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1.  Genetic and Epigenetic Regulation in Age-related Macular Degeneration 
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the older population worldwide. While strong genetic risk factors have been associated with AMD etiology, environmental influences through epigenetic regulation are also likely to play a role. Recent advances in epigenetic studies have resulted in the development of numerous epigenetic drugs for the treatment of cancer and inflammation. Here, we review the current literature on the genetic and epigenetic mechanisms of AMD and suggest that understanding the cooperation of epigenetic and genetic mechanisms will greatly advance the clinical management of AMD.
PMCID: PMC3755470  PMID: 23997991
2.  Evaluation of a novel choanoid fluidized bed bioreactor for future bioartificial livers 
AIM: To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes.
METHODS: Encapsulated hepatocytes were placed in the constructed CFBB and circulated through Dulbecco’s Modified Eagle’s Medium (DMEM) for 12 h, and then through exchanged plasma for 6 h, and compared with encapsulated cells cultivated under static conditions in a spinner flask. Levels of alanine aminotransferase (ALT) and albumin were used to evaluate the CFBB during media circulation, whereas levels of ALT, total bilirubin (TBil), and albumin were used to evaluate it during plasma circulation. Mass transfer and hepatocyte injury were evaluated by comparing the results from the two experimental conditions. In addition, the viability and microstructure of encapsulated cells were observed in the different environments.
RESULTS: The bioartificial liver model based on a CFBB was verified by in vitro experiments. The viability of encapsulated cells accounting for 84.6% ± 3.7% in CFBB plasma perfusion was higher than the 74.8% ± 3.1% in the static culture group (P < 0.05) after 6 h. ALT release from cells was 29 ± 3.5 U/L vs 40.6 ± 3.2 U/L at 12 h (P < 0.01) in the CFBB medium circulation and static medium culture groups, respectively. Albumin secretion from cells was 234.2 ± 27.8 μg/1 × 107 cells vs 167.8 ± 29.3 μg/1 × 107 cells at 6 h (P < 0.01), 274.4 ± 34.6 μg/1 × 107 cells vs 208.4 ± 49.3 μg/1 × 107 cells (P < 0.05) at 12 h, in the two medium circulation/culture groups, respectively. Furthermore, ALT and TBil levels were 172.3 ± 24.1 U/L vs 236.3 ± 21.5 U/L (P < 0.05), 240.1 ± 23.9 μmol/L vs 241.9 ± 31.4 μmol/L (P > 0.05) at 6 h in the CFBB plasma perfusion and static plasma culture groups, respectively. There was no significant difference in albumin concentration between the two experimental plasma groups at any time point. The microstructure of the encapsulated hepatocytes remained healthier in the CFBB group compared with the static culture group after 6 h of plasma perfusion.
CONCLUSION: The CFBB can function as a bioartificial liver based on a bioreactor. The efficacy of this novel bioreactor is promising for the study of liver failure.
doi:10.3748/wjg.v20.i22.6869
PMCID: PMC4051926  PMID: 24944477
Choanoid; Fluidized bed; Bioreactor; Immortalized human hepatocytes; Bioartificial liver
4.  Distinguishing Nontuberculous Mycobacteria from Multidrug-Resistant Mycobacterium tuberculosis, China 
Emerging Infectious Diseases  2014;20(6):1060-1062.
doi:10.3201/eid2006.130700
PMCID: PMC4036753  PMID: 24856951
tuberculosis; Mycobacterium tuberculosis; nontuberculous Mycobacteria; multidrug-resistant Mycobacterium tuberculosis; China; tuberculosis and other mycobacteria; MDR TB
5.  Lophomonas blattarum infection presented as acute exacerbation of chronic obstructive pulmonary disease 
Journal of Thoracic Disease  2014;6(6):E73-E76.
