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1.  ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY 
Traffic (Copenhagen, Denmark)  2015;17(1):40-52.
The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein–coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin1 and β-arrestin2, indicating that activated S1PR1 follows the canonical route of endocytosis for GPCR's.
doi:10.1111/tra.12343
PMCID: PMC4688054  PMID: 26481905
G-protein coupled receptors; vesicular traffic; down regulation; clathrin
2.  Enhanced pro‐protein convertase subtilisin/kexin type 9 expression by C‐reactive protein through p38MAPK‐HNF1α pathway in HepG2 cells 
Abstract
Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.
doi:10.1111/jcmm.12931
PMCID: PMC5134380  PMID: 27633999
C‐reactive protein; pro‐protein convertase subtilisin/kexin type 9; mitogen‐activated protein kinase
3.  Safety and immunogenicity of a new 13-valent pneumococcal conjugate vaccine versus a licensed 7-valent pneumococcal conjugate vaccine: a study protocol of a randomised non-inferiority trial in China 
BMJ Open  2016;6(10):e012488.
Introduction
The invasive pneumococcal diseases (IPDs) caused by Streptococcus pneumoniae pose an enormous threat to children under 5 years of age. However, routine use of pneumococcal conjugate vaccines could aid in reducing the incidence of IPDs. The purpose of this clinical trial is to assess the non-inferiority of the investigational 13-valent pneumococcal conjugate vaccine (PCV13) to the currently licensed 7-valent pneumococcal conjugate vaccine (PCV7).
Methods and analysis
1040 infants will receive a three-dose series of either PCV13 or PCV7 at ages 3, 4 and 5 months, respectively, and a booster dose at 12–15 months. Primary end points are the percentage of participants reaching a serotype-specific IgG concentration of ≥0.35 µg/mL and the IgG antibody geometric mean concentrations (GMCs) measured 30 days after the primary immunisation. Secondary end points include the percentage of vaccine recipients reaching a serotype-specific IgG concentration threshold of 1.0 µg/mL, the percentage of participants reaching the pneumococcal opsonophagocytic assay (OPA) titre threshold of 1:8, and the geometric mean titres (GMTs) of OPA measured 30 days after primary and booster doses. The number of standard IgG responders and IgG GMCs measured 30 days after the booster immunisation will also be determined. To evaluate differences between two groups, the sequential testing of the non-inferiority of PCV13 for the seven common serotypes and its effectiveness in treating the six additional serotypes will be performed.
Ethics and dissemination
Ethics approvals have been granted by the Ethics Committees at the three provinces involved in this study: Shanxi, Henan and Hebei. The trial will be reported in accordance with the CONSORT guidance.
Trial registration number
NCT02736240.
doi:10.1136/bmjopen-2016-012488
PMCID: PMC5073664  PMID: 27798013
Pneumococcal conjugate vaccine; Sequential test; Non-inferiority
4.  Dynamic Phosphoproteome Analysis of Seedling Leaves in Brachypodium distachyon L. Reveals Central Phosphorylated Proteins Involved in the Drought Stress Response 
Scientific Reports  2016;6:35280.
Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant’s abiotic stress response through phosphorylation modification.
doi:10.1038/srep35280
PMCID: PMC5066223  PMID: 27748408
5.  Chronic leucine supplementation improves lipid metabolism in C57BL/6J mice fed with a high-fat/cholesterol diet 
Food & Nutrition Research  2016;60:10.3402/fnr.v60.31304.
Background
Leucine supplementation has been reported to improve lipid metabolism. However, lipid metabolism in adipose tissues and liver has not been extensively studied for leucine supplementation in mice fed with a high-fat/cholesterol diet (HFCD).
Design
C57BL/6J mice were fed a chow diet, HFCD, HFCD supplemented with 1.5% leucine (HFCD+1.5% Leu group) or 3% leucine (HFCD+3% Leu group) for 24 weeks. The body weight, peritoneal adipose weight, total cholesterol (TC), triglyceride in serum and liver, and serum adipokines were analyzed. In addition, expression levels of proteins associated with hepatic lipogenesis, adipocyte lipolysis, and white adipose tissue (WAT) browning were determined.
