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1.  Association between Family History Risk Categories and Prevalence of Diabetes in Chinese Population 
PLoS ONE  2015;10(2):e0117044.
To investigate the association between different family history risk categories and prevalence of diabetes in the Chinese population.
The family history of diabetes was obtained from each subject, and an oral glucose tolerance test was performed for measuring the fasting and postload glucose and insulin levels based on a national representative cross-sectional survey of 46,239 individuals (age ≥ 20 years) in the 2007–2008 China National Diabetes and Metabolism Disorders Study. The family history risk categories of diabetes were high, moderate, and average (FH2 and FH1: at least two generations and one generation of first-degree relatives with diabetes, respectively; FH0: no first-degree relatives with diabetes).
The age- and gender-adjusted prevalence rates of diabetes were 32.7% (95% confidence interval (CI): 26.4–39.7%) in FH2, 20.1% (95% CI: 18.2–22.1%) in FH1, and 8.4% (95% CI: 7.9–8.9%) in FH0 (P < 0.0001). The calculated homeostatic model assessment-estimated insulin resistance (HOMA-IR), Matsuda insulin sensitivity index (ISI), and insulinogenic index (ΔI30/ΔG30) values showed significant trending changes among the three risk categories, with the most negative effects in FH2. Multivariate logistic regression analysis showed that the odds ratios of having diabetes were 6.16 (95% CI: 4.46–8.50) and 2.86 (95% CI: 2.41–3.39) times higher in FH2 and FH1, respectively, than in FH0 after adjustment for classical risk factors for diabetes.
Family history risk categories of diabetes have a significant, independent, and graded association with the prevalence of this disease in the Chinese population.
PMCID: PMC4321835  PMID: 25664814
2.  Early B-cell factor 3 (EBF3) is a novel tumor suppressor gene with promoter hypermethylation in pediatric acute myeloid leukemia 
Pediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear.
Twelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis.
EBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells.
In this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details.
Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.
PMCID: PMC4311429  PMID: 25609158
Early B-cell factor 3; Pediatric acute myeloid leukemia; Methylation; Tumor suppressor; Real-time PCR array
3.  Relation of Hypothyroidism and Incident Atrial Fibrillation (from the Framingham Heart Study) 
American heart journal  2013;167(1):10.1016/j.ahj.2013.10.012.
Hyperthyroidism has a well-described association with atrial fibrillation (AF). However, the relation of hypothyroidism to AF has had limited investigation. Hypothyroidism is associated with cardiovascular risk factors, subclinical cardiovascular disease and overt cardiovascular disease, all of which predispose to AF. We investigated 10-year incidence of AF in a community-dwelling cohort.
Among 6,653 Framingham heart Study participants, 5,069 participants, 52% woman, mean age 57±12, were eligible after excluding those with missing thyroid stimulating hormone (TSH), TSH <0.45 μU/L (hyperthyroid), TSH >19.9 μU/L or prevalent AF. TSH was categorized by range (≥0.45 to <4.5, 4.5 to <10.0, 10.0 to ≤19.9 μU/L) and by quartiles. We examined the associations between TSH and 10-year risk of AF using multivariable-adjusted Cox proportional hazards analysis.
Over 10-year follow-up, we observed 277 cases of incident AF. A 1-standard deviation (SD) increase in TSH was not associated with increased risk of AF (hazard ratio 1.01, 95% confidence interval 0.90 to 1.14, p=0.83). In categorical analysis, employing TSH ≥0.45 to <4.5 μU/L as the referent (equivalent to euthyroid state), we found no significant association between hypothyroidism and 10-year AF risk. Comparing the highest (2.6
In conclusion, we did not identify a significant association between hypothyroidism and 10-year risk of incident AF in a community-based study.
PMCID: PMC3868014  PMID: 24332151
Atrial fibrillation; hypothyroidism; risk factors; cohort study
Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.
PMCID: PMC4307303  PMID: 25574601
RO3280; pediatric acute myeloid leukemia (AML); polo-like kinase 1 (PLK1); apoptosis; oncogene target
Scientific Reports  2014;4:7167.
Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.
PMCID: PMC4241513  PMID: 25418599
Papillary thyroid cancer (PTC) is the most common epithelial thyroid tumor, accounting for more than 80% of all thyroid cancers. Although PTC shows an indolent character and excellent prognosis, patients with aggressive characteristics are more likely to have a disease recurrence and die in the end. The aim of this study was to analyze BRAFV600E mutation and methylation levels of CpG sites in the promoters of CDH1, DAPK, RARβ and RUNX3 genes in a cohort of PTCs, and investigate their association with tumor recurrence. In this study, we used pyrosequencing method to individually quantified methylation levels at multiple CpG sites within each gene promoter, and detect BRAFV600E mutation in 120 PTCs and 23 goiter tissues as normal control. Moreover, appropriate cut-off values for each CpG site were set up to predict disease recurrence. Our data showed that overall average methylation levels of CDH1 and RUNX3 genes were significantly higher in PTCs than that in control subjects. Conversely, overall average methylation levels of DAPK promoter were significantly lower in PTCs than that in control subjects. Moreover, BRAFV600E mutation and overall average methylation levels of all these genes were not significant difference between recurrent and non-recurrent cases. However, we found that hypermethylation of RUNX3 at CpG sites -1397, -1406, -1415 and -1417 significantly increased the risk of of disease recurrence by using appropriate site-specific cut-off values. Collectively, our findings suggest RUNX3 site-specific hypermethylation may offer value in predicting or monitoring postoperative recurrence of PTC patients.
PMCID: PMC4266707  PMID: 25520863
Papillary thyroid cancer (PTC); tumor recurrence; RUNX3; promoter hypermethylation; pyrosequencing
PLoS ONE  2014;9(11):e112626.
Antibiotic-resistant bacteria and genes are recognized as new environmental pollutants that warrant special concern. There were few reports on veterinary antibiotic-resistant bacteria and genes in China. This work systematically analyzed the prevalence and distribution of sulfonamide resistance genes in soils from the environments around poultry and livestock farms in Jiangsu Province, Southeastern China. The results showed that the animal manure application made the spread and abundance of antibiotic resistance genes (ARGs) increasingly in the soil. The frequency of sulfonamide resistance genes was sul1 > sul2 > sul3 in pig-manured soil DNA and sul2 > sul1 > sul3 in chicken-manured soil DNA. Further analysis suggested that the frequency distribution of the sul genes in the genomic DNA and plasmids of the SR isolates from manured soil was sul2 > sul1 > sul3 overall (p<0.05). The combination of sul1 and sul2 was the most frequent, and the co-existence of sul1 and sul3 was not found either in the genomic DNA or plasmids. The sample type, animal type and sampling time can influence the prevalence and distribution pattern of sulfonamide resistance genes. The present study also indicated that Bacillus, Pseudomonas and Shigella were the most prevalent sul-positive genera in the soil, suggesting a potential human health risk. The above results could be important in the evaluation of antibiotic-resistant bacteria and genes from manure as sources of agricultural soil pollution; the results also demonstrate the necessity and urgency of the regulation and supervision of veterinary antibiotics in China.
PMCID: PMC4236111  PMID: 25405870
PLoS ONE  2014;9(11):e111737.
A monoclonal antibody (McAb) against non-structural protein (NSP) 3B of foot-mouth-disease virus (FMDV) (3B4B1) was generated and shown to recognize a conserved epitope spanning amino acids 24–32 of 3B (GPYAGPMER) by peptide screening ELISA. This epitope was further shown to be a unique and predominant B cell epitope in 3B2, as sera from animals infected with different serotypes of FMDV blocked the ability of McAb 3B4B1 to bind to NSP 2C3AB. Also, a polyclonal antibody against NSP 2C was produced in a rabbit vaccinated with 2C epitope regions expressed in E. coli. Using McAb 3B4B1 and the 2C polyclonal antibody, a solid-phase blocking ELISA (SPB-ELISA) was developed for the detection of antibodies against NSP 2C3AB to distinguish FMDV-infected from vaccinated animals (DIVA test). The parameters for this SPB-ELISA were established by screening panels of sera of different origins. Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal. This test showed a similar performance as the commercially available PrioCHECK NS ELISA. This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.
