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author:("Yang, zhikong")
1.  Selection of competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for patients undergoing preimplantation genetic screening: a prospective study with sibling oocytes 
BMC Medical Genomics  2014;7:38.
Recent advances in time-lapse monitoring in IVF treatment have provided new morphokinetic markers for embryonic competence. However, there is still very limited information about the relationship between morphokinetic parameters, chromosomal compositions and implantation potential. Accordingly, this study aimed at investigating the effects of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing on pregnancy and implantation outcomes for patients undergoing preimplantation genetic screening (PGS).
A total of 1163 metaphase II (MII) oocytes were retrieved from 138 PGS patients at a mean age of 36.6 ± 2.4 years. These sibling MII oocytes were then randomized into two groups after ICSI: 1) Group A, oocytes (n = 582) were cultured in the time-lapse system and 2) Group B, oocytes (n = 581) were cultured in the conventional incubator. For both groups, whole genomic amplification and array CGH testing were performed after trophectoderm biopsy on day 5. One to two euploid blastocysts within the most predictive morphokinetic parameters (Group A) or with the best morphological grade available (Group B) were selected for transfer to individual patients on day 6. Ongoing pregnancy and implantation rates were compared between the two groups.
There were significant differences in clinical pregnancy rates between Group A and Group B (71.1% vs. 45.9%, respectively, p = 0.037). The observed implantation rate per embryo transfer significantly increased in Group A compared to Group B (66.2% vs. 42.4%, respectively, p = 0.011). Moreover, a significant increase in ongoing pregnancy rates was also observed in Group A compared to Group B (68.9% vs. 40.5%. respectively, p = 0.019). However, there was no significant difference in miscarriage rate between the time-lapse system and the conventional incubator (3.1% vs. 11.8%, respectively, p = 0.273).
This is the first prospective investigation using sibling oocytes to evaluate the efficiency of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for PGS patients. Our data clearly demonstrate that the combination of these two advanced technologies to select competent blastocysts for transfer results in improved implantation and ongoing pregnancy rates for PGS patients.
PMCID: PMC4077552  PMID: 24954518
Time-lapse monitoring; Array CGH; PGS; Ploidy; Implantation; Miscarriage
2.  Long term exposure to L-arginine accelerates endothelial cell senescence through arginase-II and S6K1 signaling 
Aging (Albany NY)  2014;6(5):369-379.
L-arginine supplementation is proposed to improve health status or as adjunct therapy for diseases including cardiovascular diseases. However, controversial results and even detrimental effects of L-arginine supplementation are reported. We investigate potential mechanisms of L-arginine-induced detrimental effects on vascular endothelial cells. Human endothelial cells were exposed to a physiological (0.1 mmol/L) or pharmacological (0.5 mmol/L) concentration of L-arginine for 30 minutes (acute) or 7 days (chronic). The effects of L-arginine supplementation on endothelial senescence phenotype, i.e., levels of senescence-associated beta-galactosidase, expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, eNOS-uncoupling, arginase-II expression/activity, and mTORC1-S6K1 activity were analyzed. While acute L-arginine treatment enhances endothelial NO production accompanied with superoxide production and activation of S6K1 but no up-regulation of arginase-II, chronic L-arginine supplementation causes endothelial senescence, up-regulation of the adhesion molecule expression, and eNOS-uncoupling (decreased NO and enhanced superoxide production), which are associated with S6K1 activation and up-regulation of arginase-II. Silencing either S6K1 or arginase-II inhibits up-regulation/activation of each other, prevents endothelial dysfunction, adhesion molecule expression, and senescence under the chronic L-arginine supplementation condition. These results demonstrate that S6K1 and arginase-II form a positive circuit mediating the detrimental effects of chronic L-arginine supplementation on endothelial cells.
PMCID: PMC4069264  PMID: 24860943
Arginase-II; endothelium; L-arginine; Senescence; mTOR; adhesion molecules
3.  Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles 
In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles.
