collected off the waters of Taiwan. Although caryophyllene-type sesquiterpenes are rarely found in marine organisms, compounds of this type could be principal components of R. antipathies. The structures of new Compounds 1 and 2 were determined by analysis of their spectroscopic data, including 1D and 2D NMR experiments. Caryophyllene 1 and 2 were evaluated in terms of their anti-inflammatory activity by examining their inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.
PMCID: PMC4200752  PMID: 25192289
Rumphella antipathies; sesquiterpene; caryophyllene; rumphellol; superoxide anion; elastase
16.  Structure Elucidation and Cytotoxic Evaluation of New Polyacetylenes from a Marine Sponge Petrosia sp. 
The sponge Petrosia sp. yielded five polyacetylenic compounds (1–5), including two new polyacetylenes, petrosianynes A (1) and B (2). The structures of these compounds were elucidated by detailed spectroscopic analysis and by comparison with the physical and spectral data of related known analogues. Compounds 1–5 exhibited significant cytotoxic activity against a limited panel of cancer cell lines.
PMCID: PMC4200862  PMID: 25238415
sponge; Petrosia; polyacetylene
17.  Rho GTPase-Activating Protein 35 rs1052667 Polymorphism and Osteosarcoma Risk and Prognosis 
BioMed Research International  2014;2014:396947.
The Rho GTPase-activating protein 35 (ARHGAP35), an important Rho family GTPase-activating protein, may be associated with tumorigenesis of some tumors. Here, we investigated the relationship between an important polymorphic variant at 3′-UTR of this gene (rs1052667) and osteosarcoma risk and prognosis. This hospital-based case-control study, including 247 osteosarcoma patients and 428 age-, sex-, and race-matched healthy controls, was conducted in Guangxi population. Genotypes were tested using TaqMan PCR technique. We found a significant difference in the frequency of rs1052667 genotypes between cases and controls. Compared with the homozygote of rs1052667 C alleles (rs1052667-CC), the genotypes with rs1052667 T alleles (namely, rs1052667-CT or -TT) increased osteosarcoma risk (odds ratios: 2.41 and 7.35, resp.). Moreover, rs1052667 polymorphism was correlated with such pathological features of osteosarcoma as tumor size, tumor grade, and tumor metastasis. Additionally, this polymorphism also modified the overall survival and recurrence-free survival of osteosarcoma cases. Like tumor grade, ARHGAP35 rs1052667 polymorphism was an independent prognostic factor influencing the survival of osteosarcoma. These results suggest that ARHGAP35 rs1052667 polymorphism may be associated with osteosarcoma risk and prognosis.
PMCID: PMC4124850  PMID: 25136583
18.  A Systematic Review of Home-Based Childhood Obesity Prevention Studies 
Pediatrics  2013;132(1):e193-e200.
Childhood obesity is a global epidemic. Despite emerging research about the role of the family and home on obesity risk behaviors, the evidence base for the effectiveness of home-based interventions on obesity prevention remains uncertain. The objective was to systematically review the effectiveness of home-based interventions on weight, intermediate (eg, diet and physical activity [PA]), and clinical outcomes.
We searched Medline, Embase, PsychInfo, CINAHL,, and the Cochrane Library from inception through August 11, 2012. We included experimental and natural experimental studies with ≥1-year follow-up reporting weight-related outcomes and targeting children at home. Two independent reviewers screened studies and extracted data. We graded the strength of the evidence supporting interventions targeting diet, PA, or both for obesity prevention.
We identified 6 studies; 3 tested combined interventions (diet and PA), 1 used diet intervention, 1 combined intervention with primary care and consumer health informatics components, and 1 combined intervention with school and community components. Select combined interventions had beneficial effects on fruit/vegetable intake and sedentary behaviors. However, none of the 6 studies reported a significant effect on weight outcomes. Overall, the strength of evidence is low that combined home-based interventions effectively prevent obesity. The evidence is insufficient for conclusions about home-based diet interventions or interventions implemented at home in association with other settings.
The strength of evidence is low to support the effectiveness of home-based child obesity prevention programs. Additional research is needed to test interventions in the home setting, particularly those incorporating parenting strategies and addressing environmental influences.
PMCID: PMC3691540  PMID: 23753095
child; obesity; overweight; intervention; home; BMI
19.  Systematic Review of Community-Based Childhood Obesity Prevention Studies 
Pediatrics  2013;132(1):e201-e210.
This study systematically reviewed community-based childhood obesity prevention programs in the United States and high-income countries.
We searched Medline, Embase, PsychInfo, CINAHL,, and the Cochrane Library for relevant English-language studies. Studies were eligible if the intervention was primarily implemented in the community setting; had at least 1 year of follow-up after baseline; and compared results from an intervention to a comparison group. Two independent reviewers conducted title scans and abstract reviews and reviewed the full articles to assess eligibility. Each article received a double review for data abstraction. The second reviewer confirmed the first reviewer’s data abstraction for completeness and accuracy.