The present case described a 70-year-old male who was initially diagnosed and treated as acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Ultimately Lophomonas blattarum (L. blattarum), a rare protozoan causing opportunistic infection, was found in suction sputum smear. Bronchoscopy showed a lot of purulent sputum in airways, diffusely swelling and friable mucus on bronchus. After single tinidazole treatment, symptoms and image showed marked improvement. It indicates though in the untraditional immunocompromised case, the suspect of opportunistic diseases is necessary, especially in the cases failed to improvement under empirical treatment. It also supports the tinidazole treatment is efficacy in L. blattarum infection.
doi:10.3978/j.issn.2072-1439.2014.03.40
PMCID: PMC4073364  PMID: 24977031
Lophomonas blattarum infection; chronic obstructive pulmonary disease (COPD)
6.  Overexpression of IL-17RC associated with ocular sarcoidosis 
Background
Sarcoidosis is a chronic inflammatory disease with a systemic granulomatous disorder affecting multiple organs including the eye. Both CD4+ T cell and macrophage have been linked to the pathogenesis of the disease.
Methods
The expression of IL-17RC was measured using FACS,immunohistochemistry and real-time PCR. Serum level of IL-17 was detected using ELISA.
Results
An elevated expression of IL-17RC on CD8+ T cells in peripheral blood was found in patients with ocular sarcoidosis as compared to healthy controls. Interestingly, we found a significant increase in the serum level of IL-17 in patients with ocular sarcoidosis as compared to healthy controls, which may be responsible for the induction of IL-17RC on CD8+ cells. In addition, IL-17RC appeared only in the retinal tissue of the patient with clinically active sarcoidosis.
Conclusions
Our results suggested a potential involvement of IL-17RC+CD8+ T cells in pathogenesis of ocular sarcoidosis.
doi:10.1186/1479-5876-12-152
PMCID: PMC4059456  PMID: 24885153
Ocular sarcoidosis; IL-17RC; CD8
7.  Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells 
The Scientific World Journal  2014;2014:480387.
Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.
doi:10.1155/2014/480387
PMCID: PMC4055124  PMID: 24971375
8.  Design Challenges and Guidelines for Persuasive Technologies that Facilitate Healthy Lifestyles 
Many diseases and health risks are the result of unhealthy lifestyles and technology could be used as an intervention. However, designing healthy lifestyle technologies is challenging, as the technology should be able to influence user behavior. In this case study, the design and evaluation process of a persuasive healthy lifestyle assistance technology was investigated. The iterative design and evaluation process included: contextual inquiry, storyboarding, concept generation, paper prototyping, video prototyping, interactive prototyping and user testing. Several design challenges are identified and guidelines are described for designing a technological intervention to encourage healthy lifestyles.
PMCID: PMC4028115  PMID: 24855501
Persuasive technology; case study; lifestyle; health; wellness
9.  Practising medicine in China: an unsolved dilemma 
doi:10.1258/jrsm.2011.100379
PMCID: PMC3089872  PMID: 21558101
10.  Death decoy receptor overexpression and increased malignancy risk in colorectal cancer 
AIM: To evaluate human epidermal growth factor receptor 2 (HER2) and death decoy receptor (DcR3) as colorectal cancer prognostic indicators.
METHODS: Colorectal carcinoma specimens from 300 patients were analyzed by immunohistochemistry to detect the staining patterns of HER2 and DcR3. Classification of HER2 staining was carried out using the United States Food and Drug Administration semi-quantitative scoring system, with scores of 0 or 1+ indicating a tumor-negative (normal expression) status and scores of 2+ and 3+ indicating a tumor-positive (overexpression) status. Classification of DcR3 was carried out by quantitating the percentage of positive cells within the stained section, with < 10% indicating a tumor-negative status and ≥ 10% indicating a tumor-positive status. Correlation of the HER2 and DcR3 staining status with clinicopathological parameters [age, sex, tumor size, differentiation, and the tumor, node, metastasis (pTNM) classification] and survival was statistically assessed.
