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1.  Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase on Cardiac Fibrosis 
PLoS ONE  2014;9(4):e94092.
Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2−/−) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2−/− and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2−/− but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2−/− than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis.
PMCID: PMC3981766  PMID: 24718617
2.  Spatial and Seasonal CH4 Flux in the Littoral Zone of Miyun Reservoir near Beijing: The Effects of Water Level and Its Fluctuation 
PLoS ONE  2014;9(4):e94275.
Wetlands, and especially their littoral zones, are considered to be CH4 emissions hotspots. The recent creation of reservoirs has caused a rapid increase in the area of the world’s littoral zones. To investigate the effects of water depth and water level fluctuation on CH4 fluxes, and how these are coupled with vegetation and nutrients, we used static closed chamber and gas chromatography techniques to measure CH4 fluxes in the littoral zone of a large reservoir near Beijing, China, from November 2011 to October 2012. We found that CH4 flux decreased significantly along a transect from open water to dry land, from 3.1 mg m−2 h−1 at the deep water site to approximately 1.3 mg m−2 h−1 at the shallow water site, and less than 0.01 mg m−2 h−1 in the non-flooded area. Water level influenced CH4 flux by affecting soil properties including soil redox potential, soil carbon and nitrogen, and bulk density. The largest emission of all was from the seasonally flooded site after a flooding event (up to 21.1 mg m−2 h−1), which may have been caused by vegetation decomposition. Submerged sites had greater emissions, while the driest site had lower emissions. Immediately after the monthly measurements had been made, we removed the aboveground vegetation to enable an assessment of the gas transportation per unit of biomass. Removal of biomass decreased emissions by up to 53%. These results indicated the dominant effect of water depth on CH4 flux through effects of soil conditions, plant species composition and distribution. This study suggests that temporally flooded wetlands, including littoral zones, contribute significantly to the global CH4 burden. However, the current challenge is to capture their spatial extent and temporal variation in the fluxes.
PMCID: PMC3978027  PMID: 24710279
3.  Mechanisms and Metabolic Implications of Regional Differences among Fat Depots 
Cell metabolism  2013;17(5):644-656.
Fat distribution is closely linked to metabolic disease risk. Distribution varies with sex, genetic background, disease state, certain drugs and hormones, development, and aging. Preadipocyte replication and differentiation, developmental gene expression, susceptibility to apoptosis and cellular senescence, vascularity, inflammatory cell infiltration, and adipokine secretion vary among depots, as do fatty-acid handling and mechanisms of enlargement with positive-energy and loss with negative-energy balance. How interdepot differences in these molecular, cellular, and pathophysiological properties are related is incompletely understood. Whether fat redistribution causes metabolic disease or whether it is a marker of underlying processes that are primarily responsible is an open question.
PMCID: PMC3942783  PMID: 23583168
4.  Anti-VEGF Agents with or without Antimetabolites in Trabeculectomy for Glaucoma: A Meta-Analysis 
PLoS ONE  2014;9(2):e88403.
We aimed to evaluate the intraoperative application of antimetabolites compared with anti-vascular endothelial growth factor (VEGF) agents with or without antimetabolites in trabeculectomy (Trab) for glaucoma.
Relevant studies were selected through extensive search using PubMed, EMBASE, the Cochrane Library, and Web of Science databases in August 2013. The primary efficacy estimate was measured using weighted mean difference of the percentage of intraocular pressure reduction (IOPR%) from baseline to end-point, and the secondary efficacy estimates were odds ratio (OR) and 95% confidence interval (CI) for complete success rate and qualified success rate. ORs were also used to measure the tolerability estimate for adverse events. Meta-analyses of fixed or random effects models were conducted using RevMan software 5.2 to pool the results of the studies included. Heterogeneity was assessed using Chi2 test and the I2 measure.