First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups.
There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05).
While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings.
PMCID: PMC3766007  PMID: 23937723
aCGH; Trophectoderm biopsy; Cryopreservation; Implantation
4.  Arginase‐II Induces Vascular Smooth Muscle Cell Senescence and Apoptosis Through p66Shc and p53 Independently of Its l‐Arginine Ureahydrolase Activity: Implications for Atherosclerotic Plaque Vulnerability 
Vascular smooth muscle cell (VSMC) senescence and apoptosis are involved in atherosclerotic plaque vulnerability. Arginase‐II (Arg‐II) has been shown to promote vascular dysfunction and plaque vulnerability phenotypes in mice through uncoupling of endothelial nitric oxide synthase and activation of macrophage inflammation. The function of Arg‐II in VSMCs with respect to plaque vulnerability is unknown. This study investigated the functions of Arg‐II in VSMCs linking to plaque vulnerability.
Methods and Results
In vitro studies were performed on VSMCs isolated from human umbilical veins, whereas in vivo studies were performed on atherosclerosis‐prone apolipoprotein E‐deficient (ApoE−/−) mice. In nonsenescent VSMCs, overexpressing wild‐type Arg‐II or an l‐arginine ureahydrolase inactive Arg‐II mutant (H160F) caused similar effects on mitochondrial dysfunction, cell apoptosis, and senescence, which were abrogated by silencing p66Shc or p53. The activation of p66Shc but not p53 by Arg‐II was dependent on extracellular signal‐regulated kinases (ERKs) and sequential activation of 40S ribosomal protein S6 kinase 1 (S6K1)—c‐Jun N‐terminal kinases (JNKs). In senescent VSMCs, Arg‐II and S6K1, ERK‐p66Shc, and p53 signaling levels were increased. Silencing Arg‐II reduced all these signalings and cell senescence/apoptosis. Conversely, silencing p66Shc reduced ERK and S6K1 signaling and Arg‐II levels and cell senescence/apoptosis. Furthermore, genetic ablation of Arg‐II in ApoE−/− mice reduced the aforementioned signaling and apoptotic VSMCs in the plaque of aortic roots.
Arg‐II, independently of its l‐arginine ureahydrolase activity, promotes mitochondrial dysfunction leading to VSMC senescence/apoptosis through complex positive crosstalk among S6K1‐JNK, ERK, p66Shc, and p53, contributing to atherosclerotic vulnerability phenotypes in mice.
PMCID: PMC3828809  PMID: 23832324
apoptosis; arginase; p53; p66Shc; vascular smooth muscle cells
5.  A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13 
PLoS ONE  2013;8(6):e65256.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is increasing in frequency in the U.S. The major reason for the low postoperative survival rate of HCC is widespread intrahepatic metastasis or invasion, and activation of TGFβ signaling is associated with the invasive phenotype. This study aims at determining the novel function of miR-127 in modulating HCC migration. Overexpression of miR-127 inhibits HCC cell migration, invasion and tumor growth in nude mice. MiR-127 directly represses matrix metalloproteinase 13 (MMP13) 3′UTR activity and protein expression, and diminishes MMP13/TGFβ-induced HCC migration. In turn, TGFβ decreases miR-127 expression by enhancing c-Jun-mediated inhibition of miR-127 promoter activity. In contrast, p53 transactivates miR-127 promoter and induces miR-127 expression, which is antagonized by c-Jun. The inhibition of miR-127 by c-Jun is through TGFβ-mediated ERK and JNK pathways. The lower miR-127 expression shows a negative correlation with the higher MMP13 expression in a subset of human HCC specimens. This is the first report elucidating a feedback regulation between miR-127 and the TGFβ/c-Jun cascade in HCC migration via MMP13 that involves a crosstalk between the oncogene c-Jun and tumor suppressor p53.
PMCID: PMC3676458  PMID: 23762330
6.  In Vivo Measurement of Oxygenation Changes after Stroke Using Susceptibility Weighted Imaging Filtered Phase Data 
PLoS ONE  2013;8(5):e63013.
