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1.  MicroRNA-133a Regulates Insulin-like Growth Factor-1 Receptor Expression and Vascular Smooth Muscle Cell Proliferation in Murine Atherosclerosis 
Atherosclerosis  2013;232(1):171-179.
Objective
MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis.
Methods and Results
Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE−/− aortas. ApoE−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein-deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3’UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE−/− VSMC than in WT cells.
Conclusion
MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1 stimulated VSMC survival and growth.
doi:10.1016/j.atherosclerosis.2013.11.029
PMCID: PMC4334121  PMID: 24401233
MicroRNA; Insulin-like growth factor; Artery; Smooth muscle cell; Atherosclerosis
2.  Gastric metastasis from small cell lung cancer: A case report 
Small cell lung cancer (SCLC) represents a group of highly malignant tumors that give rise to early and widespread metastases at the time of diagnosis. The preferential metastatic sites are the brain, liver, adrenal glands, bone, and bone marrow. However, metastases of the gastrointestinal system, especially the stomach, are rare; most cases of stomach metastasis are asymptomatic and, as a result, are usually only discovered at autopsy. We report a case of gastric metastasis originating from SCLC. The patient was a 66-year-old man admitted to our hospital due to abdominal pain. He underwent gastroscopy, with the pathological report of the tissue biopsy proving it to be a small cell cancer. Immunohistochemistry was positive for CD56, synaptophysin, and pan-cytokeratin. These results confirmed the diagnosis of gastric metastasis of a neuroendocrine small cell carcinoma from the lung.
doi:10.3748/wjg.v21.i5.1684
PMCID: PMC4316115  PMID: 25663792
Gastric metastasis; Small cell lung cancer; Immunohistochemistry
3.  Absolute quantification of UGT1A1 in various tissues and cell lines using isotope-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities 
Uridine5′-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) method. Standard addition method was used to validate the assay to account for the matrix effect. 17β-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200–0.0195 nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R2 =0.85) and in microsomes prepared from cell lines (R2 =0.95). The current isotope-labeled peptides were not necessary as the designated standard for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines.
doi:10.1016/j.jpba.2013.08.024
PMCID: PMC4010308  PMID: 24055854
UGT1A1; absolute quantification; LC-MS/MS; microsomes; glucuronidation
4.  Validation of IMP Dehydrogenase Inhibitors in a Mouse Model of Cryptosporidiosis 
Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.
doi:10.1128/AAC.02075-13
PMCID: PMC3957894  PMID: 24366728
5.  The effect of radiation dose on mouse skeletal muscle remodeling 
Radiology and Oncology  2014;48(3):247-256.
Background
The purpose of this study was to determine the effect of two clinically relevant radiation doses on the susceptibility of mouse skeletal muscle to remodeling.
Materials and methods.
Alterations in muscle morphology and regulatory signaling were examined in tibialis anterior and gastrocnemius muscles after radiation doses that differed in total biological effective dose (BED). Female C57BL/6 (8-wk) mice were randomly assigned to non-irradiated control, four fractionated doses of 4 Gy (4x4 Gy; BED 37 Gy), or a single 16 Gy dose (16 Gy; BED 100 Gy). Mice were sacrificed 2 weeks after the initial radiation exposure.
Results
The 16 Gy, but not 4x4 Gy, decreased total muscle protein and RNA content. Related to muscle regeneration, both 16 Gy and 4x4 Gy increased the incidence of central nuclei containing myofibers, but only 16 Gy increased the extracellular matrix volume. However, only 4x4 Gy increased muscle 4-hydroxynonenal expression. While both 16 Gy and 4x4 Gy decreased IIB myofiber mean cross-sectional area (CSA), only 16 Gy decreased IIA myofiber CSA. 16 Gy increased the incidence of small diameter IIA and IIB myofibers, while 4x4 Gy only increased the incidence of small diameter IIB myofibers. Both treatments decreased the frequency and CSA of low succinate dehydrogenase activity (SDH) fibers. Only 16 Gy increased the incidence of small diameter myofibers having high SDH activity. Neither treatment altered muscle signaling related to protein turnover or oxidative metabolism.