Nine studies enrolling a total of 349 patients were included. The weighted mean difference of IOPR% from baseline was 7.23 (95% CI: 2.57–11.89) for antimetabolites vs. anti-VEGF agents and 3.96 (95% CI: −4.18–12.10) for antimetabolites vs. anti-VEGF agents plus antimetabolites. The pooled ORs comparing antimetabolites with anti-VEGF agents were 2.37 (95% CI: 0.78, 7.21) for the complete success rate and 1.93 (95% CI: 0.52, 7.16) for qualified success rate. The pooled ORs comparing antimetabolites with anti-VEGF agents plus antimetabolites were 1.43 (95% CI: 0.48, 4.29) for the complete success rate and 2.11 (95% CI: 0.12, 37.72) for qualified success rate. The rates of adverse events did not significantly differ between antimetabolites and anti-VEGF agents, with pooled ORs of 0.86 (0.28–2.69) for bleb leakage, 3.01 (0.45–20.10) for choroidal effusion, 0.96 (0.23–3.98) for flat anterior chamber, and 0.90 (0.12–6.60) for hypotony. Further, the rates of adverse events were similar between antimetabolites and anti-VEGF agents plus antimetabolites, with pooled ORs of 0.40 (0.08–2.00) and 8.00 (0.93–68.59) for bleb leakage and hypotony, respectively.
In comparison with anti-VEGF agents, antimetabolites were more effective in lowering IOP in Trab, while the intraoperative application of these two types of agents did not indicate statistically significant differences in the complete success rate, qualified success rate, or incidence of adverse events.
PMCID: PMC3921170  PMID: 24523890
5.  Cell-size control 
Cell Cycle  2014;13(5):693-694.
PMCID: PMC3979902  PMID: 24526119
Cdr2 kinase; Pom1 kinase; cell division; cell size; fission yeast; mitotic entry
6.  Atractylenolide-I Sensitizes Human Ovarian Cancer Cells to Paclitaxel by Blocking Activation of TLR4/MyD88-dependent Pathway 
Scientific Reports  2014;4:3840.
Paclitaxel, a known TLR4 ligand, leads to activation of TLR4/MyD88-dependent pathway that mediates chemoresistance and tumor progression in epithelial ovarian carcinoma (EOC). Atractylenolide-I (AO-I), a novel TLR4-antagonizing agent, inhibits TLR4 signaling by interfering with the binding of LPS or paclitaxel to membrane TLR4 of human leukocytes. In this study, AO-I was found to attenuate paclitaxel-induced protein expression of IL-6, VEGF and survivin, and to enhance early apoptosis and growth inhibition in MyD88+ EOC cells; AO-I was shown to fit into the hydrophobic pocket of human MD-2 and to partially overlap with the binding site of paclitaxel by docking simulations, suggesting that AO-I may block the MD-2-mediated TLR4/MyD88-dependent paclitaxel signaling in MyD88+ EOC cells. Therefore, AO-I could significantly sensitize the response of MyD88+ EOC cells to paclitaxel by blocking MD-2-mediated TLR4/MyD88 signaling, and that AO-I-paclitaxel combination could be a promising strategy for the treatment of EOC with a functional TLR4/MyD88/NF-κB pathway.
PMCID: PMC3899591  PMID: 24452475
7.  Cellular senescence and the senescent secretory phenotype: therapeutic opportunities 
Aging is the largest risk factor for most chronic diseases, which account for the majority of morbidity and health care expenditures in developed nations. New findings suggest that aging is a modifiable risk factor, and it may be feasible to delay age-related diseases as a group by modulating fundamental aging mechanisms. One such mechanism is cellular senescence, which can cause chronic inflammation through the senescence-associated secretory phenotype (SASP). We review the mechanisms that induce senescence and the SASP, their associations with chronic disease and frailty, therapeutic opportunities based on targeting senescent cells and the SASP, and potential paths to developing clinical interventions.
PMCID: PMC3582125  PMID: 23454759
8.  Is there a difference in cognitive development between preschool singletons and twins born after intracytoplasmic sperm injection or in vitro fertilization?*  
Objective: To explore whether there exist differences in cognitive development between singletons and twins born after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Methods: A total of 566 children were recruited for the study, including 388 children (singletons, n=175; twins, n=213) born after IVF and 178 children (singletons, n=87; twins, n=91) born after ICSI. The cognitive development was assessed using the Chinese-Wechsler Intelligence Scale for Children (C-WISC). Results: For all pre-term offspring, all the intelligence quotient (IQ) items between singletons and twins showed no significant differences no matter if they were born after IVF or ICSI. There was a significant difference in the cognitive development of IVF-conceived full-term singletons and twins. The twins born after IVF obtained significantly lower scores than the singletons in verbal IQ (containing information, picture & vocabulary, arithmetic, picture completion, comprehension, and language), performance IQ (containing maze, visual analysis, object assembly, and performance), and full scale IQ (P<0.05). The cognitive development of full-term singletons and twins born after ICSI did not show any significant differences. There was no significant difference between the parents of the singletons and twins in their characteristics where data were collected, including the age of the mothers, the current employment status, the educational backgrounds, and areas of residence. There were also no consistent differences in the duration of pregnancy, sex composition of the children, age, and height between singletons and twins at the time of our study although there existed significant differences between the two groups in the sex composition of the full-term children born after ICSI (P<0.05). Conclusions: Compared to the full-term singletons born after IVF, the full-term twins have lower cognitive development. The cognitive development of full-term singletons and twins born after ICSI did not show any significant differences. For all pre-term offspring, singletons and twins born after IVF or ICSI, the results of the cognitive development showed no significant differences.