Background and Purpose
Cerebral blood oxygenation level is critical for following the evolution of stroke patients. The purpose of this study was to investigate the feasibility of measuring changes in blood oxygen levels for patients with acute stroke using SWI and to compare these changes with the patient's recovery over time.
Materials and Methods
A total 30 MRI scans was performed on 10 acute ischemic stroke patients. Every patient was followed at three time points: less than 24 hours; 2–3 weeks after stroke and 2 months after stroke. Both MRI scan and NIH stroke scale (NIHSS) were acquired for each patient at all three time points. Oxygen saturation changes were derived from phase values differences (Δφ) measured over 10 veins from each hemisphere for all 10 patients over 3 time points. The correlation of oxygen saturation and NIHSS was further evaluated.
The stroke affected side of the brain showed moderate (r = −0.62) to strong (r = −0.70) correlation between the oxygenation change and NIHSS change. The oxygen saturation change from the normal side of the brain had essentially no association with recovery (r = −0.02 and−0.31). The results suggest that increases in oxygen saturation correspond to improved outcome and reductions in oxygen saturation correspond to worse outcome.
High resolution SWI provided a novel method to measure changes in oxygenation change of the human brain in vivo. By using the phase values from the veins, both spatial and temporal information can be found that relates to patient outcome post stroke.
PMCID: PMC3652854  PMID: 23675450
7.  An autoregulatory feedback loop between Mdm2 and SHP that fine tunes Mdm2 and SHP stability 
Febs Letters  2012;586(8):1135-1140.
Mdm2 is a crucial negative regulator of the tumor suppressor function of p53. However, little is known about Mdm2 protein stability regulation by other tumor suppressors. Nuclear receptor small heterodimer partner (SHP, NROB2) functions as a tumor suppressor in liver cancer. We show here a surprising finding of a feedback regulatory loop between SHP and Mdm2. SHP stabilizes Mdm2 protein by abrogating Mdm2 self-ubiquitination, and Mdm2 in turn attenuates SHP protein levels under p53-deficient conditions. Such cross-regulation critically depends on the physical interaction of SHP with Mdm2 through the SHP K170 residue. The Mdm2-SHP interplay represents a novel component of Mdm2 signaling that is likely to dictate Mdm2 activity and function.
PMCID: PMC3350641  PMID: 22575647
nuclear receptor; small heterodimer partner; protein stability; Mdm2
8.  δ- and γ-Tocopherols, but not α-Tocopherol, Inhibit Colon Carcinogenesis in Azoxymethane-Treated F344 Rats 
The cancer preventive activity of vitamin E has been extensively discussed, but the activities of specific forms of tocopherols have not received sufficient attention. Herein, we compared the activities of δ-tocopherol (δ-T), γ-T and α-T in a colon carcinogenesis model. Male F344 rats, 7 weeks old, were given 2 weekly subcutaneous injections of azoxymethane (AOM) each at a dose of 15 mg/kg body weight. Starting 1 week before the AOM injection, the animals were maintained on a modified AIN76A diet, or the same diet containing 0.2% of δ-T, γ-T, α-T or a γ-T-rich mixture of tocopherols (γ-TmT), until the termination of the experiment at 8 weeks after the second AOM injection. δ-T treatment showed the strongest inhibitory effect, decreasing the numbers of aberrant crypt foci by 62%. γ-T and γ-TmT were also effective, but α-T was not. Immunohistochemical analysis showed that δ-T and γ-T treatments reduced the levels of 4-hydroxynonenal and nitrotyrosine and the expression of cyclin D1 in the colon, preserved the expression of PPAR-γ, and decreased the serum levels of prostaglandin E2 and 8-isoprostane. Supplementation with 0.2% δ-T, γ-T or α-T increased the respective levels of tocopherols and their side-chain degradation metabolites in the serum and colon tissues. Rather high concentrations of δ-T and γ-T and their metabolites were found in colon tissues. Our study provides the first evidence for the much higher cancer preventive activity of δ-T and γ-T than α-T in a chemically-induced colon carcinogenesis model. It further suggests that δ-T is more effective than γ-T.