Nine community-based studies were included; 5 randomized controlled trials and 4 non–randomized controlled trials. One study was conducted only in the community setting, 3 were conducted in the community and school setting, and 5 were conducted in the community setting in combination with at least 1 other setting such as the home. Desirable changes in BMI or BMI z-score were found in 4 of the 9 studies. Two studies reported significant improvements in behavioral outcomes (1 in physical activity and 1 in vegetable intake).
The strength of evidence is moderate that a combined diet and physical activity intervention conducted in the community with a school component is more effective at preventing obesity or overweight. More research and consistent methods are needed to understand the comparative effectiveness of childhood obesity prevention programs in the community setting.
PMCID: PMC3691541  PMID: 23753099
childhood obesity; community-based; prevention
20.  The Effects of Choline on Hepatic Lipid Metabolism, Mitochondrial Function and Antioxidative Status in Human Hepatic C3A Cells Exposed to Excessive Energy Substrates 
Nutrients  2014;6(7):2552-2571.
Choline plays a lipotropic role in lipid metabolism as an essential nutrient. In this study, we investigated the effects of choline (5, 35 and 70 μM) on DNA methylation modifications, mRNA expression of the critical genes and their enzyme activities involved in hepatic lipid metabolism, mitochondrial membrane potential (Δψm) and glutathione peroxidase (GSH-Px) in C3A cells exposed to excessive energy substrates (lactate, 10 mM; octanoate, 2 mM and pyruvate, 1 mM; lactate, octanoate and pyruvate-supplemented medium (LOP)). Thirty five micromole or 70 μM choline alone, instead of a low dose (5 μM), reduced hepatocellular triglyceride (TG) accumulation, protected Δψm from decrement and increased GSH-Px activity in C3A cells. The increment of TG accumulation, reactive oxygen species (ROS) production and Δψm disruption were observed under LOP treatment in C3A cells after 72 h of culture, which were counteracted by concomitant treatment of choline (35 μM or 70 μM) partially via reversing the methylation status of the peroxisomal proliferator-activated receptor alpha (PPARα) gene promoter, upregulating PPARα, carnitine palmitoyl transferase-I (CPT-I) and downregulating fatty acid synthase (FAS) gene expression, as well as decreasing FAS activity and increasing CPT-I and GSH-Px activities. These findings provided a novel insight into the lipotropic role of choline as a vital methyl-donor in the intervention of chronic metabolic diseases.
PMCID: PMC4113756  PMID: 25010553
choline; hepatic lipid metabolism; mitochondria; proliferator-activated receptor alpha; methylation
21.  Establishment of a chronic left ventricular aneurysm model in rabbit 
To establish a cost-effective and reproducible procedure for induction of chronic left ventricular aneurysm (LVA) in rabbits.
Acute myocardial infarction (AMI) was induced in 35 rabbits via concomitant ligation of the left anterior descending (LAD) coronary artery and the circumflex (Cx) branch at the middle portion. Development of AMI was confirmed by ST segment elevation and akinesis of the occluded area. Echocardiography, pathological evaluation, and agar intra-chamber casting were utilized to validate the formation of LVA four weeks after the surgery. Left ventricular end systolic pressure (LVESP) and diastolic pressure (LVEDP) were measured before, immediately after and four weeks after ligation. Dimensions of the ventricular chamber, thickness of the interventricular septum (IVS) and the left ventricular posterior wall (LVPW) left ventricular end diastolic volume (LVEDV), systolic volume (LVESV), and ejection fraction (EF) were recorded by echocardiogram.
Thirty one (88.6%) rabbits survived myocardial infarction and 26 of them developed aneurysm (83.9%). The mean area of aneurysm was 33.4% ± 2.4% of the left ventricle. LVEF markedly decreased after LVA formation, whereas LVEDV, LVESV and the thickness of IVS as well as the dimension of ventricular chamber from apex to mitral valve annulus significantly increased. LVESP immediately dropped after ligation and recovered to a small extent after LVA formation. LVEDP progressively increased after ligation till LVA formation. Areas in the LV that underwent fibrosis included the apex, anterior wall and lateral wall but not IVS. Agar intra-chamber cast showed that the bulging of LV wall was prominent in the area of aneurysm.
Ligation of LAD and Cx at the middle portion could induce development of LVA at a mean area ratio of 33.4% ± 2.4% which involves the apex, anterior wall and lateral wall of the left ventricle.
PMCID: PMC4076457  PMID: 25009567
Myocardial infarction; Left ventricular aneurysm; Animal model; Rabbit; Intra-chamber cast
22.  Pregnane-Type Steroids from the Formosan Soft Coral Scleronephthya flexilis 
Three pregnane-type steroids, including a new metabolite, 3β-methoxy-5,20-pregnadiene (1) along with two known analogues, 3β-acetoxy-5,20-pregnadiene (2) and 5α-pregna-1,20-dien-3-one (3) were isolated from the soft coral Scleronephthya flexilis. Standard spectroscopic techniques were used to determine the structure of new steroid 1. The absolute stereochemistry of steroid 2 was confirmed by X-ray diffraction analysis. Steroid 3 exhibited potent activity against MOLT-4 tumor cells.