PMCID: PMC3891118  PMID: 24390744
Cognitive development; Intelligence quotient (IQ); In vitro fertilization (IVF); Intracytoplasmic sperm injection (ICSI); Singleton; Twins
9.  Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the regulation of fission yeast cytokinesis 
Molecular Biology of the Cell  2013;24(20):3187-3204.
Previous results showed that putative Rho-GEF Gef2 regulates division-site positioning during early cytokinesis in fission yeast. Here Nod1 is identified as a binding partner of Gef2. The two proteins form a complex to regulate division-site positioning and contractile-ring maintenance. In addition, Gef2 binds to GTPases Rho1, Rho4, and Rho5 in vitro.
Cytokinesis is the last step of the cell-division cycle, which requires precise spatial and temporal regulation to ensure genetic stability. Rho guanine nucleotide exchange factors (Rho GEFs) and Rho GTPases are among the key regulators of cytokinesis. We previously found that putative Rho-GEF Gef2 coordinates with Polo kinase Plo1 to control the medial cortical localization of anillin-like protein Mid1 in fission yeast. Here we show that an adaptor protein, Nod1, colocalizes with Gef2 in the contractile ring and its precursor cortical nodes. Like gef2∆, nod1∆ has strong genetic interactions with various cytokinesis mutants involved in division-site positioning, suggesting a role of Nod1 in early cytokinesis. We find that Nod1 and Gef2 interact through the C-termini, which is important for their localization. The contractile-ring localization of Nod1 and Gef2 also depends on the interaction between Nod1 and the F-BAR protein Cdc15, where the Nod1/Gef2 complex plays a role in contractile-ring maintenance and affects the septation initiation network. Moreover, Gef2 binds to purified GTPases Rho1, Rho4, and Rho5 in vitro. Taken together, our data indicate that Nod1 and Gef2 function cooperatively in a protein complex to regulate fission yeast cytokinesis.
PMCID: PMC3806657  PMID: 23966468
10.  Liver Kinase B1 Is Required for Thromboxane Receptor-Dependent Nuclear Factor-κB Activation and Inflammatory Responses 
Arteriosclerosis, thrombosis, and vascular biology  2013;33(6):10.1161/ATVBAHA.113.301296.
Thromboxane A2 receptor (TPr) has been reported to trigger vascular inflammation. Nuclear factor κ B (NF-κB) is a known transcription factor. The aims of the present study were to determine the contributions of NF-κB activation to TPr-triggered vascular inflammation and elucidate the mechanism(s) underlying TPr activation of NF-κB.
Approach and Results
The effects of TPr activators, I-BOP and U46619, on NF-κB activation, phosphorylation of rhoA/ rho-associated kinases and liver kinase B1, cell adhesion and migration, proliferation, and endothelium-dependent vasorelaxation were assayed in cultured human umbilical vein endothelial cells, human monocytes, or isolated mouse aortas. Exposure of human umbilical vein endothelial cells to TPr agonists I-BOP and U46619 induced dose-dependent and time-dependent phosphorylation of inhibitor of κB α in parallel with aberrant expression of inflammatory markers cyclooxygenase-2, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Inhibition of NF-κB by pharmacological or genetic means abolished TPr-triggered expression of inflammatory markers. Consistently, exposure of human umbilical vein endothelial cells to either I-BOP or U46619 significantly increased phosphorylation of inhibitor of κB α, IkappaB kinase, rhoA, rho-associated kinases, and liver kinase B1. Pretreatment of human umbilical vein endothelial cells with the TPr antagonist SQ29548 or rho-associated kinases inhibitor Y27632 or silencing of the LKB1 gene blocked TPr-enhanced phosphorylation of inhibitor of κB α and its upstream kinase, IkappaB kinase. Finally, exposure of isolated mouse aortas to either U46619 or I-BOP enhanced NF-κB activation and vascular inflammation in parallel with reduced endothelium-dependent relaxation in intact vessels.