Conclusions
Collectively, these results demonstrate that radiation dose differentially affects muscle remodeling, and these effects appear to be related to fiber type and oxidative metabolism.
doi:10.2478/raon-2014-0025
PMCID: PMC4110081  PMID: 25177239
extracellular matrix; irradiation; oxidative metabolism; regeneration; skeletal muscle
6.  Mutual Regioselective Inhibition of Human UGT1A1-Mediated Glucuronidation of Four Flavonoids 
Molecular pharmaceutics  2013;10(8):2891-2903.
UDP-glucuronosyltransferase (UGT) 1A1-catalyzed glucuronidation is an important elimination pathway of flavonoids, and mutually inhibitory interactions may occur when two or more flavonoids are co-administered. Our recent research suggested that glucuronidation of flavonoids displayed distinct positional preferences, but whether this will lead to the mutually regioselective inhibition of UGT1A1-mediated glucuronidation of flavonoids is unknown. Therefore, we chose three monohydroxyflavone isomers 3-hydroxyflavone (3HF), 7-hydroxyflavone (7HF), 4′-hydroxyflavone (4′HF) and one trihydroxyflavone 3,7,4′-trihydroxyflavone (3,7,4′THF) as the model compounds to characterize the possible mutually regioselective inhibition of glucuronidation using expressed human UGT1A1. Apparent kinetic parameters [e.g., reaction velocity (V), Michaelis-Menten constant (Km), maximum rate of metabolism (Vmax), concentration at which inhibitor achieve 50% inhibition or IC50] and the Lineweaver-Burk plots were used to evaluate the apparent kinetic mechanisms of inhibition of glucuronidation. The results showed that UGT1A1-mediated glucuronidation of three monohydroxyflavones (i.e., 3HF, 7HF and 4′HF) and 3,7,4′THF was mutually inhibitory, and the mechanisms of inhibition appeared to be the mixed-typed inhibition. Specifically, the inhibitory effects displayed certain positional preference. Glucuronidation of 3HF was more easily inhibited by 3,7,4′THF than that of 7HF or 4′HF. Compared to 7-O-glucuronidation of 3,7,4′THF, 3-O-glucuronidation of 3,7,4′THF was more inhibited by 3HF and 4′HF, whereas glucuronidation at both 3-OH and 7-OH positions of 3,7,4′THF was more easily inhibited by 7HF than by 3HF and 4′HF. In conclusion, 3HF, 7HF, 4′HF and 3,7,4′THF were both substrates and inhibitors of UGT1A1, and they exhibited mutually regioselective inhibition of UGT1A1-mediated glucuronidation via a mixed-type inhibitory mechanism.
doi:10.1021/mp300599t
PMCID: PMC3953455  PMID: 23786524
flavonoids; glucuronidation; inhibition; regioselective; UGT1A1
7.  MicroRNA Expression in Salivary Supernatant of Patients with Pancreatic Cancer and Its Relationship with ZHENG 
BioMed Research International  2014;2014:756347.
In traditional Chinese medicine (TCM), diagnosis and prescriptions are based on the signs and symptoms which are recognized as ZHENG. The cornerstone of TCM is to differentially treat one ZHENG from others, which is also known as syndrome differentiation, and this relies on the gathering of clinical information through inspection, auscultation and olfaction, inquiry, and palpation. However, the biomolecular basis of the ZHENG remains unclear. In this study, the expressions of 384 cancer-related miRNAs in salivary supernatant of patients with pancreatic cancer were assessed by miRNA polymerase chain reaction (PCR) array, and the different expression patterns of miRNA in three different groups of ZHENG were studied with use of real-time quantitative PCR (qRT-PCR). Some miRNAs were found to be specifically expressed in some ZHENGs, for instance, miR-17, miR-21, and miR-181b in Shi-Re ZHENG and miR-196a in Pi-Xu ZHENG. This indicates that these miRNAs may play important roles in different ZHENG condition. Therefore, this study to some extent revealed the molecular basis of TCM ZHENG in pancreatic cancer.
doi:10.1155/2014/756347
PMCID: PMC4122139  PMID: 25126577
8.  Obesity Increases the Risk of Chest Wall Pain from Thoracic Stereotactic Body Radiation Therapy 
Purpose
Stereotactic body radiation therapy (SBRT) is increasingly being used to treat thoracic tumors. We sought here to identify dose-volume parameters that predict chest wall toxicity (pain and skin reactions) in patients receiving thoracic SBRT.