Results
Mice in the HFCD group developed obesity and deteriorated lipid metabolism. Compared with HFCD, leucine supplementation lowered weight gain and TC levels in circulation and the liver without changing energy intake. The decrease in body fat was supported by histological examination in the WAT and liver. Furthermore, serum levels of proinflammatory adipokines, such as leptin, IL-6, and tumor necrosis factor-alpha, were significantly decreased by supplemented leucine. At the protein level, leucine potently decreased the hepatic lipogenic enzymes (fatty acid synthase and acetyl-coenzyme A carboxylase) and corresponding upstream proteins. In epididymal WAT, the reduced expression levels of two major lipases by HFCD, namely phosphorylated hormone-sensitive lipase and adipose triglyceride lipase, were reversed when leucine was supplemented. Uncoupling protein 1, β3 adrenergic receptors, peroxisome proliferator-activated receptor g coactivator-1α, and fibroblast growth factor 21 were involved in the thermogenic program and WAT browning. Leucine additionally upregulated their protein expression in both WAT and interscapular brown adipose tissue.
Conclusion
This study demonstrated that chronic leucine supplementation reduced the body weight and improved the lipid profile of mice fed with a HFCD. This beneficial effect was ascribed to hepatic lipogenesis, adipocyte lipolysis, and WAT browning.
doi:10.3402/fnr.v60.31304
PMCID: PMC5018683  PMID: 27616737
leucine; lipid metabolism; brown adipose tissue; white adipose tissue; WAT browning
6.  Long-term Follow-up of 5 Survivors after the First Outbreak of Human Infections with Avian Influenza A(H7N9) Virus in Shanghai, China 
Chinese Medical Journal  2016;129(17):2128-2130.
doi:10.4103/0366-6999.189061
PMCID: PMC5009600  PMID: 27569243
H7N9 Virus; High-resolution Computed Tomography; Prognosis; Pulmonary Function Tests
7.  HDL subfractions and very early CAD: novel findings from untreated patients in a Chinese cohort 
Scientific Reports  2016;6:30741.
Coronary artery disease (CAD) in very young individuals is a rare disease associated with poor prognosis. However, the role of specific lipoprotein subfractions in very young CAD patients (≤45 years) is not established yet. A total of 734 consecutive CAD subjects were enrolled and were classified as very early (n = 81, ≤45), early (n = 304, male: 45–55; female: 45–65), and late (n = 349, male: >55; female: >65) groups. Meanwhile, a group of non-CAD subjects were also enrolled as controls (n = 56, ≤45). The lipoprotein separation was performed using Lipoprint System. As a result, the very early CAD patients have lower large high-density lipoprotein (HDL) subfraction and higher small low-density lipoprotein (LDL) subfraction (p < 0.05). Although body mass index was inversely related to large HDL subfraction, overweight did not influence its association with very early CAD. In the logistic regression analysis, large HDL was inversely [OR 95% CI: 0.872 (0.825–0.922)] while small LDL was positively [1.038 (1.008–1.069)] related to very early CAD. However, after adjusting potential confounders, the association was only significant for large HDL [0.899 (0.848–0.954)]. This study firstly demonstrated that large HDL subfraction was negatively related to very early CAD suggestive of its important role in very early CAD incidence.
doi:10.1038/srep30741
PMCID: PMC4973286  PMID: 27489174
8.  Doxorubicin Resistance in Breast Cancer is Driven by Light at Night Induced Disruption of the Circadian Melatonin Signal 
Journal of pineal research  2015;59(1):60-69.
