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1.  Human intestinal transporter database: QSAR modeling and virtual profiling of drug uptake, efflux and interactions 
Pharmaceutical research  2012;30(4):996-1007.
Membrane transporters mediate many biological effects of chemicals and play a major role in pharmacokinetics and drug resistance. The selection of viable drug candidates among biologically active compounds requires the assessment of their transporter interaction profiles.
Using public sources, we have assembled and curated the largest, to our knowledge, human intestinal transporter database (>5,000 interaction entries for >3,700 molecules). This data was used to develop thoroughly validated classification Quantitative Structure-Activity Relationship (QSAR) models of transport and/or inhibition of several major transporters including MDR1, BCRP, MRP1-4, PEPT1, ASBT, OATP2B1, OCT1, and MCT1.
Results & Conclusions
QSAR models have been developed with advanced machine learning techniques such as Support Vector Machines, Random Forest, and k Nearest Neighbors using Dragon and MOE chemical descriptors. These models afforded high external prediction accuracies of 71–100% estimated by 5-fold external validation, and showed hit retrieval rates with up to 20-fold enrichment in the virtual screening of DrugBank compounds. The compendium of predictive QSAR models developed in this study can be used for virtual profiling of drug candidates and/or environmental agents with the optimal transporter profiles.
PMCID: PMC3596480  PMID: 23269503
membrane transport proteins; ADMET; drug transport; permeability; efflux
2.  The Discovery of Novel Antimalarial Compounds Enabled by QSAR-based Virtual Screening 
Quantitative structure–activity relationship (QSAR) models have been developed for a dataset of 3133 compounds defined as either active or inactive against P. falciparum. Since the dataset was strongly biased towards inactive compounds, different sampling approaches were employed to balance the ratio of actives vs. inactives, and models were rigorously validated using both internal and external validation approaches. The balanced accuracy for assessing the antimalarial activities of 70 external compounds was between 87% and 100% depending on the approach used to balance the dataset. Virtual screening of the ChemBridge database using QSAR models identified 176 putative antimalarial compounds that were submitted for experimental validation, along with 42 putative inactives as negative controls. Twenty five (14.2%) computational hits were found to have antimalarial activities with minimal cytotoxicity to mammalian cells, while all 42 putative inactives were confirmed experimentally. Structural inspection of confirmed active hits revealed novel chemical scaffolds, which could be employed as starting points to discover novel antimalarial agents.
PMCID: PMC3644566  PMID: 23252936
Antimalarial activity; quantitative structure–activity relationships; virtual screening; experimental confirmation
3.  Transactivation of Atg4b by C/EBPβ Promotes Autophagy To Facilitate Adipogenesis 
Molecular and Cellular Biology  2013;33(16):3180-3190.
Autophagy is a highly conserved self-digestion pathway involved in various physiological and pathophysiological processes. Recent studies have implicated a pivotal role of autophagy in adipocyte differentiation, but the molecular mechanism for its role and how it is regulated during this process are not clear. Here, we show that CCAAT /enhancer-binding protein β (C/EBPβ), an important adipogenic factor, is required for the activation of autophagy during 3T3-L1 adipocyte differentiation. An autophagy-related gene, Atg4b, is identified as a de novo target gene of C/EBPβ and is shown to play an important role in 3T3-L1 adipocyte differentiation. Furthermore, autophagy is required for the degradation of Klf2 and Klf3, two negative regulators of adipocyte differentiation, which is mediated by the adaptor protein p62/SQSTM1. Importantly, the regulation of autophagy by C/EBPβ and the role of autophagy in Klf2/3 degradation and in adipogenesis are further confirmed in mouse models. Our data describe a novel function of C/EBPβ in regulating autophagy and reveal the mechanism of autophagy during adipocyte differentiation. These new insights into the molecular mechanism of adipose tissue development provide a functional pathway with therapeutic potential against obesity and its related metabolic disorders.
PMCID: PMC3753907  PMID: 23754749
4.  Fetal deficiency of Lin28 programs life-long aberrations in growth and glucose metabolism 
Stem cells (Dayton, Ohio)  2013;31(8):1563-1573.
LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO can be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.
PMCID: PMC3775935  PMID: 23666760
Lin28a; Lin28b; dwarfism; growth; glucose metabolism; diabetes; let-7; mTOR
Journal of statistical research  2012;46(2):157-186.