PMCID: PMC3324634  PMID: 22366914
Tocopherols; tocopherol metabolites; colon carcinogenesis; ACF
9.  Involvement of GMRP1, a novel mediator of Akt pathway, in brain damage after intracerebral hemorrhage 
GMRP1, also known as BTBD10, has been reported to inhibit apoptosis of neuronal and islet beta cells via Akt pathway. The present study attempted to investigate whether GMRP1 and its mediated Akt pathway were involved in brain injury of rats after intracerebral hemorrhage (ICH). Rat models of ICH had been established successfully. Western blotting was used to investigate the levels of GMRP1 protein in caudate nuclei tissues of hemorrhagic and contralateral sides at 6 h, day 1, day 3, day 5, day 7 after ICH. Phosphorylation of Akt was determined in caudate nuclei mentioned above. TUNEL assay was used to measure the cell apoptosis. GMRP1 protein levels, as well as phosphorylations of Akt, significantly decreased in caudate nuclei of hemorrhagic side, compared with those of contralateral side at day 1, day 3 after ICH. Enhanced cell apoptosis was observed in hemorrhagic side by TUNEL assay. We presented here evidence that decreased GMRP1-mediated Akt pathway contributed to cell apoptosis in hemorrhagic side, suggesting that GMRP1 played an important role in brain damage after ICH.
PMCID: PMC3544232  PMID: 23330007
GMRP1; intracerebral hemorrhage; apoptosis; Akt
10.  Mdm2 is a Novel Activator of ApoCIII Promoter Which is Antagonized by p53 and SHP Inhibition 
We examined the effect of Mdm2 on regulation of the ApoCIII promoter and its cross-talk with p53 and nuclear receptor SHP. Overexpression of Mdm2 markedly enhanced ApoCIII promoter activity by HNF4α. A direct association of Mdm2 protein with the HNF4α protein was observed by co-immunoprecipitation. Ectopic expression of p53 decreased HNF4α activation of the ApoCIII promoter and antagonized the effect of Mdm2. Co-expression of SHP further strengthened p53 inhibition and abolished Mdm2 activation of the ApoCIII promoter. Mdm2 inhibited p53-mediated enrichment of HNF4α to the ApoCIII promoter while simultaneously reducing p53 binding and increasing recruitment of SHP to the ApoCIII promoter. The results from this study implicate a potentially important function of Mdm2 in regulation of lipoprotein metabolism.
PMCID: PMC3259241  PMID: 22197810
nuclear receptor; promoter activity; tumor suppressor; oncogene; lipoprotein
11.  Arginase: The Emerging Therapeutic Target for Vascular Oxidative Stress and Inflammation 
Oxidative stress and inflammation in the vascular wall are essential mechanisms of atherosclerosis and vascular dysfunctions associated with risk factors such as metabolic diseases, aging, hypertension, etc. Evidence has been provided that activation of the vascular endothelial cells in the presence of the risk factors promotes oxidative stress and vascular inflammatory responses, leading to acceleration of atherosclerotic vascular disease. Increasing number of studies from recent years demonstrates that uncoupling of endothelial nitric oxide synthase (eNOS), whereby the enzyme eNOS produces detrimental amount of superoxide anion O2− instead the vasoprotective nitric oxide (NO⋅), plays a critical role in vascular dysfunction under various pathophysiological conditions and in aging. The mechanisms of eNOS-uncoupling seem multiple and complex. Recent research provides emerging evidence supporting an essential role of increased activity of arginases including arginase-I and arginase-II in causing eNOS-uncoupling, which results in vascular oxidative stress and inflammatory responses, and ultimately leading to vascular diseases. This review article will summarize the most recent findings on the functional roles of arginases in vascular diseases and/or dysfunctions and the underlying mechanisms in relation to oxidative stress and inflammations. Moreover, regulatory mechanisms of arginases in the vasculature are reviewed and the future perspectives of targeting arginases as therapeutic options in vascular diseases are discussed.