Methods
We screened a database of patients treated with SBRT between August 2004 and August 2008 to find patients with pulmonary tumors within 2.5 cm of the chest wall. All patients received a total dose of 50 Gy in four daily 12.5-Gy fractions. Toxicity was scored according to the NCI-CTCAE V3.0.
Results
Of 360 patients in the database, 265 (268 tumors) had tumors within <2.5 cm of the chest wall; 104 (39%) developed skin toxicity (any grade); 14 (5%) developed acute pain (any grade), and 45 (17%) developed chronic pain (grade 1 in 22 cases [49%] and grade 2 or 3 in 23 cases [51%]). Both skin toxicity and chest wall pain were associated with the V30 or volume of the chest wall receiving 30Gy. Body mass index (BMI) was also strongly associated with the development of chest pain: patients with BMI ≥ 29 had almost twice the risk of chronic pain (P = 0.03). Among patients with BMI over 29, diabetes mellitus was a significant contributing factor to the development of chest pain.
Conclusion
Safe use of SBRT with 50 Gy in 4 fractions for lesions close to the chest wall requires consideration of the chest wall volume receiving 30 Gy and the patient’s BMI and diabetic state.
doi:10.1016/j.ijrobp.2010.04.022
PMCID: PMC4093830  PMID: 20542388
Stereotactic Body Radiation Therapy; toxicity
9.  Aberrant Frequency of IL-10-Producing B Cells and Its Association with Treg/Th17 in Adult Primary Immune Thrombocytopenia Patients 
BioMed Research International  2014;2014:571302.
Background. Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients. Methods. Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19+IL-10+ B cells, CD3+CD4+IL-17+ Th17 cells, and CD4+CD25hiFoxp3+ Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR. Results. The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells. Conclusions. The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.
doi:10.1155/2014/571302
PMCID: PMC4098883  PMID: 25057496
10.  Late acyltransferase genes lpxX and lpxL jointly contribute to the biological activities of Moraxella catarrhalis 
Journal of Medical Microbiology  2013;62(Pt 6):807-812.
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.
doi:10.1099/jmm.0.056846-0
PMCID: PMC3709554  PMID: 23475908
11.  Use of a sensitive and robust UPLC-MS/MS method to determine the gender-dependent pharmacokinetics in rats of emodin and its glucuronide 
The purpose of this research was to set up a sensitive and consistent UPLC-UV and UPLCMS/MS method to analyze emodin and its glucuronidated metabolite, and to determine how gender differences affect its pharmacokinetic behaviors. In addition, a breast cancer resistance protein inhibitor dipyridamole was used to test how significant the absolute oral biovailabilty of emodin or its glucuronide is increased. A sensitive and fast UPLC-MS/MS method was successfully applied to determine emodin and its metabolite in male and female SD rat plasma. The absolute oral bioavailability of emodin was extremely low whether in male rats (7.5%) and female rats (5%). Following a single intravenous injection of 4 mg/kg emodin, the emodin plasma concentration-time data fit for a good two-compartment model either in male or female SD rats. The t1/2α were 13.26±6.28min (male rats) and 13.52±7.28min (female rats). The t1/2β were 187.38±0.16min (male rats) and 118.50±83.09min (female rats). Emodin showed significant gender differences in i.v. PK profiles with higher AUC values in male (422.71 ± 163.40 mg*μg/ml) than female (282.52 ± 98.42 