Chemotherapeutic resistance, particularly to doxorubicin (Dox), represents a major impediment to successfully treating breast cancer and is linked to elevated tumor metabolism and tumor over-expression and/or activation of various families of receptor- and non-receptor-associated tyrosine kinases. Disruption of circadian time structure and suppression of nocturnal melatonin production by dim light exposure at night (dLEN), as occurs with shift work, and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of an array of diseases, including breast cancer. Melatonin inhibits human breast cancer growth via mechanisms that include the suppression of tumor metabolism and inhibition of expression or phospho-activation of the receptor kinases AKT and ERK1/2 and various other kinases and transcription factors. We demonstrate in tissue-isolated estrogen receptor alpha-positive (ERα+) MCF-7 human breast cancer xenografts, grown in nude rats maintained on a light/dark cycle of LD 12:12 in which dLEN is present during the dark phase (suppressed endogenous nocturnal melatonin), a significant shortening of tumor latency-to-onset, increased tumor metabolism and growth, and complete intrinsic resistance to Dox therapy. Conversely, a LD 12:12dLEN environment incorporating nocturnal melatonin replacement resulted in significantly lengthened tumor latency-to-onset, tumor regression, suppression of nighttime tumor metabolism, and kinase and transcription factor phosphorylation, while Dox sensitivity was completely restored. Melatonin acts as both a tumor metabolic inhibitor and circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to Dox and drive tumor regression indicating that dLEN-induced circadian disruption of nocturnal melatonin production contributes to a complete loss of tumor sensitivity to Dox chemotherapy.
doi:10.1111/jpi.12239
PMCID: PMC4490975  PMID: 25857269
Melatonin; Doxorubicin; Circadian; Breast; Warburg
9.  Relationship of lipid and lipoprotein ratios with coronary severity in patients with new on-set coronary artery disease complicated with type 2 diabetics 
Background
Diabetes mellitus (DM) is associated with coronary artery disease (CAD) progression. Although previous studies have demonstrated the association of lipid and lipoprotein ratios with CAD, no data are currently available concerning the relationship between lipid and lipoprotein ratios and the severity of new on-set CAD in diabetics. Therefore, the aim of the present study was to investigate the usefulness of lipid and lipoprotein ratios in predicting the severity of CAD in patients with type 2 DM (T2DM).
Methods
A total of 380 consecutive T2DM patients with new on-set CAD were enrolled in the present study. Then, they were classified into the three groups according to Gensini score (GS) tertiles. The relationship between lipid and lipoprotein ratios currently used and the GS was investigated.
Results
Positive correlations of natural log-transformed GS (lnGS) with apolipoprotein B to apoA-I ratio (apoB/apoA-I), non-high-density lipoprotein cholesterol to apoA-I ratio (non-HDL-C/apoA-I), and low-density lipoprotein cholesterol to apoA-I ratio (LDL-C/apoA-I) were found (r = 0.18, 0.13, 0.12, respectively, all P < 0.05). Multivariate logistic analysis indicated apoB/apoA-I as the strongest predictor for high GS (OR = 5.67, 95% CI: 1.45–23.92, P = 0.003). Area under receivers operating characteristic curve of apoB/apoA-I was 0.63 (95% CI: 0.60–0.66, P = 0.001) for predicting high GS. The optimal cutoff value of apoB/apoA-I to predict high GS was 0.72 with the sensitivity of 61.2% and the specificity of 62.1%.
Conclusions
Lipid and lipoprotein ratios might be useful for predicting the severity of new on-set CAD in T2DM patients, and the apoB/apoA-I appeared as the most significant predictor in this population.
doi:10.11909/j.issn.1671-5411.2016.08.007
PMCID: PMC5067430  PMID: 27781059
Coronary artery disease; Lipid; Lipoprotein; Type 2 diabetes mellitus
10.  Efficacy of Intra-aortic Balloon Pump before versus after Primary Percutaneous Coronary Intervention in Patients with Cardiogenic Shock from ST-elevation Myocardial Infarction 
Chinese Medical Journal  2016;129(12):1400-1405.