TPr stimulation instigates aberrant inflammation and endothelial dysfunction via rho-associated kinases/liver kinase B1/IkappaB kinase-dependent NF-κB activation in vascular endothelial cells.
PMCID: PMC3868917  PMID: 23539217
11.  ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis 
PLoS ONE  2013;8(12):e83787.
PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia.
PMCID: PMC3866170  PMID: 24358311
12.  Ginsenoside Rd promotes glutamate clearance by up-regulating glial glutamate transporter GLT-1 via PI3K/AKT and ERK1/2 pathways 
Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has been showed to protect against ischemic cerebral damage both in vitro and in vivo. However, the underlying mechanism of Rd is largely unknown. Excessive extracellular glutamate causes excitatory toxicity, leading to cell death, and neurodegenerative processes after brain ischemia. The clearance of extracellular glutamate by astrocytic glutamate transporter GLT-1 is essential for neuronal survival after stroke. Here we investigated the effects of Rd on the levels of extracellular glutamate and the expression of GLT-1 in vivo and in vitro. After rat middle cerebral artery occlusion, Rd significantly increased the mRNA and protein expression levels of GLT-1, and reduced the burst of glutamate as revealed by microdialysis. Consistently, specific glutamate uptake by cultured astrocytes was elevated after Rd exposure. Furthermore, we showed that Rd increased the levels of phosphorylated protein kinase B (PKB/Akt) and phospho-ERK1/2 (p-ERK1/2) in astrocyte culture after oxygen–glucose deprivation. Moreover, the effect of Rd on GLT-1 expression and glutamate uptake can be abolished by PI3K/AKT agonist LY294002 or ERK1/2 inhibitor PD98059. Taken together, our findings provide the first evidence that Rd can promote glutamate clearance by up-regulating GLT-1 expression through PI3K/AKT and ERK1/2 pathways.
PMCID: PMC3858668  PMID: 24376419
ginsenoside Rd; GLT-1; astrocyte; glutamate; PI3K/AKT; ERK1/2
13.  Successful interventional radiological management of postoperative complications of laparoscopic distal pancreatectomy 
During the past decade, laparoscopic distal pancreatectomy (LDP) has gained increasing acceptance in the surgical community as a viable treatment option for distal pancreatic lesions. However, the possible complication of post-LDP pancreatic leakage remains a challenge, because it may lead to a series of events resulting in intraperitoneal abscess formation, sepsis, pseudoaneurysm formation, and occasional fatal hemorrhage. Dealing with these complications is extremely difficult and not much experience has been reported to date. We report a case involving the aforementioned post-LDP complications successfully managed by interventional radiological techniques while avoiding reoperation. We conclude that these management options are attractive, safe and minimally invasive alternatives to standard protocols.
PMCID: PMC3857473  PMID: 24363541
Interventional radiology; Pancreatectomy; Laparoscopy; Postoperative complications
14.  Inactivation of the EP3 receptor attenuates the Angiotensin II pressor response via decreasing arterial contractility 
The present studies aimed at elucidating the role of prostaglandin E2 (PGE2) receptor subtype 3 (EP3) in regulating blood pressure.
Methods and Results
Mice bearing a genetic disruption of the EP3 gene (EP3−/−) exhibited reduced baseline mean arterial pressure monitored by both tail-cuff and carotid arterial catheterization. The pressor responses induced by EP3 agonists M&B28767 and sulprostone were markedly attenuated in EP3−/− mice, while the reduction of BP induced by PGE2 was comparable in both genotypes. Vasopressor effect of acute or chronic infusion of angiotensin II (AngII) was attenuated in EP3−/− mice. AngII–induced vasoconstriction in mesenteric arteries decreased in EP3−/− group. In mesenteric arteries from wild type mice, AngII–induced vasoconstriction was inhibited by EP3 selective antagonist DG-041 or L798106. The expression of Arhgef-1 is attenuated in EP3 deficient mesenteric arteries. EP3 antagonist DG-041 diminished AngII-induced phosphorylation of MLC20 and MYPT1 in isolated mesenteric arteries. Furthermore, in vascular smooth muscle cells (VSMCs), AngII induced intracellular Ca2+ increase was potentiated by EP3 agonist sulprostone, while inhibited by DG-041.