We introduce the Interactive Decision Committee method for classification when high-dimensional feature variables are grouped into feature categories. The proposed method uses the interactive relationships among feature categories to build base classifiers which are combined using decision committees. A two-stage or a single-stage 5-fold cross-validation technique is utilized to decide the total number of base classifiers to be combined. The proposed procedure is useful for classifying biochemicals on the basis of toxicity activity, where the feature space consists of chemical descriptors and the responses are binary indicators of toxicity activity. Each descriptor belongs to at least one descriptor category. The support vector machine, the random forests, and the tree-based AdaBoost algorithms are utilized as classifier inducers. Forward selection is used to select the best combinations of the base classifiers given the number of base classifiers. Simulation studies demonstrate that the proposed method outperforms a single large, unaggregated classifier in the presence of interactive feature category information. We applied the proposed method to two toxicity data sets associated with chemical compounds. For these data sets, the proposed method improved classification performance for the majority of outcomes compared to a single large, unaggregated classifier.
PMCID: PMC3887560  PMID: 24415822
Chemical toxicity; Decision committee method; Ensemble; Ensemble feature selection; QSAR modeling; Statistical learning
6.  Krüppel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression 
PLoS ONE  2013;8(12):e82449.
Krüppel-like factor 6 (KLF6) is a tumor suppressor gene and play a role in the regulation of cell proliferation and apoptosis. After the peripheral nerve injury (PNI), the microenvironment created by surrounding Schwann cells (SCs) is a critical determinant of its regenerative potential. In this study, we examined the effects of KLF6 on SCs responses during PNI. Both KLF6 mRNA and protein expression levels were upregulated in the injured sciatic nerve, and immunofluorescence results showed that many KLF6-positive cells simultaneously expressed the SC markers S-100 and p75NTR. The apoptosis inducers TNFα and cisplatin upregulated KLF6 expression in primary cultured SCs and the SC line RSC96. Although KLF6 overexpression exacerbated cisplatin- and TNFα-induced apoptosis, expression levels of the apoptosis regulators Bcl2 and Bax were not significantly affected in either KLF6-overexpressing or KLF6-depleted RSC96 cells. Realtime PCR arrays and qRT-PCR demonstrated that KLF6 overexpression upregulated four pro-apoptotic genes, FAS, TNF, TNFSF12, and PYCARD, and inhibited expression of the anti-apoptotic IL10 gene expression. Further analysis revealed that FAS protein expression was positively correlated with KLF6 expression in SCs. These data suggest that KLF6 upregulation may render SCs more vulnerable to apoptosis after injury via upregulating FAS expression.
PMCID: PMC3853331  PMID: 24324791
7.  ANRIL/CDKN2B-AS shows two-stage clade-specific evolution and becomes conserved after transposon insertions in simians 
Many long non-coding RNA (lncRNA) genes identified in mammals have multiple exons and functional domains, allowing them to bind to polycomb proteins, DNA methyltransferases, and specific DNA sequences to regulate genome methylation. Little is known about the origin and evolution of lncRNAs. ANRIL/CDKN2B-AS consists of 19 exons on human chromosome 9p21 and regulates the expression of three cyclin-dependent kinase inhibitors (CDKN2A/ARF/CDKN2B).
ANRIL/CDKN2B-AS originated in placental mammals, obtained additional exons during mammalian evolution but gradually lost them during rodent evolution, and reached 19 exons only in simians. ANRIL lacks splicing signals in mammals. In simians, multiple transposons were inserted and transformed into exons of the ANRIL gene, after which ANRIL became highly conserved. A further survey reveals that multiple transposons exist in many lncRNAs.
ANRIL shows a two-stage, clade-specific evolutionary process and is fully developed only in simians. The domestication of multiple transposons indicates an impressive pattern of “evolutionary tinkering” and is likely to be important for ANRIL’s structure and function. The evolution of lncRNAs and that of transposons may be highly co-opted in primates. Many lncRNAs may be functional only in simians.
PMCID: PMC3831594  PMID: 24225082
8.  A network of heterochronic genes including Imp1 regulates temporal changes in stem cell properties 
eLife  2013;2:e00924.