Background:
Previous studies showed that patients with cardiogenic shock (CS) from ST-elevation acute myocardial infarction (STEMI) supported by intra-aortic balloon pump (IABP) before primary percutaneous coronary intervention (PCI) decreased the risk of in-hospital mortality than patients who received IABP after PCI. However, little evidence is available on the optimal order of IABP insertion and primary PCI. The aim of this study was to investigate the impact of the sequence of IABP support and PCI and its association with major adverse cardiac and cerebrovascular events (MACCEs).
Methods:
Data were obtained from 218 consecutive patients with CS due to STEMI in Beijing Anzhen Hospital between 2008 and 2014, who were treated with IABP and PCI. The patients were divided into two groups: Group A in whom IABP received before PCI (n = 106) and Group B in whom IABP received after PCI (n = 112). We evaluated the myocardial perfusion using myocardial blush grade and resolution of ST-segment elevation. The primary endpoint was 12-month risk of MACCE.
Results:
Most baseline characteristics were similar in patients between the two groups. However, patients received IABP before PCI were associated with a delay of door-to-balloon time (DBT) and higher troponin I level (P < 0.05). However, myocardial perfusion was significantly improved in patients treated with IABP before PCI (P < 0.05). Overall, IABP support before PCI was not associated with significantly lower risk of MACCE (P > 0.05). In addition, risk of all-cause mortality, bleeding, and acute kidney injury (AKI) was similar between two groups (P > 0.05). Multivariate analysis showed that DBT (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.1–4.8, P = 0.04), IABP support after PCI (OR 5.7, 95% CI 2.7–8.4, P = 0.01), and AKI (OR 7.4, 95% CI 4.9–10.8, P = 0.01) were the independent predictors of mortality at 12-month follow-up.
Conclusions:
Early IABP insertion before primary PCI is associated with improved myocardial perfusion although DBT increases. IABP support before PCI does not confer a 12-month clinical benefit when used for STEMI with CS.
doi:10.4103/0366-6999.183428
PMCID: PMC4910361  PMID: 27270533
Acute Myocardial Infarction; Cardiogenic Shock; Intra-aortic Balloon Counterpulsation; Mortality; Percutaneous Coronary Intervention
11.  The Gαi AND Gαq Proteins Mediate the Effects of Melatonin on Steroid/Thyroid Hormone Receptor Transcriptional Activity and Breast Cancer Cell Proliferation 
Journal of pineal research  2008;45(4):476-488.
Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors (ERα and RARα, but not ERβ) in MCF-7, T47D and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the Gαi2, Gαi3, Gαq and Gαll proteins, and via activation of Gαi2 proteins, melatonin suppresses forskolin-induced cyclic AMP (cAMP) production, while melatonin activation of Gαq, is able to inhibit phospholipid hydrolysis and ATP’s induction of inositol triphosphate (IP3) production in MCF-7 breast cancer cells. Employing dominant-negative (DN) and dominant-positive (DP) forms of these G proteins we demonstrate that Gαi2 proteins mediate the suppression of estrogen-induced ERα transcriptional activity by melatonin, while the Gq protein mediates the enhancement of retinoid-induced RARα transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both Gαi2 and Gαq proteins.
doi:10.1111/j.1600-079X.2008.00620.x
PMCID: PMC4879591  PMID: 18705646
melatonin; G proteins; estrogen receptor alpha; breast cancer
12.  GCN5 modulates osteogenic differentiation of periodontal ligament stem cells through DKK1 acetylation in inflammatory microenvironment 
Scientific Reports  2016;6:26542.