PMCID: PMC3679468  PMID: 23781221
arginase; eNOS; superoxide; adhesion molecules; signal transduction pathway
12.  Regulation of miR-200c by Nuclear Receptors PPARα, LRH-1 and SHP 
We investigated regulation of miR-200c expression by nuclear receptors. Ectopic expression of miR-200c inhibited MHCC97H cell migration, which was abrogated by the synergistic effects of PPARα and LRH-1 siRNAs. The expression of miR-200c was decreased by PPARα/LRH-1 siRNAs and increased by SHP siRNAs, and overexpression of the receptors reversed the effects of their respective siRNAs. SHP siRNAs also drastically enhanced the ability of the LRH-1 agonist RJW100 to induce miR-200c and downregulate ZEB1 and ZEB2 proteins. Co-expression of PPARα and LRH-1 moderately transactivated the miR-200c promoter, which was repressed by SHP co-expression. RJW100 caused strong activation of the miR-200c promoter. This is the first report to demonstrate that miR-200c expression is controlled by nuclear receptors.
PMCID: PMC3248804  PMID: 22100809
nuclear receptor; microRNA; transcription; cell migration
13.  Increasing Glucose 6-Phosphate Dehydrogenase Activity Restores Redox Balance in Vascular Endothelial Cells Exposed to High Glucose 
PLoS ONE  2012;7(11):e49128.
Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.
PMCID: PMC3501497  PMID: 23185302
14.  Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis 
Period2 (Per2) is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial nitric oxide (NO) production in response to insulin in the mPer2Brdm1 mice. We show that mPer2Brdm1 mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated NO release in the aortas in both active and inactive phases of the animals. As compared to wild type (WT) mice, the mPer2Brdm1 mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2Brdm1 mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to WT controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated NO release, independently of obesity.
PMCID: PMC3429102  PMID: 22934083
blood vessel; circadian gene; glucose; glycogen; insulin; nitric oxide; obesity; signaling
15.  Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis 
Macrophage‐mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg‐II) in macrophage function remains elusive. This study characterizes the role of Arg‐II in macrophage inflammatory responses and its impact on obesity‐linked type II diabetes mellitus and atherosclerosis.
Methods and Results
In human monocytes, silencing Arg‐II decreases the monocytes’ adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low‐density lipoprotein or lipopolysaccharides, as evaluated by real‐time quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg‐II–deficient (Arg‐II−/−) mice express lower levels of lipopolysaccharide‐induced proinflammatory mediators than do macrophages of wild‐type mice. Importantly, reintroducing Arg‐II cDNA into Arg‐II−/− macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N‐acetylcysteine prevents the Arg‐II–mediated inflammatory responses. Moreover, high‐fat diet–induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg‐II−/− mice. Accordingly, Arg‐II−/− mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)–deficient mice with Arg‐II deficiency (ApoE−/−Arg‐II−/−) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE−/−Arg‐II−/− than ApoE−/−Arg‐II+/+ monocytes infiltrate into the plaque of ApoE−/−Arg‐II+/+ mice. Conversely, recipient ApoE−/−Arg‐II−/− mice accumulate fewer donor monocytes than do recipient ApoE−/−Arg‐II+/+ animals.
Arg‐II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg‐II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.)
PMCID: PMC3487353  PMID: 23130157
atherosclerosis; diabetes mellitus, type 2; inflammation; macrophages
16.  Cross-Regulation of Protein Stability by p53 and Nuclear Receptor SHP 
PLoS ONE  2012;7(6):e39789.
We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.
PMCID: PMC3380837  PMID: 22737255
17.  Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation 
During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation.
First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation.
Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis).
While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.