PMCID: PMC4219772  PMID: 25369323
Molecular Medicine Reports  2014;11(1):121-126.
Intrahepatic T helper (Th)17 cytokine and serum interleukin (IL)-17 levels in patients with hepatitis B are positively correlated with the progression of liver cirrhosis (LC). IL-35 can significantly inhibit the differentiation of Th17 cells and the synthesis of IL-17. The present study aimed to investigate the function and expression of IL-17 and IL-35 in the blood of patients with hepatitis B-related LC. The levels of IL-17 and IL-35 in the peripheral blood of 30 patients with chronic hepatitis B (CHB), 79 with LC, 14 with chronic severe hepatitis B (CSHB), and 20 normal controls were detected by ELISA. Quantitative polymerase chain reaction was used to evaluate Epstein-Barr virus-induced gene 3 (EBI3), forkhead box (FOX)P3 and IL-17 mRNA expression levels in peripheral blood mononuclear cells (PBMCs). Western blotting was used to determine protein expression. The liver function of patients and normal controls was measured. EBI3, IL-17 and FOXP3 mRNA expression levels in PBMCs from patients with LC, CHB and CSHB were higher than those in cells from the controls. IL-17 mRNA levels differed significantly according to the Child-Pugh classification and exhibited an upward trend over time in contrast to a downward trend for EBI3 and FOXP3 mRNA. The changes in protein expression in the peripheral blood were consistent with the changes in mRNA expression. Serum IL-17 levels were positively correlated with total bilirubin (TBIL), alanine aminotransferase (ALT) and Child-Pugh grade, and were negatively correlated with albumin. These observed differences were significant. Serum IL-35 levels were negatively correlated with albumin, but not with Child-Pugh grade, ALT and TBIL. IL-17 and IL-35 may be critically involved in the pathogenesis of hepatitis B-related LC.
PMCID: PMC4237084  PMID: 25323532
hepatitis B; liver cirrhosis; interleukin-35; interleukin-17
Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2′-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382 may be considered a putative tumor suppressor gene in pediatric AML. However, further studies focusing on the mechanisms responsible for ZNF382 downregulation in pediatric leukemia are required.
PMCID: PMC4214337  PMID: 25319049
zinc finger protein 382; pediatric acute myeloid leukemia; methylation; tumor suppressor
PLoS ONE  2014;9(10):e108555.
The long-term application of excessive chemical fertilizers has resulted in the degeneration of soil quality parameters such as soil microbial biomass, communities, and nutrient content, which in turn affects crop health, productivity, and soil sustainable productivity. The objective of this study was to develop a rapid and efficient solution for rehabilitating degraded cropland soils by precisely quantifying soil quality parameters through the application of manure compost and bacteria fertilizers or its combination during maize growth. We investigated dynamic impacts on soil microbial count, biomass, basal respiration, community structure diversity, and enzyme activity using six different treatments [no fertilizer (CK), N fertilizer (N), N fertilizer + bacterial fertilizer (NB), manure compost (M), manure compost + bacterial fertilizer (MB), and bacterial fertilizer (B)] in the plowed layer (0–20 cm) of potted soil during various maize growth stages in a temperate cropland of eastern China. Denaturing gradient electrophoresis (DGGE) fingerprinting analysis showed that the structure and composition of bacterial and fungi communities in the six fertilizer treatments varied at different levels. The Shannon index of bacterial and fungi communities displayed the highest value in the MB treatments and the lowest in the N treatment at the maize mature stage. Changes in soil microorganism community structure and diversity after different fertilizer treatments resulted in different microbial properties. Adding manure compost significantly increased the amount of cultivable microorganisms and microbial biomass, thus enhancing soil respiration and enzyme activities (p<0.01), whereas N treatment showed the opposite results (p<0.01). However, B and NB treatments minimally increased the amount of cultivable microorganisms and microbial biomass, with no obvious influence on community structure and soil enzymes. Our findings indicate that the application of manure compost plus bacterial fertilizers can immediately improve the microbial community structure and diversity of degraded cropland soils.