Periodontal ligament stem cells (PDLSCs) from periodontitis patients showed defective osteogenic differentiation. However, the mechanism of impaired osteogenic differentiation of PDLSCs in inflammatory microenvironments is still unclear. In this study, we found that inflammation in the microenvironment resulted in downregulation of histone acetyltransferase GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Previous study showed activated Wnt/β-cateinin pathway of PDLSCs resulted in defective osteogenic differentiation. Here we found knockdown of GCN5 decreased the expression of DKK1, an inhibitor of Wnt/β-cateinin pathway, thus activated Wnt/β-catenin pathway of PDLSCs. Mechanistically, GCN5 regulated DKK1 expression by acetylation of Histone H3 lysine 9 (H3K9) and Histone H3 lysine 14 (H3K14) at its promoter region. Interestingly, we found that in vivo injection of aspirin rescued the periodontitis of rats through inhibiting inflammation and upregulating GCN5 expression. Furthermore, aspirin treatment of PDLSCs upregulated GCN5 expression and increased osteogenic differentiation of PDLSCs. In conclusion, GCN5 plays a protective role in periodontitis through acetylation of DKK1 and applying drugs targeting GCN5, such as aspirin, could be a new approach for periodontitis treatment.
doi:10.1038/srep26542
PMCID: PMC4877597  PMID: 27216891
13.  Molecular deficiency (ies) in MT1 melatonin signaling pathway underlies the melatonin-unresponsive phenotype in MDA-MB-231 human breast cancer cells 
Journal of pineal research  2014;56(3):246-253.
Melatonin, has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its anti-proliferative effects have been well-studied in MCF-7 human breast cancer cells and several other estrogen receptor α (ERα)-positive human breast cancer cell lines. However, the MDA-MB-231 breast cancer cell line, an ERα negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin’s growth-suppressive effect in vitro. Here we examined the effect of melatonin on the cell proliferation of several ERα-negative breast cancer cell lines including MDA-MB-231, BT-20 and SK-BR-3 cells. Although the MT1 G-protein-coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK-BR-3 cells without having any significant effect on the growth of MDA-MB-231 and BT-20 cells. We confirmed that the MT1-associated Gα proteins are expressed in MDA-MB-231 cells. Further studies demonstrated that the melatonin-unresponsiveness in MDA-MB-231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.
doi:10.1111/jpi.12117
PMCID: PMC4868402  PMID: 24372669
Melatonin; melatonin receptor (MT1)
14.  Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9 
Scientific Reports  2016;6:25029.
CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.
doi:10.1038/srep25029
PMCID: PMC4844959  PMID: 27113799
15.  Chimerism in piglets developed from aggregated cloned embryos 
FEBS Open Bio  2016;6(4):285-302.
Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP‐cell derived embryos with two tdTomato‐cell derived embryos at the 4‐cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP‐expressing cells, and this phenomenon was possibly due to the hyper‐methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity‐related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell.
doi:10.1002/2211-5463.12037
PMCID: PMC4821359  PMID: 27239442
aggregation; chimerism; development; somatic cell nuclear transfer
16.  CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts 
Scientific Reports  2016;6:22024.
Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for in vivo studies of artificial lens safety and drug interactions. Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment.
doi:10.1038/srep22024
PMCID: PMC4766569  PMID: 26912477
17.  Increasing Pain Sensation Eliminates the Inhibitory Effect of Depression on Evoked Pain in Rats 
Although previous studies have suggested that depression may be associated with inhibition of evoked pain but facilitation of spontaneous pain, the mechanisms underlying these relationships are unclear. The present study investigated whether the difference between evoked and spontaneous pain on sensory (descending inhibition) and affective (avoidance motivation) components contributes to the divergent effects of depression on them. Depressive-like behavior was produced in male Wistar rats by unpredictable chronic mild stress (UCMS). Tone-laser conditioning and formalin-induced conditioned place avoidance (F-CPA) were used to explore avoidance motivation in evoked and spontaneous pain, respectively. Behavioral pharmacology experiments were conducted to examine descending inhibition of both evoked (thermal stimulation) and spontaneous pain behavior (formalin pain). The results revealed that the inhibitory effect of depression on evoked pain was eliminated following repeated thermal stimuli. Avoidance behavior in the tone-laser conditioning task was reduced in UCMS rats, relative to controls. However, avoidance motivation for formalin pain in the UCMS group was similar to controls. 5-HT1A receptor antagonism interfered with inhibition of pain responses over time. The present study demonstrated that the inhibitory effect of depression on evoked pain dissipates with increased nociception and that the sensory-discriminative and affective-motivational components of pain are jointly involved in the divergent effects of depression on pain.
doi:10.3389/fnbeh.2016.00183
PMCID: PMC5039174  PMID: 27733820
unpredictable chronic mild stress; pain; serotonin; descending inhibition; avoidance motivation
18.  Melatonin: an Inhibitor of Breast Cancer 
Endocrine-related cancer  2015;22(3):R183-R204.