Activation of the EP3 receptor raises baseline blood pressure and contributes to AngII-dependent hypertension at least partially via enhancing Ca2+ sensitivity and intracellular calcium concentration in VSMCs. Selective targeting of the EP3 receptor may represent a potential therapeutic target for the treatment of hypertension.
PMCID: PMC3565847  PMID: 23065824
EP3; angiotensin II; hypertension; vasoconstriction; calcium
15.  Prevalence of Overweight and Obesity and Their Associations with Socioeconomic Status in a Rural Han Chinese Adult Population 
PLoS ONE  2013;8(11):e79946.
The purpose of this study is to describe the prevalence of overweight, general obesity, and abdominal obesity and examine their associations with socioeconomic status in a rural Chinese adult population.
This cross-sectional study was performed on 15,236 participants ≥ 35 years of age (6,313 men [41.4%] and 8,923 women [58.6%]). Each participant’s weight, height, waist circumference (WC), and hipline circumference (HC) were measured, and demographic and socioeconomic data were collected using questionnaires.
The mean body mass index (BMI) values were 23.31 ± 2.96 and 23.89 ± 3.23 kg m-2 and the mean WC values were 79.13 ± 8.43 and 79.54 ± 8.27 cm for men and women, respectively. The age-standardized prevalence rates of overweight (BMI ≥ 24.0 kg m-2), general obesity (BMI ≥ 28.0 kg m-2), and abdominal obesity (WC ≥ 85 cm for men and ≥ 80 cm for women) were 32.0%, 6.7%, and 27.0% for men and 35.1%, 9.7%, and 48.3% for women, respectively. All gender differences were statistically significant (p < 0.001). In addition, the age-specific prevalence rates of general and abdominal obesity slowly decreased among men but sharply increased among women as age increased (p < 0.001). In subsequent logistic regression analysis, educational level was negatively associated with both general obesity and abdominal obesity among women but positively associated with abdominal obesity among men. No significant correlation was found between obesity and income.
These results suggest a high prevalence of obesity which might differ by gender and age, and an inverse association among women and a mixed association among men noted between education and obesity in our locality. Preventive and therapeutic programs are warranted to control this serious public health problem. The gender-specific characteristics of populations at high-risk of developing obesity should be taken into consideration when designing interventional programs.
PMCID: PMC3818265  PMID: 24224024
16.  Morphological characteristics of Schatzker type IV tibial plateau fractures: a computer tomography based study 
International Orthopaedics  2012;36(11):2355-2360.
The objective of this study was to evaluate the morphological characteristics of Schatzker type IV tibial plateau fractures.
A retrospective analysis of radiographic and computed tomographic data of tibial plateau Schatzker type IV fractures from January 2010 to December 2011 was conducted in a level 1 trauma centre. The medial fracture angle (MFA), surface area percentage (SAP), and medial fracture height (MFH) were measured on CT images using the Picture Archiving and Communication System.
Based on the location of fracture and the MFA, 75 cases of Schatzker type IV fracture were divided into three subtypes: anteromedial fracture (seven cases, 9.3 %), total medial plateau fracture (36 cases, 48 %), and posteromedial fracture (32 cases, 42.7 %). The anteromedial fracture was located on the anterior part of the medial plateau, the average MFA was positive 47.5°, the SAP was 38.3 % and the MFH was 41.6 mm. The total medial plateau fracture usually involved the entire medial plateau, the mean value of MFA was 81.2°, the SAP was 53.9 % and the MFH was 64.0 mm. The posteromedial fracture was located on the posterior part of the medial plateau, the MFA was negative 42.5°, the SAP was 32.4 % and the MFH was 44.8 mm.
The direction and location of Schatzker type IV fractures are highly variable. Proper operative approach and fixation method should be selected based on the morphological characteristics of individual medial plateau fractures.
PMCID: PMC3479292  PMID: 22915237
17.  Insulin receptor substrate signaling suppresses neonatal autophagy in the heart 
The Journal of Clinical Investigation  2013;123(12):5319-5333.
The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.
PMCID: PMC3859408  PMID: 24177427
18.  Comprehensive analysis of the percentage of surface receptors and cytotoxic granules positive natural killer cells in patients with pancreatic cancer, gastric cancer, and colorectal cancer 
Digestive malignancies, especially pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer (CRC), still occur at persistently high rates, and disease progression in these cancers has been associated with tumor immunosurveillance escape. Natural killer (NK) cell dysfunction may be responsible for this phenomenon, however, the exact relationship between tumor immunosurveillance escape in digestive malignancies and NK cell dysfunction remains unclear.