RESULTS: Tumor-positive status for HER2 and DcR3 was found in 18.33% and 58.33% of the 300 colorectal carcinoma specimens, respectively. HER2 tumor-positive status showed a significant correlation with tumor size (P = 0.003) but not with other clinicopathological parameters. DcR3 tumor-positive status showed a significant correlation with tumor differentiation (P < 0.001), pTNM stage (P < 0.001), and lymph node metastasis (P < 0.001). However, correlation coefficient analysis did not indicate that a statistically significant correlation exists between tumor-positive status for the HER2 and DcR3 overexpression (P = 0.236). Patients with specimens classified as DcR3-overexpressing had a significantly worse overall survival (OS) rate than those without DcR3 overexpression (median OS: 42.11 vs 61.21 mo; HR = 50.27, 95%CI: 44.90-55.64, P < 0.001). HER2 overexpression had no significant impact on median OS (35.10 mo vs 45.25 mo; HR = 44.40, 95%CI: 39.32-49.48, P = 0.344). However, patients with specimens classified as both HER2- and DcR3-overexpressing had a significantly poorer median OS than those with only HER2 overexpression (31.80 mo vs 52.20 mo; HR = 35.10, 95%CI: 22.04-48.16, P = 0.006).
CONCLUSION: HER2 overexpression is not an independent prognostic marker of colorectal cancer, but DcR3 overexpression is highly correlated with lymph node metastasis and poor OS.
doi:10.3748/wjg.v20.i15.4440
PMCID: PMC3989983  PMID: 24764685
Colorectal carcinoma; Human epidermal growth factor receptor 2; Death decoy receptor; Immunohistochemistry; Prognosis
11.  Transcriptome Analysis of the Portunus trituberculatus: De Novo Assembly, Growth-Related Gene Identification and Marker Discovery 
PLoS ONE  2014;9(4):e94055.
Background
The swimming crab, Portunus trituberculatus, is an important farmed species in China, has been attracting extensive studies, which require more and more genome background knowledge. To date, the sequencing of its whole genome is unavailable and transcriptomic information is also scarce for this species. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for major tissues of Portunus trituberculatus by the Illumina paired-end sequencing technology.
Results
Total RNA was isolated from eyestalk, gill, heart, hepatopancreas and muscle. Equal quantities of RNA from each tissue were pooled to construct a cDNA library. Using the Illumina paired-end sequencing technology, we generated a total of 120,137 transcripts with an average length of 1037 bp. Further assembly analysis showed that all contigs contributed to 87,100 unigenes, of these, 16,029 unigenes (18.40% of the total) can be matched in the GenBank non-redundant database. Potential genes and their functions were predicted by GO, KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literature, many putative genes with fundamental roles in growth and muscle development, including actin, myosin, tropomyosin, troponin and other potentially important candidate genes were identified for the first time in this specie. Furthermore, 22,673 SSRs and 66,191 high-confidence SNPs were identified in this EST dataset.
Conclusion
The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in Portunus trituberculatus. The data will also instruct future functional studies to manipulate or select for genes influencing growth that should find practical applications in aquaculture breeding programs. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will be essential for accelerating aquaculture breeding programs with this species.
doi:10.1371/journal.pone.0094055
PMCID: PMC3983128  PMID: 24722690
12.  Prognostic impact of gastrointestinal bleeding and expression of PTEN and Ki-67 on primary gastrointestinal stromal tumors 
Background
Prognostic indicators for gastrointestinal stromal tumors (GISTs) are under investigation. The latest risk classification criteria may still have room for improvement. This study aims to investigate prognostic factors for primary GISTs from three aspects, including clinicopathological parameters, immunohistochemical (IHC) expression of PTEN, and Ki-67 labeling index (LI), and attempts to find valuable predictors for the malignancy potential of primary GISTs.
Methods
Tumor samples and clinicopathological data from 84 patients with primary GISTs after R0 resection were obtained. Immunohistochemical analysis was performed based on tissue microarray (TMA) to estimate expression of PTEN and Ki-67 in tumor cells.