Stem cell properties change over time to match the changing growth and regeneration demands of tissues. We showed previously that adult forebrain stem cell function declines during aging because of increased expression of let-7 microRNAs, evolutionarily conserved heterochronic genes that reduce HMGA2 expression. Here we asked whether let-7 targets also regulate changes between fetal and adult stem cells. We found a second let-7 target, the RNA binding protein IMP1, that is expressed by fetal, but not adult, neural stem cells. IMP1 expression was promoted by Wnt signaling and Lin28a expression and opposed by let-7 microRNAs. Imp1-deficient neural stem cells were prematurely depleted in the dorsal telencephalon due to accelerated differentiation, impairing pallial expansion. IMP1 post-transcriptionally inhibited the expression of differentiation-associated genes while promoting the expression of self-renewal genes, including Hmga2. A network of heterochronic gene products including Lin28a, let-7, IMP1, and HMGA2 thus regulates temporal changes in stem cell properties.
eLife digest
Stem cells are found throughout the body, and play key roles in promoting tissue growth during fetal development, and in maintaining tissues in the adult. When stem cells divide, they can either give rise to more stem cells, or they can generate specialized cells required for tissue function. However, the properties of stem cells must change over time to match the changing growth and regeneration demands of tissues.
A previous study by Nishino et al. has shown that expression of a micro RNA molecule called let-7 increases throughout adulthood, and this reduces the activity of stem cells in older animals. Now, Nishino et al. report that let-7, and other genes it regulates, also control the dramatic changes that occur in the properties of stem cells between fetal development and adulthood. Whereas stem cells in the fetal forebrain undergo rapid division and are capable of generating many different cell types, stem cells in the adult forebrain divide less often and can generate only a few specific types of cell. While Nishino et al. performed their study on stem cells in the brain, their results are likely to apply also to stem cells in other tissues.
Nishino et al. show that let-7 regulates the production of an RNA binding protein called IMP1. Mice with stem cells that lack IMP1 have a smaller cerebral cortex than normal mice because their stem cells undergo fewer rounds of division before committing to become brain cells. Additional experiments revealed that IMP1 inhibits the expression of genes that trigger stem cells to commit to specific fates and promotes the expression of genes related to self-renewal.
These results indicate that the gene that encodes IMP1 is expressed in fetal neural stem cells, but not in adult neural stem cells, and that the reduced production of this protein contributes to the developmental switch from highly proliferative neural stem cells in the fetus to the more quiescent stem cells found in adults. Further studies are likely to identify many more targets of let-7 that enable stem cells to adapt their properties to the changing needs of the organism over time.
These results are interesting because let-7-regulated networks were first discovered based on their ability to regulate the timing of developmental transitions in worms. This suggests that the mechanisms employed by mammalian tissue stem cells to regulate changes in their properties over time, are at least partly evolutionarily conserved mechanisms inherited from invertebrates.
PMCID: PMC3817382  PMID: 24192035
neural stem cell; heterochronic gene; cortical development; Mouse
9.  A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples 
Biomedical chromatography : BMC  2012;26(11):1364-1370.
A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples.
PMCID: PMC3391541  PMID: 22275222
Atomoxetine; LC-MS/MS; human plasma; in vitro; protein precipitation
10.  UPLC-PDA determination of paeoniflorin in rat plasma following the oral administration of Radix Paeoniae Alba and its effects on rats with collagen-induced arthritis 
Rheumatoid arthritis (RA) is a chronic disabling autoimmune disease with characteristics of chronic, progressive inflammatory joint synovial damage, which mainly encroaches upon the synovium of the joint. The use of traditional medicine to treat RA slows the development of RA to a certain extent; however, it often has numerous side-effects. Therefore, the focus of RA research is the identification of a new, safe and effective medicine. The aim of the present study was to use an ultra performance liquid chromatography and photo diode array (UPLC-PDA) method to detect the paeoniflorin component in a Radix Paeoniae Alba decoction and in rat plasma following the oral administration of Radix Paeoniae Alba decoction. In addition, the effects of paeoniflorin on collagen-induced arthritis (CIA) in rats were investigated. The results indicate that a UPLC-PDA method for determining the presence of paeoniflorin in the Radix Paeoniae Alba decoction was successfully established. The method was fast, simple, sensitive, precise and valid. Paeoniflorin was shown to be a bioactive component of the Radix Paeoniae Alba decoction that was absorbed into rat plasma. Paeoniflorin significantly improved the disease resistant ability of RA rats and reduced the levels of the inflammatory cytokines, IL-1β and TNF-α, thereby inhibiting inflammation and bone erosion in the rats with CIA. The observations are likely to lay the foundation for further study of the mechanism of paeoniflorin in the treatment of RA.