Percentage of the surface receptors NKG2A, KIR3DL1, NKG2D, NKp30, NKp44, NKp46, and DNAM-1, as well as the cytotoxic granules perforin and granzyme B positive NK cells were determined in patients with pancreatic cancer (n = 31), gastric cancer (n = 31), and CRC (n = 32) prior to surgery and healthy controls (n = 31) by multicolor flow cytometry. Independent t-tests or Mann-Whitney U-tests were used to compare the differences between the patient and healthy control groups, as well as the differences between patients with different pathologic features of cancer.
Percentage of NKG2D, NKp30, NKp46, and perforin positive NK cells was significantly down-regulated in patients with PC compared to healthy controls, as well as GC and CRC; reduced levels of these molecules was associated with indicators of disease progression in each malignancy (such as histological grade, depth of invasion, lymph node metastasis). On the contrary, percentage of KIR3DL1 positive NK cells was significantly increased in patients with PC, as well as GC and CRC, but was not associated with any indicators of disease progression.
Altered percentage of surface receptors and cytotoxic granules positive NK cells may play a vital role in tumor immunosurveillance escape by inducing NK cell dysfunction in patients with PC, GC, and CRC.
PMCID: PMC3854023  PMID: 24138752
Cytotoxic granules; Digestive malignancies; NK cells; Surface receptors
19.  Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging 
Aging cell  2012;11(5):741-750.
A spectral analysis approach was developed for detailed study of time-resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young's modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti-beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old versus young monkeys had elevated elasticity (21.6 kPa vs 3.5 kPa or a 612% elevation in elastic modulus) and adhesion (86 pN vs 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7×10-3 to 1.9×10-2 Hz in old and 8.4×10-4 to 1.5×10-2 in young) and showed that the amplitude of oscillation was larger (p<0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating involvement of actin-myosin driven processes. In conclusion, these data demonstrate the efficacy of time-resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real-time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests mechanisms governing elasticity and adhesion are coupled and affected differentially during aging which may link these events to changes in vascular stiffness.
PMCID: PMC3444643  PMID: 22639979
Fibronectin; integrins; vascular smooth muscle cell contractile function; Young's modulus; cytoskeleton; mechanotransduction; extracellular matrix adhesion; force measurement; atomic force microscopy
20.  Inducible Overexpression of GLUT1 Prevents Mitochondrial Dysfunction and Attenuates Structural Remodeling in Pressure Overload but Does Not Prevent Left Ventricular Dysfunction 
Increased glucose transporter 1 (GLUT1) expression and glucose utilization that accompany pressure overload‐induced hypertrophy (POH) are believed to be cardioprotective. Moreover, it has been shown that lifelong transgenic overexpression of GLUT1 in the heart prevents cardiac dysfunction after aortic constriction. The relevance of this model to clinical practice is unclear because of the life‐long duration of increased glucose metabolism. Therefore, we sought to determine if a short‐term increase in GLUT1‐mediated myocardial glucose uptake would still confer cardioprotection if overexpression occurred at the onset of POH.
Methods and Results
Mice with cardiomyocyte‐specific inducible overexpression of a hemagglutinin (HA)‐tagged GLUT1 transgene (G1HA) and their controls (Cont) were subjected to transverse aortic constriction (TAC) 2 days after transgene induction with doxycycline (DOX). Analysis was performed 4 weeks after TAC. Mitochondrial function, adenosine triphosphate (ATP) synthesis, and mRNA expression of oxidative phosphorylation (OXPHOS) genes were reduced in Cont mice, but were maintained in concert with increased glucose utilization in G1HA following TAC. Despite attenuated adverse remodeling in G1HA relative to control TAC mice, cardiac hypertrophy was exacerbated in these mice, and positive dP/dt (in vivo) and cardiac power (ex vivo) were equivalently decreased in Cont and G1HA TAC mice compared to shams, consistent with left ventricular dysfunction. O‐GlcNAcylation of Ca2+ cycling proteins was increased in G1HA TAC hearts.
Short‐term cardiac specific induction of GLUT1 at the onset of POH preserves mitochondrial function and attenuates pathological remodeling, but exacerbates the hypertrophic phenotype and is insufficient to prevent POH‐induced cardiac contractile dysfunction, possibly due to impaired calcium cycling.