Results
The cut-off point of Ki-67 LI was determined as 1%, using a receiver operator characteristic test with a sensitivity of 71.7% and a specificity of 64.5%. Univariate analysis demonstrated the following factors as poor prognostic indicators for relapse-free survival (RFS) against a median follow-up of 40.25 months: gastrointestinal (GI) bleeding (P = 0.009), non-gastric tumor location (P = 0.001), large tumor size (P = 0.022), high mitotic index (P < 0.001), high cellularity (P = 0.012), tumor rupture (P = 0.013), absent or low expression of PTEN (P = 0.036), and Ki-67 LI >1% (P = 0.043). Gastrointestinal bleeding (hazard ratio, 3.85; 95% confidence interval, 1.63 to 9.10; P = 0.002) was a negative independent risk predictor in multivariate analysis, in addition to tumor size (P = 0.023), and mitotic index (P = 0.002). In addition, GI bleeding showed a good ability to predict recurrence potential, when included in our re-modified risk stratification criteria.
Conclusions
This study suggests that GI bleeding is an independent predictor of poor prognosis for RFS in primary GISTs. Expression of PTEN and Ki-67 are correlated with high risk potential and may predict early recurrence in univariate analysis.
doi:10.1186/1477-7819-12-89
PMCID: PMC3991912  PMID: 24712384
gastrointestinal bleeding; gastrointestinal stromal tumors; immunohistochemistry; Ki-67 labeling index; prognosis; PTEN; tissue microarray
13.  Synthetic microRNAs Cassette Dosing: Pharmacokinetics, Tissue Distribution and Bioactivity 
Molecular pharmaceutics  2012;9(6):1638-1644.
MicroRNAs (miRs) are deregulated in cancer and leukemia. Restoring aberrantly downregulated tumor suppressor miRs or antagonizing overexpressed oncogenic miRs in malignant cells by synthetic RNA oligonucleotides represents a potentially novel therapeutic approach in cancer and leukemia. However, given the complex networking and concurrent deregulation of miRs in malignant cells, an effective approach may require concurrent targeting of multiple miRs. Cassette dosing involves simultaneous administration of a mixture of oligonucleotides from same or different structural classes. However, information on cassette dosing pharmacokinetics, tissue distribution and bioactivity of synthetic miRs is lacking. In this study, three synthetic 2’-methoxyphosphorothioate-miRs (2’-MeOPSmiR16-1, 2’-MeOPSmiR29b and 2’-MeOPSantagomiR155) were administered i.v. to C57BL/6 mice as a mixture, each at 7.5mg/kg. Analysis of concentrations of individual miR in plasma and major organ tissues (bone marrow, spleen, liver, brain, heart, kidney and lung) was performed. The mRNA and protein levels of miR’s bio-targets were monitored sequentially after dosing up to 24 hours. Our results demonstrated that these synthetic miRs retain their different individual pharmacokinetic properties and all display three-compartmental pharmacokinetics. 2’-MeOPSmiR16-1 has the longest plasma gamma half-life of 2508 minutes and lowest total body clearance of 0.0054 L/min*kg, whereas 2’-MeOPSmiR29b has the shortest gamma half-life of 510.6 minutes and highest total body clearance of 0.042 L/min*kg. The tissue concentrations of all three 2’-MeOPS-modified miR(s)/antagomiR were measurable from 5 minutes to at least 24 hours after dosing, indicating that these concurrently delivered oligonucleotides can reach organ tissues. Importantly, there were biological activities of the concurrently administered miRs which persisted, as shown by the downregulation of specific targets in tested tissues, albeit with variations. Brain was one of the most sensitive tissues with respect to downregulation of mRNA and protein levels of four measured bio-targets (e.g. Bcl-2, Mcl-1, DNMT3a and DNMT3b) despite its relatively low miR/antagomiRs levels. We conclude that cassette dosing is applicable to 2’-MeOPS-modified synthetic miRs that are tissue-deliverable and biofunctional without any additional formulation requirement. This study supports future exploration of miR-involved combination therapies.
doi:10.1021/mp2006483
PMCID: PMC3977775  PMID: 22574727
2’-MeOPSmiRs; cassette dosing; pharmacokinetics; tissue distribution; pharmacodynamic
14.  The SLC4 Family of Bicarbonate (HCO3−) Transporters 
Molecular aspects of medicine  2013;34(2-3):159-182.