mg*μg/ml) SD rats (n=6). Emodin glucuronide was suggested a good fit for single compartmental model for the plasma emodin metabolite concentrations. The t1/2Ke were 167.40±50.91min(male rats) and 251.31±114.20min (female rats), the area under the curve (AUC0-∞, i.v.) were 2210.02 ± 950.09 mg*μg/ml and 1054.42 ± 290.31 mg*μg/ml (female rats)(n=6). There was no good fit for any PK compartmental model for the plasma concentration-time data for single dose oral administration of emodin (8mg/kg) and its metabolite. Analyzing the oral PK data using non-compartmental model, Cmax, Tmax and AUC0-∞, p.o. of emodin in male rats were: 0.31±0.094 were μg/ml, 18.00±6.71min and 65.76±34.77 mg*μg/ml respectively; whereas Cmax, Tmax and AUC0-∞, p.o. of emodin in female rats were: 0.039±0.011 μg/ml, 18.75±7.51min and 33.82±4.09 mg*μg/ml respectively. The parameters of emodin glucuronide were significant different with emodin, the Cmax, Tmax and AUC0-∞, p.o of emodin glucuronide in male rats were 6.69±1.06 μg/ml, 240min and 2261.89±655.87 mg*μg/ml respectively, in female rats, the Cmax, Tmax and AUC0-∞, p.o. were 1.81±0.58 μg/ml, 60min and 458.50±373.29 mg*μg/ml respectively. The absolute bioavailability of emodin glucuronide was 60% (male rats) and 15% (female rats). The absolute bioavailability of emodin was no significant changed (7.3%) in male rats by using dipyridamole, the bioavailability of metabolite of emodin was significant declined to 14.6%.
doi:10.1016/j.jpba.2010.12.004
PMCID: PMC4010304  PMID: 21195574
emodin; absolute oral bioavailability; pharmacokinetics; UPLC-MS/MS; emodin
12.  A validated ultra-performance liquid chromatography–tandem mass spectrometry method for the quantification of polymyxin B in mouse serum and epithelial lining fluid: application to pharmacokinetic studies 
Objectives
A rapid, sensitive and robust ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of four major polymyxin B components (polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1) in serum and epithelial lining fluid (ELF) samples.
Methods
A Waters Acquity UPLC HSS C18 column was used with 0.1% formic acid in water/acetonitrile as mobile phases. Analysis was performed in a positive ionization mode with multiple-reactions monitoring scan type. Five percent trichloroacetic acid was used to precipitate proteins in biological samples and to increase the sensitivity of detection.
Results
Our results showed a linear concentration range of 0.0065–3.2 mg/L for all the major polymyxin B components in both serum and ELF, respectively; the interday variation was <10% and the accuracy was 88%–115%. The validated method was used to characterize the pharmacokinetics (serum and ELF) of polymyxin B in mice.
Conclusions
This is the first report, to date, examining the individual pharmacokinetics of various polymyxin B components in mice. Our results revealed no considerable differences in clearances among the components. The limited exposure of polymyxin B in ELF observed was consistent with the less favourable efficacy of polymyxin B reported for the treatment of pulmonary infections. This method can be used to further examine the pharmacokinetics of polymyxin B in a variety of clinical and experimental settings.
doi:10.1093/jac/dks536
PMCID: PMC3625435  PMID: 23341128
polymyxin; UPLC-MS/MS; biological samples
13.  The Transfer-Messenger RNA-Small Protein B System Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity 
Journal of Bacteriology  2013;195(22):5064-5071.