PMCID: PMC3835233  PMID: 24052497
cardiac hypertrophy; contractile function; glucose utilization; mitochondria
21.  Association between UCP3 gene polymorphisms and nonalcoholic fatty liver disease in Chinese children 
AIM: To confirm the hypothesis that polymorphisms of the uncoupling protein 3 (UCP3) gene are associated with the occurrence of nonalcoholic fatty liver disease (NAFLD).
METHODS: A total of 250 NAFLD patients (147 males and 103 females) and 200 healthy individuals who served as controls (control, 109 males and 91 females), aged between 6 and 16 years were enrolled in this study. The four non-synonymous single nucleotide polymorphisms (SNPs) in the UCP3 gene polymorphisms of rs1726745, rs3781907, rs11235972 and rs1800849, were genotyped using MassArray. Body mass index (BMI), waist and hip circumference, blood pressure (BP), fasting blood glucose (FBG), insulin and lipid profiles were measured and B-ultrasound examination was performed in all subjects.
RESULTS: NAFLD patients showed risk factors for metabolic syndrome: elevated BMI, waist-to-hip ratio, BP, FBG, homeostasis model assessment-estimated insulin resistance, total triglyceride, total cholesterol and low-density lipoprotein-cholesterol, while decreased high-density lipoprotein-cholesterol level compared with the control group. The GG genotype distributions of rs11235972 in the NAFLD group differed significantly from that in the control group. We found that waist circumference between CC (58.76 ± 6.45 cm) and CT+TT (57.00 ± 5.59 cm), and hip circumference between CC (71.28 ± 7.84 cm) and CT+TT genotypes (69.06 ± 7.75 cm) were significantly different with and without rs1800849 variation (P < 0.05).
CONCLUSION: A higher prevalence of rs11235972 GG genotype was observed in the NAFLD group compared with the control group. No differences were observed for the other SNPs. However, there was a significant difference in body height in addition to waist and hip circumference between the CC (mutant type group) and CT+TT group with and without rs1800849 variation.
PMCID: PMC3793144  PMID: 24124336
Nonalcoholic fatty liver disease; Uncoupling protein 3; Single nucleotide polymorphisms
22.  Persistent inflammation increases GABA-induced depolarization of rat cutaneous dorsal root ganglion neurons in vitro 
Neuroscience  2012;220:330-340.
Persistent inflammation is associated with a shift in spinal GABAA signaling from inhibition to excitation such that GABAA-receptor activation contributes to inflammatory hyperalgesia. We tested the hypothesis that the primary afferent is the site of the persistent inflammation-induced shift in GABAA signaling which is due to a Na+-K+-Cl−-co-transporter (NKCC1)-dependent depolarization of the GABAA current equilibrium potential (EGABA). Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed (3 days after a subcutaneous injection of complete Freund’s adjuvant) adult male rats were studied with Ca2+ imaging, western blot and gramicidin perforated patch recording. GABA evoked a Ca2+ transient in a subpopulation of small- to medium-diameter capsaicin-sensitive cutaneous neurons. Inflammation was associated with a significant increase in the magnitude of GABA-induced depolarization as well as the percentage of neurons in which GABA evoked a Ca2+ transient. There was no detectable change in NKCC1 protein or phosphoprotein at the whole ganglia level. Furthermore, the increase in excitatory response was comparable in both HEPES- and HCO3−-buffered solutions, but was only associated with a depolarization of EGABA in HCO3−-based solution. In contrast, under both recording conditions, the excitatory response was associated with an increase in GABAA current density, a decrease in low threshold K+ current density, and resting membrane potential depolarization. Our results suggest that increasing K+ conductance in afferents innervating a site of persistent inflammation may have greater efficacy in the inhibition of inflammatory hyperalgesia than attempting to drive a hyperpolarizing shift in EGABA.
PMCID: PMC3412885  PMID: 22728089
Inflammatory pain; nociceptor sensitization; neuroplasticity
23.  Inter-observer reliability assessment of the Schatzker, AO/OTA and three-column classification of tibial plateau fractures 
The purpose of our study was to evaluate inter-observer reliability of the Three-Column classifications with conventional Schatzker and AO/OTA of Tibial Plateau Fractures.