This review discusses recent work on melatonin-mediated circadian regulation and metabolic and molecular signaling mechanisms involved in human breast cancer growth and associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1 receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin, via the MT1 receptor, suppresses ERα mRNA expression and ERα transcriptional activity. As well, melatonin regulates the transactivation of other members of the nuclear receptor super-family, estrogen metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (Warburg effect), and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways including inhibition of p38 MAPK and repression of epithelial-to-mesenchymal transition. Studies demonstrate that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models indicate that LEN induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer to drive breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms underpinning the epidemiologic demonstration of elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LEN.
doi:10.1530/ERC-15-0030
PMCID: PMC4457700  PMID: 25876649
Melatonin; Breast Cancer; Nuclear receptors; Molecular Signaling; Circadian Disruption; Genomic instability; Drug resistance
19.  Systemic Inflammatory Markers Are Closely Associated with Atherogenic Lipoprotein Subfractions in Patients Undergoing Coronary Angiography 
Mediators of Inflammation  2015;2015:235742.
Objective. To investigate the relationship between inflammatory markers and atherogenic lipoprotein subfractions. Methods. We studied 520 eligible subjects who were not receiving any lipid-lowering therapy. The inflammatory markers including white blood cell (WBC) count, high-sensitivity C-reactive protein (hs-CRP), fibrinogen, erythrocyte sedimentation rate (ESR), and D-dimer were measured. A multimarker inflammatory index was developed. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) separation processes were performed using Lipoprint System. Results. In age- and sex-adjusted analysis, several inflammatory markers (WBC count, hs-CRP, fibrinogen, and ESR) were positively related to circulating non-HDL cholesterol and remnant cholesterol (p < 0.05, all). Among lipoprotein subfractions, we observed a positive association of inflammatory markers with very low-density lipoprotein cholesterol, small LDL cholesterol, and LDL score (p < 0.05, all). Meanwhile, a negative association was detected between inflammatory markers and mean LDL particle size (p < 0.05) or large HDL cholesterol (p < 0.05). Moreover, we found that the relationships between multimarker index quartiles and small LDL cholesterol, LDL score, and mean LDL particle size were slightly stronger in patients with CAD. Conclusions. Systemic inflammatory markers are positively correlated with small LDL cholesterol and LDL score while being negatively linked with mean LDL particle size and large HDL cholesterol, highlighting the potential contribution to increased cardiovascular risk.
doi:10.1155/2015/235742
PMCID: PMC4673360  PMID: 26688615
20.  Immunohistochemistry and microsatellite instability analysis in molecular subtyping of colorectal carcinoma based on mismatch repair competency 
Mismatch repair defective (MMRd) colorectal carcinoma (CRC) is a distinct molecular phenotype of colorectal cancer, including 12% of sporadic CRC and 3% of Lynch Syndrome. In order to investigate the clinicopathological characteristics of MMRd colorectal carcinoma, and to find the most effective method for preliminary screening, 296 CRC fulfilled revised Bethesda Guideline (RB) were selected from 1450 CRCs to perform both IHC staining for MLH1, MSH2, MSH6, PMS2 and MSI analysis. Sixty-eight tumors were classified as MSI-H by MSI test. Colorectal carcinomas with MSI-H were prone to be proximal located, poorly differentiated, and relatively early staged, with infrequent metastasis to lymph node as well as to distant organs, compared with MSS ones. All of the 68 MMRd CRCs presented abnormal expression of at least one mismatch repair protein (MMRP), with 48 concurrent negative of MLH1 and PMS2, 14 concurrent negative of MSH2 and MSH6, 4 isolated negative of MSH6, 1 isolated negative of PMS2, and 1 concurrent negative of 4 MMRPs. All of the MLH1 negative tumors also showed abnormal expression of PMS2. All of the MSH2 negative cases also presented negative expression of MSH6. The sensitivity and specificity of the 2-antibody IHC test contained only PMS2 and MSH6 for screening for MMRd CRC were 100% and 98.2% respectively, exactly the same as that of the 4-antibody IHC test with all of the 4 MMRPs. The diagnostic accordance rate of the 2-antibody approach and MSI analysis was 98.6%. In conclusion, MMRd CRC has characteristic clinicopathological features different from MSS CRCs. The 2-antibody IHC approach containing MSH6 and PMS2 is the most easy and effective way to detecting MMR deficiency in CRC.
