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1.  Overexpression of 4EBP1, p70S6K, Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells 
Li, X | Li, Z | Zhou, W | Xing, X | Huang, L | Tian, L | Chen, J | Chen, C | Ma, X | Yang, Z
Cell Death & Disease  2013;4(9):e803-9.
Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.
doi:10.1038/cddis.2013.331
PMCID: PMC3789189  PMID: 24030155
overexpression; 4EBP1; p70S6K; Akt; coxsackievirus b3; apoptosis
2.  Essential Roles of Jab1 in Cell Survival, Spontaneous DNA Damage, and DNA Repair 
Oncogene  2010;29(46):6125-6137.
Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability, and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to gamma radiation–induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibited its activity, and represent a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.
doi:10.1038/onc.2010.345
PMCID: PMC3495558  PMID: 20802511
DNA repair; DNA damage; null mutation; embryonic lethality; Rad51
3.  AUC-based biomarker ensemble with an application on gene scores predicting low bone mineral density 
Bioinformatics  2011;27(21):3050-3055.
Motivation: The area under the receiver operating characteristic (ROC) curve (AUC), long regarded as a ‘golden’ measure for the predictiveness of a continuous score, has propelled the need to develop AUC-based predictors. However, the AUC-based ensemble methods are rather scant, largely due to the fact that the associated objective function is neither continuous nor concave. Indeed, there is no reliable numerical algorithm identifying optimal combination of a set of biomarkers to maximize the AUC, especially when the number of biomarkers is large.
Results: We have proposed a novel AUC-based statistical ensemble methods for combining multiple biomarkers to differentiate a binary response of interest. Specifically, we propose to replace the non-continuous and non-convex AUC objective function by a convex surrogate loss function, whose minimizer can be efficiently identified. With the established framework, the lasso and other regularization techniques enable feature selections. Extensive simulations have demonstrated the superiority of the new methods to the existing methods. The proposal has been applied to a gene expression dataset to construct gene expression scores to differentiate elderly women with low bone mineral density (BMD) and those with normal BMD. The AUCs of the resulting scores in the independent test dataset has been satisfactory.
Conclusion: Aiming for directly maximizing AUC, the proposed AUC-based ensemble method provides an efficient means of generating a stable combination of multiple biomarkers, which is especially useful under the high-dimensional settings.
Contact: lutian@stanford.edu
Supplementary Information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr516
PMCID: PMC3198577  PMID: 21908541
4.  Long-term effects of high-fat or high-carbohydrate diets on glucose tolerance in mice with heterozygous carnitine palmitoyltransferase-1a deficiency 
Nutrition & Diabetes  2011;1(8):e14-.
Background: Abnormal fatty acid metabolism is an important feature in the mechanisms of insulin resistance and β-cell dysfunction. Carnitine palmitoyltransferase-1a (CPT-1a, liver isoform) has a pivotal role in the regulation of mitochondrial fatty acid oxidation. We investigated the role of CPT-1a in the development of impaired glucose tolerance using a mouse model for CPT-1a deficiency when challenged by either a high-carbohydrate (HCD) or a high-fat diet (HFD) for a total duration of up to 46 weeks.
Methods: Insulin sensitivity and glucose tolerance were assessed in heterozygous CPT-1a-deficient (CPT-1a+/−) male mice after being fed either a HCD or a HFD for durations of 28 weeks and 46 weeks. Both glucose and insulin tolerance tests were used to investigate β-cell function and insulin sensitivity. Differences in islet insulin content and hepatic steatosis were evaluated by morphological analysis.
Results: CPT-1a+/− mice were more insulin-sensitive than CPT-1a+/+ mice when fed either HCD or HFD. The increased insulin sensitivity was associated with an increased expression of Cpt-1b (muscle isoform) in liver, as well as increased microvesicular hepatic steatosis compared with CPT-1a+/+ mice. CPT-1a+/− mice were more glucose tolerant than CPT-1a+/+ mice when fed the HCD, but there was no significant difference when fed HFD. Moreover, CPT-1a+/− mice fed HFD or HCD had fewer and smaller pancreatic islets than CPT-1a+/+ mice.