The SLC4 family consists of ten genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots—consistent with 10 – 14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3− (or a related species, such as CO3=) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1 – 3) and five Na+-coupled HCO3− transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na+ - coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na+-coupled HCO3− transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H+ or HCO3− by several epithelia, as well as the regulation of cell volume and intracellular pH.
doi:10.1016/j.mam.2012.10.008
PMCID: PMC3605756  PMID: 23506864
SLC4; Bicarbonate; carbonate; chloride; sodium; boron; exchanger; cotransporter
15.  Prognostic value of KIT/PDGFRA mutations in gastrointestinal stromal tumors: a meta-analysis 
Background
The postulated relationship between KIT/PDGFRA mutations and their prognostic value in gastrointestinal stromal tumors (GISTs) has generated intense attention during the past decade, despite the fact that a great deal of studies have been conducted on this subject. To provide a strong quantitative estimate of this postulated relationship, we carried out a meta-analysis which combined, compared, and summarized the results of existing relevant studies.
Methods
Studies were identified by searching databases and reviewing citations in relevant articles. Of 48 potentially relevant studies, we combined individual patient data from 18 studies which involved 1,487 patients with GISTs, by which we made a comparison between the positive KIT mutation subgroup and the negative KIT mutation subgroup (PDGFRA mutation and wild type). We tabulated and analyzed the patient characteristics from each study, including general information such as age and gender, histopathological parameters, and clinical follow-up outcomes.
Results
KIT mutations, compared with PDGFRA mutations and wild type, showed a marked increased risk not only for tumor size (>5 cm) but also for higher mitotic activity (>5), suggesting that KIT mutations significantly correlated with the National Comprehensive Cancer Network (NCCN) high risk or National Institutes of Health (NIH) high risk (1.74 (95% CI, 1.20 to 2.53) and 2.00 (95% CI, 1.08 to 3.68), respectively). Moreover, higher recurrence and metastasis was observed in GISTs with KIT mutations, revealing its closer correlation with clinical malignant risk (P <0.001 for each, with odds ratio (OR) of 2.06 (95%, 1.37 to 3.11) and 2.77 (95%, 1.64 to 4.67), respectively). High risk or malignant GISTs with KIT mutations had a significantly poorer prognosis, as measured by 3-year overall survival, compared to those with PDGFRA mutations and wild type (0.47 (95% CI, 0.25 to 0.90)).
Conclusions
KIT mutations, compared with PDGFRA mutations and wild type, represent a poorer prognostic marker in high risk or malignant GISTs.
doi:10.1186/1477-7819-12-71
PMCID: PMC4046004  PMID: 24674052
Gastrointestinal stromal tumors; KIT; PDGFRA; Prognosis; Meta-analysis
16.  Betel Chewing and Arecoline Affects Eotaxin-1, Asthma and Lung Function 
PLoS ONE  2014;9(3):e91889.
Background
Betel nut is commonly used in many countries. Despite evidence suggesting an association with asthma, few studies have investigated the connection between betel nut use and asthma; thus, the underlying mechanism for the association with asthma is also unclear. The aim of this study was to investigate the association between betel chewing and asthma as well as the associations of plasma arecoline (a biomarker for exposure) and eotaxin-1 (a potential mediator) with asthma and lung function.
Methods
We recruited 600 hospital-based asthmatic patients and 1200 age- and gender-matched community controls in southern Taiwan. To clarify the mechanism of action for eotaxin-1 in the association between betel chewing and asthma, we also designed an in vitro experiment to study the functional associations between arecoline exposure and eotaxin-1 levels.