PMCID: PMC4100144  PMID: 24914763
soft coral; pregnane; Scleronephthya flexilis; cytotoxicity; MOLT-4
23.  Cracking the Cytotoxicity Code: Apoptotic Induction of 10-Acetylirciformonin B is Mediated through ROS Generation and Mitochondrial Dysfunction 
Marine Drugs  2014;12(5):3072-3090.
A marine furanoterpenoid derivative, 10-acetylirciformonin B (10AB), was found to inhibit the proliferation of leukemia, hepatoma, and colon cancer cell lines, with selective and significant potency against leukemia cells. It induced DNA damage and apoptosis in leukemia HL 60 cells. To fully understand the mechanism behind the 10AB apoptotic induction against HL 60 cells, we extended our previous findings and further explored the precise molecular targets of 10AB. We found that the use of 10AB increased apoptosis by 8.9%–87.6% and caused disruption of mitochondrial membrane potential (MMP) by 15.2%–95.2% in a dose-dependent manner, as demonstrated by annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of HL 60 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by 10AB, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of 10AB. The results of a cell-free system assay indicated that 10AB could act as a topoisomerase catalytic inhibitor through the inhibition of topoisomerase IIα. On the protein level, the expression of the anti-apoptotic proteins Bcl-xL and Bcl-2, caspase inhibitors XIAP and survivin, as well as hexokinase II were inhibited by the use of 10AB. On the other hand, the expression of the pro-apoptotic protein Bax was increased after 10AB treatment. Taken together, our results suggest that 10AB-induced apoptosis is mediated through the overproduction of ROS and the disruption of mitochondrial metabolism.
PMCID: PMC4052332  PMID: 24857964
10-acetylirciformonin B; apoptosis; hexokinase; mitochondria; reactive oxygen species (ROS); topoisomerase
24.  Efficacy of novel antibacterial compounds targeting histidine kinase YycG protein 
Applied Microbiology and Biotechnology  2014;98(13):6003-6013.
Treating staphylococcal biofilm-associated infections is challenging. Based on the findings that compound 2 targeting the HK domain of Staphylococcus epidermidis YycG has bactericidal and antibiofilm activities against staphylococci, six newly synthesized derivatives were evaluated for their antibacterial activities. The six derivatives of compound 2 inhibited autophosphorylation of recombinant YycG′ and the IC50 values ranged from 24.2 to 71.2 μM. The derivatives displayed bactericidal activity against planktonic S. epidermidis or Staphylococcus aureus strains in the MIC range of 1.5–3.1 μM. All the derivatives had antibiofilm activities against the 6- and 24-h biofilms of S. epidermidis. Compared to the prototype compound 2, they had less cytotoxicity for Vero cells and less hemolytic activity for human erythrocytes. The derivatives showed antibacterial activities against clinical methicillin-resistant staphylococcal isolates. The structural modification of YycG inhibitors will assist the discovery of novel agents to eliminate biofilm infections and multidrug-resistant staphylococcal infections.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-5685-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4057637  PMID: 24737057
Staphylococcus epidermidis; Methicillin-resistant Staphylococcus aureus (MRSA); Antibacterial; Minimal inhibitory concentration (MIC); Minimal bactericidal concentration (MBC); Antibiofilm activity
25.  The Pilot Study of Fibrin with Temporomandibular Joint Derived Synovial Stem Cells in Repairing TMJ Disc Perforation 
BioMed Research International  2014;2014:454021.
TMJ disc related diseases are difficult to be cured due to the poor repair ability of the disc. TMJ-SDSCs were ideal cell sources for cartilage tissue engineering which have been widely used in hyaline cartilage regeneration. Fibrin gel has been demonstrated as a potential scaffold for neocartilage formation. The aim of this study was to repair the TMJ disc perforation using fibrin/chitosan hybrid scaffold combined with TMJ-SDSCs. Rat TMJ-SDSCs were cultured on hybrid scaffold or pure chitosan scaffolds. The cell seeding efficiency, distribution, proliferation, and chondrogenic differentiation capacity were investigated. To evaluate the in vivo repair ability of cell/scaffold construct, rat TMJ disc explants were punched with a defect to mimic TMJ disc perforation. Cell seeded scaffolds were inserted into the defect of TMJ disc explants and then were implanted subcutaneously in nude mice for 4 weeks. Results demonstrated that fibrin may improve cell seeding, proliferation, and chondrogenic induction in vitro. The in vivo experiments showed more cartilage ECM deposition in fibrin/chitosan scaffold, which suggested an enhanced reparative ability. This pilot study demonstrated that the regenerative ability of TMJ-SDSCs seeded in fibrin/chitosan scaffold could be applied for repairing TMJ disc perforation.
PMCID: PMC4009306  PMID: 24822210

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