PMCID: PMC3861030  PMID: 24348792
ultra performance liquid chromatography; Radix Paeoniae Alba; paeoniflorin; collagen-induced arthritis; rheumatoid arthritis; bizhongxiao decoction
11.  An Interactive Association of Advanced Glycation End-Product Receptor Gene Four Common Polymorphisms with Coronary Artery Disease in Northeastern Han Chinese 
PLoS ONE  2013;8(10):e76966.
Growing evidence indicates that advanced glycation end-product receptor (RAGE) might play a contributory role in the pathogenesis of coronary artery disease (CAD). To shed some light from a genetic perspective, we sought to investigate the interactive association of RAGE gene four common polymorphisms (rs1800625 or T-429C, rs1800624 or T-374A, rs2070600 or Gly82Ser, and rs184003 or G1704A) with the risk of developing CAD in a large northeastern Han Chinese population.
Methodology/Principal Findings
This was a hospital-based case-control study incorporating 1142 patients diagnosed with CAD and 1106 age- and gender-matched controls. All individuals were angiographically confirmed. Risk estimates were expressed as odds ratio (OR) and 95% confidence interval (CI). Overall there were significant differences in the genotype and allele distributions of rs1800625 and rs184003, even after the Bonferroni correction. Logistic regression analyses indicated that rs1800625 and rs184003 were associated with significant risk of CAD under both additive (OR = 1.20 and 1.23; 95% CI: 1.06–1.37 and 1.06–1.42; P = 0.006 and 0.008) and recessive (OR = 1.75 and 2.39; 95% CI: 1.28–2.40 and 1.47–3.87; P<0.001 and <0.001) models after adjusting for confounders. In haplotype analyses, haplotypes C-T-G-G and T-A-G-T (alleles in order of rs1800625, rs1800624, rs2070600 and rs184003), overrepresented in patients, were associated with 52% (95% CI: 1.19–1.87; P = 0.0052) and 63% (95% CI: 1.14–2.34; P = 0.0075) significant increases in adjusted risk for CAD. Further interactive analyses identified an overall best multifactor dimensionality reduction (MDR) model including rs1800625 and rs184003. This model had a maximal testing accuracy of 0.6856 and a cross-validation consistency of 10 out of 10 (P = 0.0016). The validity of this model was substantiated by classical Logistic regression analysis.
Our findings provided strong evidence for the potentially contributory roles of RAGE multiple genetic polymorphisms, especially in the context of locus-to-locus interaction, in the pathogenesis of CAD among northeastern Han Chinese.
PMCID: PMC3796558  PMID: 24155913
12.  Interaction of organic cation transporter 3 (SLC22A3) and amphetamine 
Journal of neurochemistry  2010;114(1):142-149.
The organic cation transporter (OCT) 3 is widely expressed in various organs in humans, and involved in the disposition of many exogenous and endogenous compounds. Several lines of evidence have suggested that OCT3 expressed in the brain plays an important role in the regulation of neurotransmission. Relative to wild-type (WT) animals, Oct3 knockout (KO) mice have displayed altered behavioral and neurochemical responses to psychostimulants such as amphetamine (AMPH) and methamphetamine. In the present study, both in vitro and in vivo approaches were utilized to explore potential mechanisms underlying the disparate neuropharmacological effects observed following AMPH exposure in Oct3 KO mice. In vitro uptake studies conducted in OCT3 transfected cells indicated that dextroamphetamine (d-AMPH) is not a substrate of OCT3. However, OCT3 was determined to be a high-capacity and low-affinity transporter for the neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5-HT). Inhibition studies demonstrated that d-AMPH exerts relatively weak inhibitory effects on the OCT3-mediated uptake of DA, NE, 5-HT, and the model OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide. The IC50 values were determined to be 41.5 ± 7.5 and 24.1 ± 7.0 μM for inhibiting DA and 5-HT uptake, respectively, while 50% inhibition of NE and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide uptake was not achieved by even the highest concentration of d-AMPH applied (100 μM). Furthermore, the disposition of d-AMPH in various tissues including the brain, liver, heart, kidney, muscle, intestine, spleen, testis, uterus, and plasma were determined in both male and female Oct3 KO and WT mice. No significant difference was observed between either genotypes or sex in all tested organs and tissues. Our findings suggest that OCT3 is not a prominent factor influencing the disposition of d-AMPH. Additionally, based upon the inhibitory potency observed in vitro, d-AMPH is unlikely to inhibit the uptake of monoamines mediated by OCT3 in the brain. Differentiated neuropharmacological effects of AMPHs noted between Oct3 KO and WT mice appear to be due to the absence of Oct3 mediated uptake of neurotransmitters in the KO mice.