Extraintestinal pathogenic Escherichia coli (ExPEC) is capable of colonizing outside of the intestinal tract and evolving into a systemic infection. Avian pathogenic E. coli (APEC) is a member of the ExPEC group and causes avian colibacillosis. Transfer-mRNA-small protein B (tmRNA-SmpB)-mediated trans-translation is a bacterial translational control system that directs the modification and degradation of proteins, the biosynthesis of which has stalled or has been interrupted, facilitating the rescue of ribosomes stalled at the 3′ ends of defective mRNAs that lack a stop codon. We found that disruption of one, or both, of the smpB or ssrA genes significantly decreased the virulence of the APEC strain E058, as assessed by chicken infection assays. Furthermore, the mutants were obviously attenuated in colonization and persistence assays. The results of quantitative real-time reverse transcription-PCR analysis indicated that the transcription levels of the transcriptional regulation gene rfaH and the virulence genes kpsM, chuA, and iss were significantly decreased compared to those of the wild-type strain. Macrophage infection assays showed that the mutant strains reduced the replication and/or survival ability in the macrophage HD11 cell line compared to that of the parent strain, E058. However, no significant differences were observed in ingestion by macrophages and in chicken serum resistance between the mutant and the wild-type strains. These data indicate that the tmRNA-SmpB system is important in the pathogenesis of APEC O2 strain E058.
doi:10.1128/JB.00628-13
PMCID: PMC3811600  PMID: 24013628
14.  Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium 
Toxicological Sciences  2012;132(2):379-389.
Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5′-ACGTG-3′) exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10–100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)–treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at −387/−383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.
doi:10.1093/toxsci/kfs327
PMCID: PMC3595521  PMID: 23161664
lysyl oxidase; cobalt; cadmium; hypoxia; hypoxia-response element; hypoxia-inducible factor (HIF)-1α.
15.  Comparative risk of high-grade histopathology diagnosis following a CIN1 finding in endocervical curettage vs. cervical biopsy 
Objective
No evidence-based clinical management recommendations exist for women with an endocervical curettage (ECC) cervical intraepithelial neoplasia (CIN) grade 1 (CIN1) result when the concurrent cervical biopsy is not high-grade. For women with these pathology findings, we assessed their short-term risk of high-grade histopathology diagnosis in the Calgary Health Region where ECC was routinely performed.
Materials and Methods
We analyzed pathology and colposcopy reports from 1902 referral colposcopies where both ECC and biopsies were normal or CIN1. We calculated the short-term risk of CIN2 or more severe (CIN2+) detected 12–24 months after colposcopy. Pearson chi-square tests or Fisher’s exact tests were used to compare risks of a CIN2+ diagnosis between combinations of test results and strata of risk factors.
Results
The short-term risk of CIN2+ was the same following a CIN1 biopsy and CIN1 ECC (4.9% of 1389 vs. 5.0% of 359, respectively, P=.37). Compared to low-grade referral cytology, the risk of CIN2+ following high-grade cytology was elevated significantly for CIN1 ECC (13.3% vs. 3.3%, P<.01) and non-significantly for CIN1 biopsy (7.1% vs. 4.6%, P=.12).
Conclusions
Following low-grade cytology, the short-term risk of a high-grade histologic diagnosis in women with either CIN1 ECC or biopsy is equivalent, suggesting similar management. A CIN1 ECC may warrant different management in the context of high-grade referral cytology.
doi:10.1097/LGT.0b013e3182630c41
PMCID: PMC3608705  PMID: 23343702
cervical intraepithelial neoplasia; colposcopy; curettage; diagnosis; endocervical sampling
16.  CypA, a Gene Downstream of HIF-1α, Promotes the Development of PDAC 
PLoS ONE  2014;9(3):e92824.
Hypoxia-inducible factor-1α (HIF-1α) is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC). In response to tumor hypoxia, cyclophilin A (CypA) is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE) in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.
doi:10.1371/journal.pone.0092824
PMCID: PMC3963943  PMID: 24662981
17.  A New Strategy to Rapidly Evaluate Kinetics of Glucuronide Efflux by Breast Cancer Resistance Protein (BCRP/ABCG2) 
Pharmaceutical research  2012;29(11):3199-3208.
Purpose
The efflux transporter breast cancer resistance protein (BCRP/ABCG2) plays an important role in excretion of anionic drugs and metabolites including glucuronides in humans.
Methods
In this article, our recently published cell model (i.e., HeLa cells over-expressing UGT1A9 (HeLa1A9)) is used to determine the kinetic parameters of BCRP-mediated transport of glucuronides.