Conclusions: CPT-1a deficiency preserved insulin sensitivity when challenged by long-term feeding of either diet. Furthermore, CPT-1a-deficient mice had distinct phenotypes dependent on the diet fed demonstrating that both diet and genetics collectively have a role in the development of impaired glucose tolerance.
doi:10.1038/nutd.2011.11
PMCID: PMC3251273  PMID: 22229081
carnitine palmitoyltransterase-1a deficiency; mouse model; glucose tolerance; fatty acid oxidation disorders
5.  Calibrating parametric subject-specific risk estimation 
Biometrika  2010;97(2):389-404.
Summary
For modern evidence-based medicine, decisions on disease prevention or management strategies are often guided by a risk index system. For each individual, the system uses his/her baseline information to estimate the risk of experiencing a future disease-related clinical event. Such a risk scoring scheme is usually derived from an overly simplified parametric model. To validate a model-based procedure, one may perform a standard global evaluation via, for instance, a receiver operating characteristic analysis. In this article, we propose a method to calibrate the risk index system at a subject level. Specifically, we developed point and interval estimation procedures for t-year mortality rates conditional on the estimated parametric risk score. The proposals are illustrated with a dataset from a large clinical trial with post-myocardial infarction patients.
doi:10.1093/biomet/asq012
PMCID: PMC3412577  PMID: 23049123
Cardiovascular diseases; Cox model; Nonparametric functional estimation; Risk index; ROC analysis; Survival analysis
6.  Regularized Estimation for the Accelerated Failure Time Model 
Biometrics  2009;65(2):394-404.
Summary
In the presence of high dimensional predictors, it is challenging to develop reliable regression models that can be used to accurately predict future outcomes. Further complications arise when the outcome of interest is an event time which is often not fully observed due to censoring. In this paper, we develop robust prediction models for event time outcomes by regularizing the Gehan’s estimator for the AFT model (Tsiatis, 1990) with LASSO penalty. Unlike existing methods based on the inverse probability weighting and the Buckley and James estimator (Buckely and James, 1979), the proposed approach does not require additional assumptions about the censoring and always yields a solution that is convergent. Furthermore, the proposed estimator leads to a stable regression model for prediction even if the AFT model fails to hold. To facilitate the adaptive selection of the tuning parameter, we detail an efficient numerical algorithm for obtaining the entire regularization path. The proposed procedures are applied to a breast cancer dataset to derive a reliable regression model for predicting patient survival based on a set of clinical prognostic factors and gene signatures. Finite sample performances of the procedures are evaluated through a simulation study.
doi:10.1111/j.1541-0420.2008.01074.x
PMCID: PMC3073158  PMID: 18573133
AFT model; LASSO regularization; linear programming
7.  Identifying subjects who benefit from additional information for better prediction of the outcome variables 
Biometrics  2008;65(3):894-902.
SUMMARY
Suppose that we are interested in using new bio- or clinical-markers to improve prediction or diagnosis of the patient’s clinical outcome in addition to the conventional markers. The incremental value from the new markers is typically assessed by averaging across patients in the entire study population. However, when measuring the new markers is costly or invasive, an overall improvement does not justify measuring the new markers in all patients. A more practical strategy is to utilize the patient’s conventional markers to decide whether the new markers are needed for improving prediction of his/her health outcomes. In this article, we propose inference procedures for the incremental values of new markers across various subgroups of patients classified by the conventional markers. The resulting point and interval estimates can be quite useful for medical decision makers seeking to balance the predictive or diagnostic value of new markers against their associated cost and risk. Our proposals are theoretically justified and illustrated empirically with two real examples.
doi:10.1111/j.1541-0420.2008.01125.x
PMCID: PMC2921328  PMID: 18945268
Biomarker; Cardiovascular events; Diagnosis; K-fold crossvalidation; Prediction accuracy; Subgroup analysis
8.  DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers 
British Journal of Cancer  2009;100(4):663-669.
Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours.
doi:10.1038/sj.bjc.6604888
PMCID: PMC2653732  PMID: 19156137
tumour suppressor gene (TSG); DLEC1; CpG island; methylation; colon and gastric cancers
9.  Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model 
BMC Cancer  2008;8:306.
Background
Bone marrow-derived stromal cells (BMSCs) are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor.
Methods
Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1). The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models.
Results
BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors.
Conclusion
We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.
doi:10.1186/1471-2407-8-306
PMCID: PMC2580769  PMID: 18947384

Results 1-9 (9)