PMCID: PMC3775896  PMID: 20402963
amphetamine; inhibitor; monoamine neurotransmitters; organic cation transporter 3; substrate
13.  Reversal Effects of Pantoprazole on Multidrug Resistance in Human Gastric Adenocarcinoma Cells by Down-Regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 Signaling Pathway In Vitro and In Vivo 
Journal of cellular biochemistry  2012;113(7):2474-2487.
To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO2 atmosphere at 37°C. Adriamycin (ADR)-resistant cells were cultured with addition of 0.8 μg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK-8 Assay was performed to evaluate the cytotoxicity of anti-tumor drugs; BCECF-AM pH-sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H+-ATPases (V-ATPases), mTOR, HIF-1α, P-glycoprotein (P-gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 μg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P <0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose-dependently (P <0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose-dependently. After 24-h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non-PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 μg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V-ATPases, mTOR, HIF-1α, P-gp, and MRP1, and alter intracellular expressions in parent and ADR-resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti-tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti-tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway.
PMCID: PMC3762681  PMID: 22396185
14.  Damped propagation of cell polarization explains distinct PCP phenotypes of epithelial patterning 
Scientific Reports  2013;3:2528.
During epithelial patterning in metazoans cells are polarized in the plane of a tissue, a process referred to as planar cell polarity (PCP). Interactions between a few molecules produce distinct phenotypes in diverse tissues in animals from flies to humans and make PCP tightly associated with tissue and organ growth control. An interesting question is whether these phenotypes share common traits. Previous computational models revealed how PCP signalling determines cell polarization in some specific contexts. We have developed a computational model, examined PCP signalling in varied molecular contexts, and revealed how details of molecular interactions and differences in molecular contexts affect the direction, speed, and propagation of cell polarization. The main finding is that damped propagation of cell polarization can generate rich variances in phenotypes of domineering non-autonomy and error correction in different contexts. These results impressively demonstrate how simple molecular interactions cause distinct, yet inherently analogous, developmental patterning.
PMCID: PMC3759060  PMID: 23995897
15.  Enantiospecific determination of dl-methylphenidate and dl-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: Application to human ethanol interactions 
In humans, concomitant dl-methylphenidate (dl-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite dl-ethylphenidate (dl-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between d- and l-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic – pharmacodynamic correlations regarding the MPH – ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneously analysis of d- and l-MPH as well as d- and l-EPH concentration from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r2 > 0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol was included, and the implications of this pharmacokinetic drug interaction were discussed.
PMCID: PMC3733562  PMID: 21402502
methylphenidate; ethylphenidate; ethanol; enantiomer; LC-MS/MS; drug interaction
16.  Antitumor agents 294. Novel E-ring-modified camptothecin–4β-anilino-4′-O-demethylepipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents 
Bioorganic & medicinal chemistry  2012;20(14):4489-4494.
Two conjugates (1 and 2) of camptothecin (CPT) and 4β-anilino-4′-O-demethylepipodophyllotoxin were previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this current study, two novel conjugates (1E and 2E) with an open E-ring in the CPT moiety were first synthesized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo I-linked DNA (TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analogs. This lower religation potency resulted in decreased formation of protein-linked DNA breaks (PLDBs), and hence, less cell growth inhibition. In addition to their impact on topo I, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo II-induced DNA cleavage. The novel structures of 1E and 2E may present scaffolds for further development of dual function topo I and II inhibitors with improved pharmacological profiles and physicochemical properties.
PMCID: PMC3389137  PMID: 22698783
Topoisomerase; cytotoxicity; camptothecin (CPT); etoposide (VP-16); epipodophyllotoxin; conjugates
17.  Influence of Threonine Metabolism on S-adenosyl-methionine and Histone Methylation 
Science (New York, N.Y.)  2012;339(6116):222-226.
Threonine is the only amino acid critically required for the pluripotency of mouse embryonic stem cells (mESCs) but the detailed mechanism remains unclear. We found that threonine (Thr) and S-adenosyl-methionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation. Isotope labeling of mESCs revealed that Thr provides a substantial fraction of both the cellular glycine (Gly) and the acetyl-coenzyme A (CoA) needed for SAM synthesis. Depletion of Thr from the culture medium or threonine dehydrogenase (Tdh) from mESCs decreased accumulation of SAM and decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth, and increased differentiation. Thus abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate.