Results
After incubation of the aglycone with the cells, a steady-state (i.e., zero-order or near zero-order) excretion of its glucuronide is rapidly achieved and then maintained. Kinetic profiling with different (intracellular) glucuronide concentrations and their corresponding excretion rates is enabled by varying the concentration of the aglycone, which allows for the determination of kinetic parameters responsible for BCRP-mediated efflux of glucuronides. This approach was validated theoretically using a cellular pharmacokinetic model incorporating various enzymatic and transporter-mediated kinetic processes. It was also validated experimentally in that kinetic parameters of efflux of glucuronides of 6-hydroxyflavone and 4-methylumberiferone in the HeLa1A9 cell model were shown to be consistent with those derived with BCRP-overexpressing membrane vesicles.
Conclusion
This study provides a new strategy for rapidly evaluating the kinetics of glucuronide efflux by BCRP.
doi:10.1007/s11095-012-0817-2
PMCID: PMC3953450  PMID: 22752253
BCRP; efflux; glucuronidation; glucuronide; UGT1A9
18.  Peripheral dose measurements in cervical cancer radiotherapy: a comparison of volumetric modulated arc therapy and step-and-shoot IMRT techniques 
Purpose
The aim of this study was to investigate the peripheral doses resulting from volumetric modulated arc therapy (VMAT) and intensity modulated radiotherapy (IMRT) techniques in cervical cancer radiotherapy.
Methods
Nine patients with cervical cancer had treatment planned with both VMAT and IMRT. A specially designed phantom was used for this study, with ion chambers placed at interest points approximating the position of the breast, thyroid, and lens. The peripheral doses at the phantom interest points were measured and compared between the VMAT and IMRT techniques.
Results
VMAT provides a potential dosimetric advantage compared with IMRT. The mean (± standard deviation) peripheral dose to the breast point for 1 fraction (2 Gy) during VMAT measured 5.13 ± 0.96 mGy, compared with 9.04 ± 1.50 mGy for IMRT. At the thyroid and lens interest points, the mean (± standard deviation) peripheral dose during VMAT was 2.19 ± 0.33 and 2.16 ± 0.28 mGy, compared with 7.07 ± 0.76 and 6.97 ± 0.91 mGy for IMRT, respectively. VMAT reduced the monitor units used by 28% and shortened the treatment delivery time by 54% compared with IMRT.
Conclusion
While the dosimetric results are similar for both techniques, VMAT results in a lower peripheral dose to the patient and reduces the monitor-unit usage and treatment delivery time compared with IMRT.
doi:10.1186/1748-717X-9-61
PMCID: PMC3996072  PMID: 24555547
Cervical cancer; Volumetric modulated arc therapy; Intensity modulated radiation therapy; Peripheral dose
19.  A comparative study between Embosphere® and conventional transcatheter arterial chemoembolization for treatment of unresectable liver metastasis from GIST 
Objective
Transcatheter arterial chemoembolization (TACE) is a standard treatment for hepatocellular carcinoma (HCC) and/or some unresectable liver metastasis tumors. Hypervascular liver metastatic lesions such as metastasis from gastrointestinal stromal tumor (GIST) are an indication for transcatheter arterial embolization (TAE). The purpose of this study was to evaluate the efficacy and safety of Embosphere®-TAE (Embo-TAE) in comparison with conventional TACE (cTACE) for the treatment of liver metastasis from GIST.
Methods
A total of 45 patients who underwent TACE between Aug 2008 and Feb 2013 were enrolled. Patients with GIST who underwent TAE with Embosphere® (n=19) were compared with controls who received cTACE (n=26). The primary end points were treatment response and treatment-related adverse events. The secondary end points were progression-free survival (PFS) and overall survival (OS).
Results
The treatment response of Embo-TAE group was significantly higher than that of the cTACE group (P<0.001). The PFS was significantly better in the Embosphere®-group than in the cTACE group (56.6 and 42.1 weeks, respectively; P=0.003). However, there was no statistically significant difference in liver toxicity between the two groups (P>0.05). The median OS in the Embo-TAE group was longer than that in the cTACE group (74.0 weeks, 95% CI: 68.2-79.8 vs. 61.7 weeks, 95% CI: 56.2-67.2 weeks) (unadjusted P=0.045). The use of Embo-TAE significantly reduced the risk of death in patients with GIST with liver metastases according to the Cox proportional hazards regression model [hazard ratio (HR): 0.149; 95% CI: 0.064-0.475].