Results
A significant association was found between asthma and current betel chewing (adjusted odds ratio 2.05, 95% CI = 1.12–3.76), which was independent of potential confounders but was attenuated following adjustment for eotaxin-1. Arecoline and eotaxin-1 levels were positively correlated (Spearman r = 0.303, p = 0.02), while arecoline and arecaidine were negatively correlated with lung function. Functionally, arecoline alone does not induce eotaxin-1 release in vitro from dermal and gingival fibroblasts. However, in the presence of IL-4 and TNF-alpha, arecoline at 100 μg/ml induced more eotaxin-1 release than arecoline at 0 μg/ml (2700±98 pg/ml vs 1850±142 pg/ml, p = 0.01 in dermal fibroblast cells, and 1489±78 pg/ml vs 1044±95 pg/ml, p = 0.03 in gingival fibroblast cells, respectively).
Conclusion
Betel chewing is associated with asthma in this population, with arecoline induction of eotaxin-1 supported as a plausible causal pathway.
doi:10.1371/journal.pone.0091889
PMCID: PMC3962362  PMID: 24658613
17.  Identification of a 24-Gene Prognostic Signature That Improves the European LeukemiaNet Risk Classification of Acute Myeloid Leukemia: An International Collaborative Study 
Journal of Clinical Oncology  2013;31(9):1172-1181.
Purpose
To identify a robust prognostic gene expression signature as an independent predictor of survival of patients with acute myeloid leukemia (AML) and use it to improve established risk classification.
Patients and Methods
Four independent sets totaling 499 patients with AML carrying various cytogenetic and molecular abnormalities were used as training sets. Two independent patient sets composed of 825 patients were used as validation sets. Notably, patients from different sets were treated with different protocols, and their gene expression profiles were derived using different microarray platforms. Cox regression and Kaplan-Meier methods were used for survival analyses.
Results
A prognostic signature composed of 24 genes was derived from a meta-analysis of Cox regression values of each gene across the four training sets. In multivariable models, a higher sum value of the 24-gene signature was an independent predictor of shorter overall (OS) and event-free survival (EFS) in both training and validation sets (P < .01). Moreover, this signature could substantially improve the European LeukemiaNet (ELN) risk classification of AML, and patients in three new risk groups classified by the integrated risk classification showed significantly (P < .001) distinct OS and EFS.
Conclusion
Despite different treatment protocols applied to patients and use of different microarray platforms for expression profiling, a common prognostic gene signature was identified as an independent predictor of survival of patients with AML. The integrated risk classification incorporating this gene signature provides a better framework for risk stratification and outcome prediction than the ELN classification.
doi:10.1200/JCO.2012.44.3184
PMCID: PMC3595425  PMID: 23382473
18.  LC–MS/MS quantification of a neuropeptide fragment kisspeptin-10 (NSC 741805) and characterization of its decomposition product and pharmacokinetics in rats 
The kisspeptins are critical regulators of mammalian reproduction. Kisspeptin-10 (45YNWNSFGLRF-NH254, kisspeptin-112–121 or metastin 45–54, NSC 741805), an active fragment of kisspeptin, has been shown to be a potent stimulator of gonadotropin-releasing hormone and secretion of luteinizing hormone in both rodents and primates. This shorter peptide fragment may have clinical utility potential and it is important to characterize its pharmacokinetic property. Recently, the pharmacokinetics of both kisspeptin-54 and kisspeptin-10 were characterized in humans using a radioimmunoassay (RIA), which measures only the immunoreactive kisspeptin (kisspeptin-IR). In this study, a highly sensitive and specific LC–MS/MS assay was developed to quantify kisspeptin-10 levels in rat plasma. The lower limit of quantitation (LLOQ) was 0.5 ng/mL, the within-day and between-day coefficient of variations (CVs) ranged from 5.2 to 15.4% and 1.3 to 14.2%, and the accuracy values ranged from 98 to 114% and 99 to 105%, respectively. With this method, stability studies demonstrated that kisspeptin-10 degraded rapidly with decomposition half-lives of 6.8 min, 2.9 min and 1.7 min at 4 °C, 25 °C, and 37 °C, respectively. The principal decomposition product was characterized as the N-terminal tyrosine deleted kisspeptin-10 46NWDSFGLRF-NH254. Pharmacokinetic study in rats showed that low ng/mL kisspeptin-10 was detected in the first few minutes, and eliminated rapidly and became undetectable 30 min after intravenous (i.v.) bolus administration of 1.0 mg/kg kisspeptin-10.