PMCID: PMC4723875  PMID: 26885030
Mismatch repair; microsatellite instability; Lynch syndrome; immunohistochemistry; molecular phenotype; CRC
21.  Association of Big Endothelin-1 with Coronary Artery Calcification 
PLoS ONE  2015;10(11):e0142458.
Background
The coronary artery calcification (CAC) is clinically considered as one of the important predictors of atherosclerosis. Several studies have confirmed that endothelin-1(ET-1) plays an important role in the process of atherosclerosis formation. The aim of this study was to investigate whether big ET-1 is associated with CAC.
Methods and Results
A total of 510 consecutively admitted patients from February 2011 to May 2012 in Fu Wai Hospital were analyzed. All patients had received coronary computed tomography angiography and then divided into two groups based on the results of coronary artery calcium score (CACS). The clinical characteristics including traditional and calcification-related risk factors were collected and plasma big ET-1 level was measured by ELISA. Patients with CAC had significantly elevated big ET-1 level compared with those without CAC (0.5±0.4 vs. 0.2±0.2, P<0.001). In the multivariate analysis, big ET-1 (Tertile 2, HR = 3.09, 95% CI 1.66–5.74, P <0.001, Tertile3 HR = 10.42, 95% CI 3.62–29.99, P<0.001) appeared as an independent predictive factor of the presence of CAC. There was a positive correlation of the big ET-1 level with CACS (r = 0.567, p<0.001). The 10-year Framingham risk (%) was higher in the group with CACS>0 and the highest tertile of big ET-1 (P<0.01). The area under the receiver operating characteristic curve for the big ET-1 level in predicting CAC was 0.83 (95% CI 0.79–0.87, p<0.001), with a sensitivity of 70.6% and specificity of 87.7%.
Conclusions
The data firstly demonstrated that the plasma big ET-1 level was a valuable independent predictor for CAC in our study.
doi:10.1371/journal.pone.0142458
PMCID: PMC4643989  PMID: 26565974
22.  Distribution of High-Density Lipoprotein Subfractions and Hypertensive Status 
Medicine  2015;94(43):e1912.
Supplemental Digital Content is available in the text
Abstract
The exact mechanisms of hypertension contributing to atherosclerosis have not been fully elucidated. Although multiple studies have clarified the association with low-density lipoprotein (LDL) subfractions, uncertainty remains about its relationship with high-density lipoprotein (HDL) subfractions. Therefore, we aimed to comprehensively determine the relationship between distribution of HDL subfractions and hypertensive status.
A total of 953 consecutive subjects without previous lipid-lowering drug treatment were enrolled and were categorized based on hypertension history (with hypertension [n = 550] or without hypertension [n = 403]). Baseline clinical and laboratory data were collected. HDL separation was performed using the Lipoprint System.