50 cases involving all kinds of the fracture patterns were collected from 278 consecutive patients with tibial plateau fractures who were internal fixed in department of Orthopedics and Trauma III in Shanghai Sixth People’s Hospital. The series were arranged randomly, numbered 1 to 50. Four observers were chosen to classify these cases. Before the research, a classification training session was held to each observer. They were given as much time as they required evaluating the radiographs accurately and independently. The classification choices made at the first viewing were not available during the second viewing. The observers were not provided with any feedback after the first viewing. The kappa statistic was used to analyze the inter-observer reliability of the three fracture classification made by the four observers.
The mean kappa values for inter-observer reliability regarding Schatzker classification was 0.567 (range: 0.513–0.589), representing “moderate agreement”. The mean kappa values for inter-observer reliability regarding AO/ASIF classification systems was 0.623 (range: 0.510–0.710) representing “substantial agreement”. The mean kappa values for inter-observer reliability regarding Three-Column classification systems was 0.766 (range: 0.706–0.890), representing “substantial agreement”.
Three-Column classification, which is dependent on the understanding of the fractures using CT scans as well as the 3D reconstruction can identity the posterior column fracture or fragment. It showed “substantial agreement” in the assessment of inter-observer reliability, higher than the conventional Schatzker and AO/OTA classifications. We finally conclude that Three-Column classification provides a higher agreement among different surgeons and could be popularized and widely practiced in other clinical centers.
PMCID: PMC3848575  PMID: 24025650
Inter-observer reliability; Tibial plateau fracture; Classification
24.  Early embryonic lethality of mice with disrupted transcription cofactor PIMT/NCOA6IP/Tgs1 gene 
Mechanisms of development  2012;129(9-12):193-207.
PIMT (also known as PIPMT/NCOA6IP/Tgs1), first isolated as a transcription coactivator PRIP (NCOA6)-interacting 96-kDa protein with RNA-binding property, possesses RNA methyltransferase activity. As a transcription coactivator binding protein, PIMT enhances the nuclear receptor transcriptional activity and its methyltransferase property is involved in the formation of the 2,2,7-trimethylguanosine cap of non-coding small RNAs, but the in vivo functions of this gene have not been fully explored. To elucidate the biological functions, we used gene targeting to generate mice with a disrupted PIMT/Tgs1 gene. Disruption of PIMT gene results in early embryonic lethality due to impairment of development around the blastocyst and uterine implantation stages. We show that PIMT is expressed in all cells of the E3.5 day blastocyst in the mouse. PIMT null mutation abolished PIMT expression in all cells of the blastocyst and caused a reduction in the expression of Oct4 and Nanog transcription factor proteins in the E3.5 blastocyst resulting in the near failure to form inner cell mass (ICM). With conditional deletion of PIMT gene, mouse embryonic fibroblasts (MEFs) exhibit defective wound healing in the scratch assay and a reduction in cell proliferation due to decreased G0/G1 transition and G2/M phase cell cycle arrest. We conclude that PIMT/NCOA6IP, which is expressed in all cells of the 3.5 day stage blastocyst, is indispensable for early embryonic development.
PMCID: PMC3503541  PMID: 22982455
PIMT/ NCOA6IP/Tgs1; Embryonic lethality; Apoptosis; Blastocyst; G2/M phase arrest; Mouse Embryonic Fibroblasts; Defective wound healing
25.  Mitochondrial DNA haplogroup associated with sperm motility in the Han population 
Asian Journal of Andrology  2013;15(5):630-633.
In this study, we aimed to determine whether the main mitochondrial DNA (mtDNA) haplogroups of the Han people have an impact on spermatozoa motility. We recruited 312 men who were consecutively admitted to two affiliated hospitals of College of Medicine, Zhejiang University from May 2011 to April 2012 as part of fertility investigations. Semen and whole blood samples were collected from the men. We determined the mtDNA haplogroups by analysing the sequences of mtDNA hypervariable segment I and testing diagnostic polymorphisms in the mtDNA coding region with DNA probes. No significant differences were found in the clinical characteristics of the mtDNA haplogroup R and non-R (P>0.05). Our results suggest that mtDNA haplogroup R is a strong independent predictor of sperm motility in the Han population, conferring a 2.97-fold (95% confidence interval: 1.74–4.48, P<0.001) decreased chance of asthenozoospermia compared with those without haplogroup R.
PMCID: PMC3881638  PMID: 23912313
asthenozoospermia; haplogroup; mitochondrial DNA (mtDNA)

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