doi:10.1016/j.jchromb.2013.02.027
PMCID: PMC3955120  PMID: 23524040
Kisspeptin-10; LC–MS/MS; Pharmacokinetics; Metabolites
19.  Deregulation of COMMD1 Is Associated with Poor Prognosis in Diffuse Large B-cell Lymphoma 
PLoS ONE  2014;9(3):e91031.
Background
Despite improved survival for the patients with diffuse large B-cell lymphoma (DLBCL), the prognosis after relapse is poor. The aim was to identify molecular events that contribute to relapse and treatment resistance in DLBCL.
Methods
We analysed 51 prospectively collected pretreatment tumour samples from clinically high risk patients treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis with high resolution array comparative genomic hybridization (aCGH) and gene expression microarrays. Major finding was validated at the protein level immunohistochemically in a trial specific tissue microarray series of 70, and in an independent validation series of 146 patients.
Results
We identified 31 genes whose expression changes were strongly associated with copy number aberrations. In addition, gains of chromosomes 2p15 and 18q12.2 were associated with unfavourable survival. The 2p15 aberration harboured COMMD1 gene, whose expression had a significant adverse prognostic impact on survival. Immunohistochemical analysis of COMMD1 expression in two series confirmed the association of COMMD1 expression with poor prognosis.
Conclusion
COMMD1 is a potential novel prognostic factor in DLBCLs. The results highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL.
Trial Registration
ClinicalTrials.gov NCT01502982
doi:10.1371/journal.pone.0091031
PMCID: PMC3953211  PMID: 24625556
20.  Identification of a Suppressor Mutation That Improves the Yields of Hexon-Modified Adenovirus Vectors 
Journal of Virology  2013;87(17):9661-9671.
We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.
doi:10.1128/JVI.00462-13
PMCID: PMC3754123  PMID: 23824800
21.  Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX 
Nature structural & molecular biology  2012;19(12):1287-1292.
Mammalian H3.3 is a variant of the canonical histone H3.1 essential for genome reprogramming in the fertilized eggs and maintenance of chromatin structure in neuronal cells. An H3.3-specific histone chaperone, DAXX, directs the deposition of H3.3 onto pericentric and telomeric heterochromatin. H3.3 differs from H3.1 by only five amino acids, yet DAXX can distinguish the two with high precision. By a combination of structural, biochemical and cell-based targeting analyses, here we show that Ala87 and Gly90 are the principal determinants of H3.3 specificity. DAXX uses a shallow hydrophobic pocket to accommodate the small, hydrophobic Ala87 of H3.3, whereas a polar binding environment in DAXX prefers Gly90 in H3.3 over the hydrophobic Met90 in H3.1. An H3.3-H4 heterodimer is bound by the histone-binding domain of DAXX, which makes extensive contacts with both H3.3 and H4.
doi:10.1038/nsmb.2439
PMCID: PMC3932182  PMID: 23142979
22.  Dual Effects of Cigarette Smoke Extract on Proliferation of Endothelial Progenitor Cells and the Protective Effect of 5-aza-2′-deoxycytidine on EPCs against the Damage Caused by CSE 
BioMed Research International  2014;2014:640752.