Plasma large HDL-cholesterol (HDL-C) and large HDL percentage were dramatically lower whereas the small HDL-C and small HDL percentage were higher in patients with hypertension (all P < 0.05). The antihypertensive drug therapy was not associated with large or small HDL subfractions (on treatment vs not on treatment, P > 0.05; combination vs single drug therapy, P > 0.05). However, the blood pressure well-controlled patients have significantly lower small HDL subfraction (P < 0.05). Moreover, large HDL-C and percentage were inversely whereas small HDL percentage was positively associated with incident hypertension after adjusting potential confounders (all P < 0.05). In the multivariate model conducted in patients with and without hypertension separately, the cardio-protective value of large HDL-C was disappeared in patients with hypertension (OR 95%CI: 1.011 [0.974–1.049]).
The distribution of HDL subfractions is closely associated with hypertensive status and hypertension may potentially impact the cardio-protective value of large HDL subfraction.
doi:10.1097/MD.0000000000001912
PMCID: PMC4985429  PMID: 26512616
23.  Risk Factors, Coronary Severity, Outcome and ABO Blood Group 
Medicine  2015;94(43):e1708.
Supplemental Digital Content is available in the text
Abstract
ABO blood type locus has been reported to have ethnic difference and to be a pivotal genetic determinant of cardiovascular risk, whereas few prospective data regarding the impact on cardiovascular outcomes are available in a large cohort of patients with angiography-proven coronary artery disease, especially from the Chinese population. The objective of this study was to assess the prognostic role of blood type in future cardiovascular events (CVEs) in Chinese Han patients undergoing coronary angiography.
The population of this prospective cohort study consisted of 3823 eligible patients, and followed annually to capture all CVEs. Baseline characteristics and ABO blood type were obtained. Cox proportional hazards models were used to evaluate the risk of ABO blood type on CVEs.
New CVEs occurred in 348 patients [263 (10.3%) non-O and 85 (7.8%) O] during a median period of 24.6 months follow-up. Significantly, non-O blood group was related to the presence and severity of coronary atherosclerosis and several risk factors including inflammatory markers. The log-rank test revealed that there was a significant difference between non-O and O blood groups in event-free survival analysis (P = 0.026). In particular, the Cox proportional hazards models revealed that non-O blood type was associated with increased CVEs risk [hazard ratio (95% confidence interval) 1.320 (1.033–1.685)], even after adjusting for potential confounders [adjusted hazard ratio (95% confidence interval) non-O: 1.289 (1.003–1.656); A: 1.083 (0.797–1.472); B: 1.481 (1.122–1.955); AB: 1.249 (0.852–1.831), respectively].
Non-O blood type is associated with future CVEs in Chinese Han patients undergoing coronary angiography.
doi:10.1097/MD.0000000000001708
PMCID: PMC4985373  PMID: 26512559
24.  RASSF8 downregulation promotes lymphangiogenesis and metastasis in esophageal squamous cell carcinoma 
Oncotarget  2015;6(33):34510-34524.
Lymphatic vessels are the major routes of human esophageal squamous cell carcinoma (ESCC) metastasis. Tumor cells secrete pro-lymphangiogenic factors to induce new lymphatic vessels, promoting lymph node metastasis. In this study, we show that RAS association domain family 8 (RASSF8) expression in ESCC clinical samples was inversely correlated with lymph node metastasis and patients survival. Tumor cells with low RASSF8 expression had higher apparent migratory ability, and promoted and lymphangiogenesis both in vitro and in vivo. RASSF8 downregulation enhanced VEGF-C expression and caused subcellular redistribution of p65 in ESCC. Our results show that RASSF8 acts as a tumor suppressor in ESCC and is a potential therapeutic target for preventing lymph node metastasis.
PMCID: PMC4741469  PMID: 26439687
tumor metastasis; RASSF8; lymphangiogenesis; esophageal cancer
25.  Global regulatory mechanism underlying the activation of an exon network required for neurogenesis 
Molecular cell  2014;56(1):90-103.
SUMMARY
The vertebrate and neural-specific SR-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells, while weakening 3′ splice site recognition and contributing to exon skipping in non-neural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3′ splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis.
doi:10.1016/j.molcel.2014.08.011
PMCID: PMC4608043  PMID: 25219497

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