Conclusions
TAE with Embosphere® showed better treatment response and delayed tumor progression compared with cTACE. There was no significant difference in treatment-related hepatic toxicities. Embo-TAE thus appears to be a feasible and promising approach in the treatment of liver metastasis from GIST.
doi:10.3978/j.issn.1000-9604.2014.02.11
PMCID: PMC3937743  PMID: 24653635
Transcatheter arterial chemoembolization (TACE); gastrointestinal stromal tumor (GIST); embolization
20.  Testosterone regulation of Akt/mTORC1/FoxO3a Signaling in Skeletal Muscle 
Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C2C12 myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C2C12 myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt/mTOR signaling. In summary, androgen withdrawal decreases muscle myofibrillar protein synthesis through Akt/mTORC1 signaling, which is independent of AMPK activation, and readily reversible by anabolic steroid administration. Acute Akt activation in C2C12 myotubes is sensitive to a high concentration of testosterone, and low concentrations of testosterone can activate mTOR signaling independent of Akt.
doi:10.1016/j.mce.2012.10.019
PMCID: PMC3529800  PMID: 23116773
Muscle; Testosterone; raptor; Akt; mTOR; AMPK; FoxO; REDD1; MuRF1; atrophy; IGF-1; castration
21.  Single Domain SmCo5@Co Exchange-coupled Magnets Prepared from Core/shell Sm[Co(CN)6]·4H2O@GO Particles: A Novel Chemical Approach 
Scientific Reports  2013;3:3542.
SmCo5 based magnets with smaller size and larger maximum energy product have been long desired in various fields such as renewable energy technology, electronic industry and aerospace science. However, conventional relatively rough synthetic strategies will lead to either diminished magnetic properties or irregular morphology, which hindered their wide applications. In this article, we present a facile chemical approach to prepare 200 nm single domain SmCo5@Co core/shell magnets with coercivity of 20.7 kOe and saturation magnetization of 82 emu/g. We found that the incorporation of GO sheets is responsible for the generation of the unique structure. The single domain SmCo5 core contributes to the large coercivity of the magnets and the exchange-coupled Co shell enhances the magnetization. This method can be further utilized in the synthesis other Sm-Co based exchange-coupled magnets.
doi:10.1038/srep03542
PMCID: PMC3868969  PMID: 24356309
22.  RfaH Promotes the Ability of the Avian Pathogenic Escherichia coli O2 Strain E058 To Cause Avian Colibacillosis 
Journal of Bacteriology  2013;195(11):2474-2480.
Avian pathogenic Escherichia coli (APEC) infection causes avian colibacillosis, which refers to any localized or systemic infection, such as acute fatal septicemia or subacute pericarditis and airsacculitis. The RfaH transcriptional regulator in E. coli is known to regulate a number of phenotypic traits. The direct effect of RfaH on the virulence of APEC has not been investigated yet. Our results showed that the inactivation of rfaH significantly decreased the virulence of APEC E058. The attenuation was assessed by in vivo and in vitro assays, including chicken infection assays, an ingestion and intracellular survival assay, and a bactericidal assay with serum complement. The virulence phenotype was restored to resemble that of the wild type by complementation of the rfaH gene in trans. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the deletion of rfaH correlated with decreased virulence of the APEC E058 strain.