PMCID: PMC3652341  PMID: 23118012
18.  Safety and efficacy of a non-polymeric paclitaxel-eluting microporous stent in real-world percutaneous coronary intervention 
At present, there is an increasing focus on stents that have a biodegradable polymer coating, rather than a permanent polymer coating. This is due to the fact that following the implantation of a drug-eluting stent (DES) with a permanent polymer coating, the continued existence of the coating may result in a foreign body reaction and delayed re-endothelialization. The aim of the present study was to evaluate the safety and efficacy of a non-polymeric paclitaxel-eluting microporous (YINYI™) stent in real-life percutaneous coronary intervention (PCI) for patients with coronary artery disease (CAD). A total of 686 YINYI™ stents were implanted in 404 patients with CAD in a PCI procedure and outpatient follow-ups were performed 1, 6, 12 and 15 months subsequent to the PCI, respectively. The observation endpoints were major adverse cardiac events (MACEs), including cardiac death, non-fatal myocardial infarction (MI), restenosis, target lesion revascularization, stent thrombosis and recurrence of angina pectoris. The average follow-up time was 15 months. The results revealed that the cumulative incidences of MACEs were as follows: mortality, 0.99%; non-fatal MI, 0.74%; restenosis, 4.0%; and target lesion revascularization, 2.7%. The results at the short- and long-term clinical follow-ups indicated that YINYI™ stents are effective and safe for use in PCI for patients with CAD.
PMCID: PMC3786829  PMID: 24137271
coronary artery disease; percutaneous coronary intervention; YINYI™ stent; non-polymeric paclitaxel-eluting microporous stent; follow-up
19.  Necrotizing myositis causes restrictive hypoventilation in a mouse model for human enterovirus 71 infection 
Virology Journal  2013;10:215.
Enterovirus 71 (EV71) infections are associated with a high prevalence of hand, foot and mouth disease (HFMD) in children and occasionally cause lethal complications. Most infections are self-limiting. However, resulting complications, including aseptic meningitis, encephalitis, poliomyelitis-like acute flaccid paralysis, and neurological pulmonary edema or hemorrhage, are responsible for the lethal symptoms of EV71 infection, the pathogenesis of which remain to be clarified.
In the present study, 2-week-old Institute of Cancer Research (ICR) mice were infected with a mouse-adapted EV71 strain. These infected mice demonstrated progressive paralysis and died within 12 days post infection (d.p.i.). EV71, which mainly replicates in skeletal muscle tissues, caused severe necrotizing myositis. Lesions in the central nervous system (CNS) and other tissues were not observed.
Necrotizing myositis of respiratory-related muscles caused severe restrictive hypoventilation and subsequent hypoxia, which could explain the fatality of EV71-infected mice. This finding suggests that, in addition to CNS injury, necrotic myositis may also be responsible for the paralysis and death observed in EV71-infected mice.
PMCID: PMC3710232  PMID: 23809248
Enterovirus 71; Skeletal muscle; Necrotizing myositis; Restrictive hypoventilation
20.  Hippo pathway genes developed varied exon numbers and coevolved functional domains in metazoans for species specific growth control 
The Hippo pathway controls growth by mediating cell proliferation and apoptosis. Dysregulation of Hippo signaling causes abnormal proliferation in both healthy and cancerous cells. The Hippo pathway receives inputs from multiple developmental pathways and interacts with many tissue-specific transcription factors, but how genes in the pathway have evolved remains inadequately revealed.
To explore the origin and evolution of Hippo pathway, we have extensively examined 16 Hippo pathway genes, including upstream regulators and downstream targets, in 24 organisms covering major metazoan phyla. From simple to complex organisms, these genes are varied in the length and number of exons but encode conserved domains with similar higher-order organization. The core of the pathway is more conserved than its upstream regulators and downstream targets. Several components, despite existing in the most basal metazoan sponges, cannot be convincingly identified in other species. Potential recombination breakpoints were identified in some genes. Coevolutionary analysis reveals that most functional domains in Hippo genes have coevolved with interacting functional domains in other genes.