PMCID: PMC4193766  PMID: 25302996
PLoS ONE  2014;9(10):e107922.
Testis-specific protein kinase 1 (Tesk1) is a serine/threonine kinase with unique structural features. In the present study, we cloned and characterized the tesk1 gene of tongue sole, Cynoglossus semilaevis. The full-length tesk1 cDNA consists of 1,672 nucleotides, encoding a 331 amino acid polypeptide with a characteristic structure composed of an N-terminal kinase domain and a C-terminal proline-rich domain. The tesk1 genomic sequence contains eight exons and seven introns. Real-time quantitative PCR revealed that tesk1 mRNA is expressed predominantly in the testis, though the level of expression varied throughout development. We used in situ hybridization to show that tesk1 mRNA is expressed in the spermatids of males and pseudo-males, but not in triploid males. Our results suggest that tongue sole Tesk1 may play a role in spermatogenesis.
PMCID: PMC4182740  PMID: 25271995
Oncotarget  2014;5(20):10048-10057.
Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.
PMCID: PMC4259404  PMID: 25301727
Gene amplification; Merkel cell carcinoma; MCV; Promoter mutations; Telomerase; TERT
PLoS ONE  2014;9(9):e107652.
Prevention intervention trials have been conducted to reduce risk of sexual transmission among people living with HIV/AIDS (PLWHA), but the findings were inconsistent. We performed a systematic review and meta-analysis to evaluate overall efficacy of prevention interventions on unprotected vaginal or anal intercourse (UVAI) among PLWHA from randomized clinical trials (RCTs).
RCTs of prevention interventions among PLWHA published as of February 2012 were identified by systematically searching thirteen electronic databases. The primary outcome was UVAI. The difference of standardized mean difference (SMD) of UVAI between study arms, defined as effect size (ES), was calculated for each study and then pooled across studies using standard meta-analysis with a random effects model.
Lower likelihood of UVAI was observed in the intervention arms compared with the control arms either with any sexual partners (mean ES: −0.22; 95% confidence interval [CI]: −0.32, −0.11) or with HIV-negative or unknown-status sexual partners (mean ES and 95% CI: −0.13 [−0.22, −0.04]). Short-term efficacy of interventions with ≤10 months of follow up was significant in reducing UVAI (1–5 months: −0.27 [−0.45, −0.10]; 6–10 months: −0.18 [−0.30, −0.07]), while long-term efficacy of interventions was weaker and might have been due to chance (11–15 months: −0.13 [−0.34, 0.08]; >15 months: −0.05 [−0.43, 0.32]).
Our meta-analyses confirmed the short-term impact of prevention interventions on reducing self-reported UVAI among PLWHA irrespective of the type of sexual partner, but did not support a definite conclusion on long-term effect. It is suggested that booster intervention sessions are needed to maintain a sustainable reduction of unprotected sex among PLWHA in future risk reduction programs.
PMCID: PMC4171502  PMID: 25243404
PLoS ONE  2014;9(9):e107636.
Distylium chinense is an evergreen shrub used for the vegetation recovery of floodplain and riparian areas in Three Gorges Reservoir Region. To clarify the morphological and physiological responses and tolerance of Distylium chinense to off-season flooding, a simulation flooding experiment was conducted during autumn and winter. Results indicated that the survival rate of seedlings was 100%, and that plant height and stem diameter were not significantly affected by flooding. Adventitious roots and hypertrophic lenticels were observed in flooded seedlings after 30 days of flooding. Flooding significantly reduced the plant biomass of roots, net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), maximum photochemical efficiency (Fv/Fm), photochemical quenching (qP), and electron transport rate (ETR) in leaves, and also affected the allocation and transport of carbohydrate and nutrients. However, D. chinense was able to maintain stable levels of Pn, Fv/Fm, qP, ETR, and nutrient content (N and P) in leaves and to store a certain amount of carbohydrate in roots over prolonged durations of flooding. Based on these results, we conclude that there is a high flooding tolerance in D. chinense, and the high survival rate of D. chinense may be attributable to a combination of morphological and physiological responses to flooding.