doi:10.1128/JB.02074-12
PMCID: PMC3676060  PMID: 23504015
23.  A Review on the Importance of Metals and Metalloids in Atmospheric Dust and Aerosol from Mining Operations 
Contaminants can be transported rapidly and over relatively long distances by atmospheric dust and aerosol relative to other media such as water, soil and biota; yet few studies have explicitly evaluated the environmental implications of this pathway, making it a fundamental but understudied transport mechanism. Although there are numerous natural and anthropogenic activities that can increase dust and aerosol emissions and contaminant levels in the environment, mining operations are notable with respect to the quantity of particulates generated, the global extent of area impacted, and the toxicity of contaminants associated with the emissions. Here we review (i) the environmental fate and transport of metals and metalloids in dust and aerosol from mining operations, (ii) current methodologies used to assess contaminant concentrations and particulate emissions, and (iii) the potential health and environmental risks associated with airborne contaminants from mining operations. The review evaluates future research priorities based on the available literature and suggest that there is a particular need to measure and understand the generation, fate and transport of airborne particulates from mining operations, specifically the finer particle fraction. More generally, our findings suggest that mining operations play an important but underappreciated role in the generation of contaminated atmospheric dust and aerosol and the transport of metal and metalloid contaminants, and highlight the need for further research in this area. The role of mining activities in the fate and transport of environmental contaminants may become increasingly important in the coming decades, as climate change and land use are projected to intensify, both of which can substantially increase the potential for dust emissions and transport.
doi:10.1016/j.scitotenv.2012.06.013
PMCID: PMC3418464  PMID: 22766428
Dust; Aerosol; Mining; Tailings; Metals and Metalloids; Arsenic and Lead
24.  Can Increases in the Cigarette Tax Rate be Linked to Cigarette Retail Prices? Solving mysteries related to the cigarette pricing mechanism in China 
Tobacco control  2011;21(6):560-562.
Objective
To explain China’s cigarette pricing mechanism and the role of the Chinese State Tobacco Monopoly Administration (STMA) on cigarette pricing and taxation.
Methods
Published government tobacco tax documentation and statistics published by the Chinese State Tobacco Monopoly Administration (STMA) are used to analyze the interrelations among industry profits, taxes, and retail price of cigarettes in China.
Results
The 2009 excise tax increase on cigarettes in China has not translated into higher retail prices because the Chinese STMA used its policy authority to ensure that retail cigarette prices did not change. The government tax increase is being collected at both the producer and wholesale levels. As a result, the 2009 excise tax increase in China has resulted in higher tax revenue for the government and lower profits for the tobacco industry, with no increase in the retail price of cigarettes for consumers.
Conclusions
Numerous studies have found that taxation is one of the most effective policy instruments for tobacco control. However, these findings come from countries that have market economies where market forces determine prices and influence how cigarette taxes are passed to the consumers in retail prices. China’s tobacco industry is not a market economy; therefore, nonmarket forces and the current Chinese tobacco monopoly system determine cigarette prices. The result is that tax increases do not necessarily get passed on to the retail price.
doi:10.1136/tobaccocontrol-2011-050027
PMCID: PMC3704211  PMID: 23076787
cigarette; pricing; taxes; China
25.  High-Density Three-Dimension Graphene Macroscopic Objects for High-Capacity Removal of Heavy Metal Ions 
Scientific Reports  2013;3:2125.
The chemical vapor deposition (CVD) fabrication of high-density three-dimension graphene macroscopic objects (3D-GMOs) with a relatively low porosity has not yet been realized, although they are desirable for applications in which high mechanical and electrical properties are required. Here, we explore a method to rapidly prepare the high-density 3D-GMOs using nickel chloride hexahydrate (NiCl2·6H2O) as a catalyst precursor by CVD process at atmospheric pressure. Further, the free-standing 3D-GMOs are employed as electrolytic electrodes to remove various heavy metal ions. The robust 3D structure, high conductivity (~12 S/cm) and large specific surface area (~560 m2/g) enable ultra-high electrical adsorption capacities (Cd2+ ~ 434 mg/g, Pb2+ ~ 882 mg/g, Ni2+ ~ 1,683 mg/g, Cu2+ ~ 3,820 mg/g) from aqueous solutions and fast desorption. The current work has significance in the studies of both the fabrication of high-density 3D-GMOs and the removal of heavy metal ions.
doi:10.1038/srep02125
PMCID: PMC3699809  PMID: 23821107

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