The two essential upstream regulators cadherins fat and dachsous may have originated in the unicellular organism Monosiga brevicollis and evolved more significantly than the core of the pathway. Genes having varied numbers of exons in different species, recombination events, and the gain and loss of some genes indicate alternative splicing and species-specific evolution. Coevolution signals explain some species-specific loss of functional domains. These results significantly unveil the structure and evolution of the Hippo pathway in distant phyla and provide valuable clues for further examination of Hippo signaling.
PMCID: PMC3620549  PMID: 23547742
21.  Beta-Cell Injury in Ncb5or-null Mice is Exacerbated by Consumption of a High-Fat Diet 
NADH-cytochrome b5 oxidoreductase (Ncb5or) in endoplasmic reticulum (ER) is involved in fatty acid metabolism, and Ncb5or−/− mice fed standard chow (SC) are insulin-sensitive but weigh less than wild type (WT) littermates. Ncb5or−/− mice develop hyperglycemia at about age 7 weeks due to β-cell dysfunction and loss associated with saturated fatty acid accumulation and manifestations of ER and oxidative stress. Here we report that when Ncb5or−/− mice born to heterozygous mothers fed a high fat (HF) diet continue to ingest HF, they weigh as much as SC-fed WT at age 5 weeks. By age 7 weeks, diabetes mellitus develops in all HF-fed vs. 68% of SC-fed Ncb5or−/− mice. Islet β-cell content in age 5-week Ncb5or−/− mice fed HF for 7 days is lower (53%) than for those fed SC (63%), and both are lower than for WT (75%, SC, vs. 69%, HF). Islet transcript levels for markers of mitochondrial biogenesis (PGC-1α) and ER stress (ATF6α) are higher in Ncb5or−/− than WT mice but not significantly affected by diet. Consuming a HF diet exacerbates Ncb5or−/− β-cell accumulation of intracellular saturated fatty acids and increases the frequency of ER distention from 11% (SC) to 47% (HF), thus accelerates β-cell injury in Ncb5or−/− mice.
PMCID: PMC3348618  PMID: 22582025
diabetes; beta-cells; lipotoxicity; high-fat diet; ER stress
22.  A discriminative analytical method for detection of CES1A1 and CES1A2/CES1A3 genetic variants 
Pharmacogenetics and genomics  2012;22(3):215-218.
Human carboxylesterase 1(hCES1), encoded by the CES1 gene, is the predominant hepatic hydrolase responsible for the metabolism of many therapeutic agents, toxins, and endogenous substances. Genetic variants of CES1 can affect hCES1 function and expression and ultimately influence clinical outcomes of hCES1 substrate drugs. CES1 gene consists of three isoforms including the functional CES1A1 and CES1A2 genes and nonfunctional pseudogene CES1A3. Natural variants of these isoforms exert differing impacts on hCES1 function. However, the existing CES1 genotyping methods are incapable of discriminating between these variants due to the high similarity among these three genes. We report the development of a novel long-range PCR-based, discriminative genotyping assay with the capacity of specifically detecting the variants among CES1A1, CES1A2, and CES1A3 genes. The comparison of the genotyping results between this novel assay and those previously reported methods highlighted the necessity of applying the discriminative genotyping assay in pharmacogentic studies involving CES1 gene.
PMCID: PMC3412537  PMID: 22237548
CES1A1; CES1A2; CES1A3; genotyping; long-range PCR
23.  Effect of Bizhongxiao decoction and its dismantled formulae on IL-1 and TNF levels in collagen-induced arthritis in rat synovial joints 
Rheumatoid arthritis (RA), a chronic autoimmune disease, affects sufferers in many different ways. Treatment of this chronic condition is particularly challenging. Traditional Chinese Medicine (TCM) provides alternatives. Bizhongxiao decoction (BZX) is a TCM complex, which has been used clinically for many years to treat RA. The purpose of this study is to compare the effects of BZX decoction and its dismantled formulae on IL-1 and TNF-1 levels in rats with RA, and to elucidate its mechanism of action.
Ninety healthy normal female SD rats were randomly divided into six groups: normal (control), model, BZX decoction, and the three dismantled formulae (I: heat-clearing and detoxication, II: dissipating dampness, and III: blood circulation promotion). Apart from the normal (control) group, the rats in each group were injected subcutaneously with bovine type II collagen and complete Freund adjuvant to establish a collagen-induced arthritis model, so that inhibition of foot swelling in the rats by BZX decoction and its dismantled formulae could be observed. Immunohistochemistry was used to assess the levels of the inflammatory cytokines IL-1 and TNF in synovial joints at various time points.