Cigarette smoke is a major public health problem associated with multitude of diseases, including pulmonary and vascular diseases. Endothelial progenitor cells (EPCs) contribute to neovascularization and play an important role in the development of these diseases. The effect of CSE on EPCs is seldom studied. The aim of the current study is to observe the effect of CSE on biological behavior of EPCs and, further, to search for potential candidate agent in protection of proliferation of EPCs against the damage caused by CSE exposure in vitro. Methods. The proliferations of EPCs isolated from bone marrow of C57BL/6J mice were assessed by MTT after incubating the EPCs with a series of concentrations of CSE (1.0%, 2.5%, 5.0%, and 10.0%) for different times (3, 6, and 24 hours) as well as with 1.0% CSE in presence of 5-AZA-CdR for 24 hours. Results. The proliferations of EPCs were significantly enhanced after 3 hours of exposure to concentrations of 1.0% and 2.5% CSE but depressed when exposed to concentrations of 5.0% and 10.0% CSE. Furthermore, the 5-AZA-CdR in concentrations of 2.0 μmol/L and 5.0 μmol/L partly protected against the depression of proliferation of EPCs caused by CSE exposure. Conclusions. The CSE showed dual effects on proliferation of EPCs isolated from mice. The 5-AZA-CdR partly protected the proliferation of EPCs against the damage caused by CSE exposure in vitro, suggesting that DNA methylation may be involved in the dysfunction of EPCs induced by CSE.
doi:10.1155/2014/640752
PMCID: PMC3947928  PMID: 24696861
23.  Clinicopathological findings in a case series of abdominopelvic solitary fibrous tumors 
Oncology Letters  2014;7(4):1067-1072.
Solitary fibrous tumors (SFTs) represent a rare type of soft tissue tumor. Extrathoracic SFTs (ESFTs) in the soft tissues of the abdominopelvic cavity are extremely rare. Between January 2002 and January 2013, 10 patients were identified with abdominopelvic SFTs at the Northern Jiangsu People’s Hospital. The clinicopathological data, treatment and follow-up results were retrospectively analyzed in this study. Patients included four females and six males, whose age ranged between 21 and 75 years (mean, 53.3 years). The maximum diameter of the tumors was 2.5–28 cm (mean, 12.7 cm). Two cases were diagnosed as malignant variants of ESFTs. R0 resection was performed in eight patients, while one patient underwent R1 resection, and one patient received palliative chemotherapy for an inoperable mass. Follow-up time ranged between 6 and 126 months (mean, 50 months). The patient with R1 resection suffered a local relapse, and the patient receiving palliative chemotherapy succumbed to the disease. The remaining eight patients remained free of disease. Abdominopelvic SFTs usually reveal an indolent process, although the majority of tumors in the present study were of giant size when diagnosed. The risk of local recurrence and metastasis correlates with tumor size and the histological status of surgical margins. The preferred treatment is complete resection followed by extended follow-up surveillance.
doi:10.3892/ol.2014.1872
PMCID: PMC3961353  PMID: 24944670
solitary fibrous tumor; spindle cell tumor; histopathology; immunohistochemistry
24.  Inhibition of Herpes Simplex Virus type 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate 
Green tea polyphenol epigallocatechin gallate (EGCG) is a strong anti-oxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for Herpes Simplex Virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of Herpes Simplex Virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 µM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.
doi:10.1016/j.fct.2012.11.006
PMCID: PMC3703635  PMID: 23182741
HSV-1; Vero cells; EGCG; palmitoyl-EGCG
25.  Sp1-mediated microRNA-182 expression regulates lung cancer progression 
Oncotarget  2014;5(3):740-753.
Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells, while represses metastasis. In this study, we found that the transcriptional activity of FOXO3 was increased, but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182, which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth, but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect, suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9, CDH9 and CD44 was increased following miR-182 knockdown. In conclusion, in the early stages of lung cancer progression, Sp1 stimulates miR-182 expression, which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages, Sp1 and miR-182 decline, thus increasing FOXO3 expression, which leads to lung metastasis.
PMCID: PMC3996653  PMID: 24519909
Sp1; miR-182; FOXO3; Lung cancer

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