PMCID: PMC3398117  PMID: 22816070
Fertilization in vitro; Comparative genomic hybridization; Preimplantation genetic diagnosis; Cryopreservation
18.  Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study 
Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET.
First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.
For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.
Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
PMCID: PMC3403960  PMID: 22551456
19.  δ-Tocopherol Is More Active than α- or γ-Tocopherol in Inhibiting Lung Tumorigenesis In Vivo 
In contrast to strong epidemiologic, preclinical, and secondary clinical evidence for vitamin E (tocopherols) in reducing cancer risk, large-scale clinical cancer-prevention trials of α-tocopherol have been negative. This vexing contrast helped spur substantial preclinical efforts to better understand and improve the antineoplastic activity of tocopherol through, for example, the study of different tocopherol forms. We previously showed that the γ-tocopherol–rich mixture (γ-TmT) effectively inhibited colon and lung carcinogenesis and the growth of transplanted lung-cancer cells in mice. We designed the present study to determine the relative activities of different forms of tocopherol in a xenograft model, comparing the anticancer activities of δ-tocopherol with those of α- and γ-tocopherols. We subcutaneously injected human lung cancer H1299 cells into NCr nu/nu mice, which then received α-, γ-, or δ-tocopherol or γ-TmT in the diet (each at 0.17% and 0.3%) for 49 days. δ-Tocopherol inhibited tumor growth most strongly. γ-Tocopherol and γ-TmT (at 0.3%) also inhibited growth significantly, but α-tocopherol did not. δ-Tocopherol also effectively decreased oxidative DNA damage and nitrotyrosine formation and enhanced apoptosis in tumor cells; again, γ-tocopherol also was active in these regards but less so, and α-tocopherol was not. Each supplemented diet increased serum levels of its tocopherol—up to 45 µM for α-tocopherol, 9.7 µM for γ-tocopherol, and 1.2 µM for δ-tocopherol; dietary γ- or δ-tocopherol, however, decreased serum α-tocopherol levels, and dietary α-tocopherol decreased serum levels of γ-tocopherol. Each dietary tocopherol also increased its corresponding side-chain–degradation metabolites, with concentrations of δ-tocopherol metabolites greater than γ-tocopherol and far greater than α-tocopherol metabolites in serum and tumors. The present study is the first in vivo assessment of δ-tocopherol in tumorigenesis and demonstrates that δ-tocopherol is more active than α- or γ-tocopherol in inhibiting tumor growth, possibly through trapping reactive oxygen and nitrogen species and inducing apoptosis; δ-tocopherol metabolites could contribute significantly to these results.
PMCID: PMC3071153  PMID: 21372040
Tocopherols; lung cancer cells; xenograft; tocopherol metabolites
20.  Perspectives of Targeting mTORC1–S6K1 in Cardiovascular Aging 
The global population aging is accelerating and age-associated diseases including cardiovascular diseases become more challenging. The underlying mechanisms of aging and age-associated cardiovascular dysfunction remain elusive. There are substantial evidences demonstrating a pivotal role of the mammalian target of rapamycin complex 1 (mTORC1) and its down-stream effector S6K1 signaling in mammalian lifespan regulation and age-related diseases such as type II diabetes mellitus and cancer. The role of mTORC1–S6K1 in age-related cardiovascular diseases is, however, largely unknown and the available experimental results are controversial. This review article primarily summarizes the most recent advances toward understanding the role of mTORC1–S6K1 in cardiovascular aging and discusses the future perspectives of targeting mTORC1–S6K1 signaling as a healthy lifespan extension modality in anti-aging and anti-cardiovascular aging.
PMCID: PMC3265748  PMID: 22291661
aging; endothelial senescence; eNOS; mTOR; S6K1; oxidative stress; rapamycin; resveratrol
21.  Metabolism of tocotrienols in animals and synergistic inhibitory actions of tocotrienols with atorvastatin in cancer cells 
Genes & Nutrition  2011;7(1):11-18.