Twenty-one days after the model was established, the levels of TNF and IL-1 were significantly higher in the model group, BZX decoction group and dismantled formula groups I, II and III than in the normal controls (P < 0.05). The levels of these cytokines were significantly higher in the model group than the BZX decoction or the three dismantled formula groups (P <0.01). At longer times, the TNF and IL-1 levels in model group rose gradually; those in the BZX decoction and dismantled formula groups were gradually reduced. The cytokine levels in the BZX decoction group were lower than in the three dismantled formula groups and continued to decline.
BZX decoction and the three dismantled formulae examined down-regulated the inflammatory factors IL-1 and TNF in collagen-induced arthritis rat models, but BZX exerted the strongest effect.
PMCID: PMC3507724  PMID: 23163934
Bizhongxiao decoction; Rheumatoid arthritis; Interleukin-1; Tumor necrosis factor
24.  Development of Diabetes in Lean Ncb5or-Null Mice is Associated with Manifestations of Endoplasmic Reticulum and Oxidative Stress in Beta Cells 
Biochimica et biophysica acta  2011;1812(11):1532-1541.
NADH-cytochrome b5 oxidoreductase (Ncb5or) is an endoplasmic reticulum (ER)-associated redox enzyme involved in fatty acid metabolism, and phenotypic abnormalities of Ncb5or−/− mice include diabetes and lipoatrophy. These mice are lean and insulin-sensitive but become hyperglycemic at age 7 weeks as a result of β-cell dysfunction and loss. Here we examine early cellular and molecular events associated with manifestations of β-cell defects in Ncb5or−/− mice. We observe lower islet β-cell content in pancreata at age 4 weeks and prominent ER distention in β-cells by age 5 weeks. Ultrastructural changes progress rapidly in severity from age 5 to 6 weeks, and their frequency rises from 10% of β-cells at 5 weeks to 33% at 6 weeks. These changes correlate temporally with the onset of diabetes. ER stress responses and lipid load in Ncb5or−/− β-cells were assessed with isolated islets from mice at age 5 weeks. Expression levels of the stress marker protein Grp78/BiP and of phosphorylated eIF2α protein were found to be reduced, although their transcript levels did not decline. This pattern stands in contrast to the canonical unfolded protein response. Ncb5or−/− β-cells also accumulated higher intracellular levels of palmitate and other free fatty acids and exhibited greater reactive oxygen species production than wild-type cells. An alloxan-susceptible genetic background was found to confer accelerated onset of diabetes in Ncb5or−/− mice. These findings provide the first direct evidence that manifestations of diabetes in lean Ncb5or−/− mice involve saturated free fatty acid overload of β-cells and ER and oxidative stress responses.
PMCID: PMC3185181  PMID: 21839170
oxidative stress; ER stress; free fatty acids; diabetes; beta-cells
25.  Attrition among Human Immunodeficiency Virus (HIV)- Infected Patients Initiating Antiretroviral Therapy in China, 2003–2010 
PLoS ONE  2012;7(6):e39414.
Mortality and morbidity from HIV have dramatically decreased in both high- and low-income countries. However, some patients may not benefit from combination antiretroviral therapy (cART) because of inadequate access to HIV care, including attrition after care initiation.
Methodology/Principal Findings
The study population included all HIV-infected patients receiving cART through the Chinese National Free Antiretroviral Treatment Program from 1 January 2003 to 31 December 2010 (n = 106,542). We evaluated retention in HIV care and used multivariable Cox proportional hazard models to identify independent factors predictive of attrition. The cumulative probability of attrition from cART initiation was 9% at 12 months, 13% at 18 months, 16% at 24 months and 24% at 60 months. A number of factors were associated with attrition, including younger age, male gender, and being single or divorced. Patients with higher CD4 cell counts at cART initiation were more likely to drop out of HIV care. The proportion of patients remaining in HIV care increased in more recent calendar years and among patients who initiated modern cART regimens.
Retention in HIV care is essential for optimizing individual and public health outcomes. Attrition, even the degree observed in our study, can lead to premature morbidity and mortality, and possibly affect further transmission of HIV and HIV resistant drug variants. Effective strategies to promote retention in HIV care programs are needed. In China these strategies may include focusing particularly on younger male patients and those with higher CD4 cell counts at therapy initiation.
PMCID: PMC3384674  PMID: 22761787

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