PMCID: PMC4164644  PMID: 25222006
American Journal of Epidemiology  2013;178(5):791-796.
Moderate-intensity exercise has attracted considerable attention because of its safety and many health benefits. Tai Chi, a form of mind-body exercise that originated in ancient China, has been gaining popularity. Practicing Tai Chi may improve overall health and well-being; however, to our knowledge, no study has evaluated its relationship with mortality. We assessed the associations of regular exercise and specifically participation in Tai Chi, walking, and jogging with total and cause-specific mortality among 61,477 Chinese men in the Shanghai Men's Health Study (2002–2009). Information on exercise habits was obtained at baseline using a validated physical activity questionnaire. Deaths were ascertained through biennial home visits and linkage with a vital statistics registry. During a mean follow-up of 5.48 years, 2,421 deaths were identified. After adjustment for potential confounders, men who exercised regularly had a hazard ratio for total mortality of 0.80 (95% confidence interval: 0.74, 0.87) compared with men who did not exercise. The corresponding hazard ratios were 0.80 (95% confidence interval: 0.72, 0.89) for practicing Tai Chi, 0.77 (95% confidence interval: 0.69, 0.86) for walking, and 0.73 (95% confidence interval: 0.59, 0.90) for jogging. Similar inverse associations were also found for cancer and cardiovascular mortality. The present study provides the first evidence that, like walking and jogging, practicing Tai Chi is associated with reduced mortality.
PMCID: PMC3755647  PMID: 23813700
Chinese; jogging; mortality; Tai Chi; walking
PLoS ONE  2014;9(8):e105200.
Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs). The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML), could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD) rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.
PMCID: PMC4136837  PMID: 25133686
Journal of Virology  2014;88(3):1484-1491.
Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 105 focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.
PMCID: PMC3911581  PMID: 24227861
Circulation  2006;114(11):1159-1168.
Cellular hypertrophy requires coordinated regulation of progrowth and antigrowth mechanisms. In cultured neonatal cardiomyocytes, Foxo transcription factors trigger an atrophy-related gene program that counters hypertrophic growth. However, downstream molecular events are not yet well defined.
Methods and Results
Here, we report that expression of either Foxo1 or Foxo3 in cardiomyocytes attenuates calcineurin phosphatase activity and inhibits agonist-induced hypertrophic growth. Consistent with these results, Foxo proteins decrease calcineurin phosphatase activity and repress both basal and hypertrophic agonist-induced expression of MCIP1.4, a direct downstream target of the calcineurin/NFAT pathway. Furthermore, hearts from Foxo3-null mice exhibit increased MCIP1.4 abundance and a hypertrophic phenotype with normal systolic function at baseline. Together, these results suggest that Foxo proteins repress cardiac growth at least in part through inhibition of the calcineurin/NFAT pathway. Given that hypertrophic growth of the heart occurs in multiple contexts, our findings also suggest that certain hypertrophic signals are capable of overriding the antigrowth program induced by Foxo. Consistent with this, multiple hypertrophic agonists triggered inactivation of Foxo proteins in cardiomyocytes through a mechanism requiring the PI3K/Akt pathway. In addition, both Foxo1 and Foxo3 are phosphorylated and consequently inactivated in hearts undergoing hypertrophic growth induced by hemodynamic stress.
This study suggests that inhibition of the calcineurin/NFAT signaling cascade by Foxo and release of this repressive action by the PI3K/Akt pathway are important mechanisms whereby Foxo factors govern cell growth in the heart.
PMCID: PMC4118290  PMID: 16952979
angiotensin; calcineurin; hypertrophy
PLoS ONE  2014;9(7):e101572.