Tocotrienols (T3s), members of the vitamin E family, exhibit potent anti-cancer, anti-oxidative, anti-inflammatory, and some other biological activities. To better understand the bioavailability and metabolism of T3s, T3s and their metabolites were identified in urine and fecal samples from mice on diet supplemented with mixed T3s using HPLC/electrochemical detection and liquid chromatography electrospray ionisation mass spectrometry (LC–ESI–MS). Whereas the short-chain metabolites carboxyethyl hydroxychromans (CEHCs) and carboxymethylbutyl hydroxychromans (CMBHCs) were the major metabolites of T3s, several new metabolites with double bonds were also identified. Similar to tocopherols, the majority of T3 metabolites were excreted as sulfate/glucuronide conjugates in mouse urine. The distribution of γ- and δ-T3 and γ-T3 metabolites were also determined in different organs as well as in urine and fecal samples from mice on diets supplemented with corresponding T3s. The synergistic anti-cancer actions of γ-T3 and atorvastatin (ATST) were studied in HT29 and HCT116 colon cancer cell lines. The combination greatly potentiated the ability of each individual agent to inhibit cancer cell growth and to induce cell cycle arrest and apoptosis. The triple combination of γ-T3, ATST, and celecoxib exhibited synergistic actions when compared with any double combination plus the third agent. Mechanistic studies revealed that the synergistic actions of γ-T3 and ATST could be attributed to their mediation of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and the subsequent inhibition of protein geranylgeranylation. It remains to be determined whether such a synergy occurs in vivo.
PMCID: PMC3250521  PMID: 21590436
Tocotrienol; Metabolites; HPLC; Atorvastatin; Synergy
22.  Regulation of microRNA expression and function by nuclear receptor signaling 
Cell & Bioscience  2011;1:31.
MicroRNAs (miRNAs) are small non-coding RNA transcripts that affect various cellular pathways by serving as regulators of gene expression at the translational and transcriptional level. Nuclear receptors (NRs) are ligand-activated transcription factors that regulate gene transcription by binding to the promoter region or by interacting with other transcription factors. NRs can regulate miRNA expression either at the transcriptional level, or through posttranscriptional maturation by interacting with miRNA processing factors. This review will summarize recent advances in knowledge of the modulation of miRNA expression by NRs. Increased understanding of the molecular basis of miRNA expression may enable new therapeutic interventions that modulate miRNA activities through NR-mediated signaling.
PMCID: PMC3192659  PMID: 21936947
miRNAs; nuclear receptors; gene regulation
23.  Correlation of change in R2* and phase with putative iron content in deep gray matter of healthy adults 
To establish a correlation between putative iron content using susceptibility weighted imaging (SWI) phase and T2* weighted magnitude values in the basal ganglia as a function of age in healthy human brains.
Materials and methods
100 healthy adults (20-69 yr.; mean = 43 yr) were evaluated for this study using a gradient echo sequence. The original magnitude and high pass filtered phase data were analyzed as proxy variables for iron content in the substantia nigra, red nucleus, globus pallidus, putamen, caudate nucleus, thalamus and pulvinar thalamus. Each structure was broken into two parts, a high iron content region and a low iron content region.
Both magnitude and phase data showed an increase in putative iron content with age. However, the high iron content region revealed two new pieces of information: both the average iron content per pixel and the area of high iron increased with age. Further, significant increase in iron uptake as a function of age was found past the age of 40.
A two region of interest analysis of iron is a much more sensitive means to evaluate iron content change over time. Contrary to the current belief that iron content increases level off with age, the putative iron deposition in region two is seen to increase with age.
PMCID: PMC2936709  PMID: 20815053
brain iron deposition; susceptibility weighted imaging; iron changes with age
25.  Suppression of Estrogen Receptor Transcriptional Activity by Connective Tissue Growth Factor 
PLoS ONE  2011;6(5):e20028.
Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.
PMCID: PMC3101213  PMID: 21629692

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