Environmental exposure to nanomaterials is inevitable, as nanomaterials have become part of our daily life now. In this study, we firstly investigated the effects of silica nanoparticles on the spermatogenic process according to their time course in male mice. 48 male mice were randomly divided into control group and silica nanoparticle group with 24 mice per group, with three evaluation time points (15, 35 and 60 days after the first dose) per group. Mice were exposed to the vehicle control and silica nanoparticles at a dosage of 20 mg/kg every 3 days, five times over a 13-day period, and were sacrificed at 15, 35 and 60 days after the first dose. The results showed that silica nanoparticles caused damage to the mitochondrial cristae and decreased the levels of ATP, resulting in oxidative stress in the testis by days 15 and 35; however, the damage was repaired by day 60. DNA damage and the decreases in the quantity and quality of epididymal sperm were found by days 15 and 35; but these changes were recovered by day 60. In contrast, the acrosome integrity and fertility in epididymal sperm, the numbers of spermatogonia and sperm in the testes, and the levels of three major sex hormones were not significantly affected throughout the 60-day period. The results suggest that nanoparticles can cause reversible damage to the sperms in the epididymis without affecting fertility, they are more sensitive than both spermatogonia and spermatocytes to silica nanoparticle toxicity. Considering the spermatogenesis time course, silica nanoparticles primarily influence the maturation process of sperm in the epididymis by causing oxidative stress and damage to the mitochondrial structure, resulting in energy metabolism dysfunction.
PMCID: PMC4086902  PMID: 25003337
Acute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear.
Eleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis.
The MT3 promoter was hypermethylated in leukemia cell lines. More CpG’s methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P < 0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P = 0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P < 0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1.
MT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.
PMCID: PMC4082423  PMID: 24962166
Metallothionein III; Pediatric acute myeloid leukemia; Methylation; Tumor suppressor
Melatonin is a powerful antioxidant. Decreased melatonin excretion has been reported to be associated with several oxidative stress-related diseases. The urinary metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), has proved to be a very reliable index of melatonin production. The present study aims to evaluate the level of urinary aMT6s in patients with type 2 diabetes mellitus and diabetic retinopathy. Urine samples were collected from 10 patients with diabetes and no diabetic retinopathy (NDR), 19 patients with nonproliferative diabetic retinopathy (NPDR), 38 patients with proliferative diabetic retinopathy (PDR), and 16 subjects without diabetes mellitus, who served as controls. The level of aMT6s in specimens was assayed by a commercial aMT6s ELISA kit, creatinine levels were also measured for each sample to get urinary aMT6s/creatinine ratio. Creatinine-adjusted urinary aMT6s values were compared among four groups. The urinary aMT6s (mean ± SD) levels were 9.95 ± 2.42, 9.90 ± 2.28, 8.40 ± 1.84 and 5.58 ± 1.33 ng/mg creatinine in the controls and in patients with NDR, NPDR, or PDR, respectively. The urinary aMT6s level of the PDR group was significantly lower than that of the control, NDR and DR groups. No significant difference was found among the control, NDR and DR groups. After adjustment for various factors (age, smoking, cancer, and coronary heart disease) that may influence the aMT6s level, the odds-ratio of urinary aMT6s comparing PDR patients to controls was 0.246 (95% confidence interval = 0.108-0.558, P = 0.001). Therefore, the urinary aMT6s level is significantly decreased in diabetic patients with PDR but not in diabetic patients without PDR, which indicates that decreased urinary aMT6s level may be associated with the pathogenesis of PDR.
PMCID: PMC4129050  PMID: 25120815
6-sulfatoxymelatonin; melatonin; diabetic retinopathy
Endocrine-Related Cancer  2014;21(3):427-434.
The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.
PMCID: PMC4045219  PMID: 24803525
TERT; telomerase; mutation; endocrine; pheochromocytoma; adrenocortical tumor
Oncology Letters  2014;8(1):345-350.
Voltage-gated sodium channels (VGSCs) are expressed not only in excitable cells but also in numerous metastatic cells, particularly in certain types of cancer cells. In some types of cancer, including prostate cancer, the expression of VGSCs is associated with cancer migration, invasion and metastasis in vivo. However, the detailed expression profiles of VGSC α subunits in normal human prostate, in prostatic hyperplasia and prostatic cancer remain controversial. In the present study, quantitative polymerase chain reaction was used to systematically detect all subtypes of VGSC α subunits in normal human prostate, benign prostatic hyperplasia (BPH) and prostate cancer cells. The expression profile of VGSC α subunits was observed to differ between these cell types. Nav1.5 was the major isoform expressed in normal human prostate tissue, while Nav1.5 and Nav1.2 were the predominant isoforms in BPH tissue. However, in PC-3 and LNCaP cells, two typical prostate cancer cell lines, Nav1.6 and Nav1.7 were abundantly expressed. By comparing the relative expression levels of Nav1.5, Nav1.6 and Nav1.7 in these cells, the mRNA levels of Nav1.6 and Nav1.7 were identified to be 6- to 27-fold higher in PC-3 and LNCaP cells than in either normal or BPH samples (P<0.05); however, Nav1.5 mRNA levels were relatively lower compared with those of Nav1.6 or Nav1.7 in all cells analyzed. To confirm whether Nav1.6 and Nav1.7 expression in cancer cells was functional, a patch-clamp technique was used to record whole-cell currents. A tetrodotoxin-sensitive sodium current was successfully recorded in PC-3 cells, but not in LNCaP cells. It was concluded that although all types of VGSC α subunits exhibited low expression levels in normal prostate and BPH cells, both Nav1.6 and Nav1.7 were significantly upregulated in the prostate cancer cell lines, suggesting these subtypes may be potential diagnostic markers and therapeutic targets for certain types of prostate cancer in humans.
PMCID: PMC4063587  PMID: 24959274
voltage-gated sodium channel; mRNA; prostate; cancer; benign prostatic hyperplasia
Atherosclerosis  2013;228(1):217-223.
Traditional clinical risk factors are associated with inflammation cross-sectionally, but associations of longitudinal variation in inflammatory biomarkers with corresponding changes in clinical risk factors are incompletely described. We sought to analyze clinical factors associated with change in inflammation in the community.
We studied 3013 Framingham Offspring (n = 2735) and Omni Cohort (n = 278) participants (mean age 59 years, 55% women, 9% ethnic/racial minority) who attended two consecutive examination cycles (mean 6.7 years apart). We selected ten inflammatory biomarkers representing distinctive biological functions: C-reactive protein (CRP), intercellular adhesion molecule-1, interleukin-6, isoprostanes, lipoprotein-associated phospholipase-2 (Lp-PLA2) activity, Lp-PLA2-mass, monocyte chemoattractant protein-1, osteoprotegerin, P-selectin, and tumor necrosis factor receptor II (TNFRII). We constructed multivariable-adjusted regression models to assess the relations of baseline, follow-up and change in clinical risk factors with change in biomarker concentrations over time.
Baseline, follow-up and change in clinical risk factors explain a moderate amount of the variation in biomarker concentrations across 2 consecutive examinations (ranging from r2 = 0.28 [TNFRII] up to 0.52 [Lp-PLA2-mass]). In multivariable models, increasing body-mass index, smoking initiation, worsening lipid profile, and increasing waist size were associated with increasing concentrations of several biomarkers. Conversely, hypercholesterolemia therapy and hormone replacement cessation were associated with decreasing concentrations of biomarkers such as CRP, Lp-PLA2-mass and activity.
Cardiovascular risk factors have different patterns of association with longitudinal change in inflammatory biomarkers and explain modest amounts of variability in biomarker concentrations. Nevertheless, a substantial proportion of longitudinal change in inflammatory markers is not explained by traditional risk factors.
PMCID: PMC3650714  PMID: 23489346
Biological markers; Longitudinal studies; Inflammation

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