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1.  Mammospheres from murine mammary stem cell-enriched basal cells: clonal characteristics and repopulating potential 
Stem cell research  2013;10(3):396-404.
Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that > 90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs.
doi:10.1016/j.scr.2013.01.007
PMCID: PMC3622180  PMID: 23466563
Mammary stem cell; Mammosphere; Lineage differentiation; In vivo repopulation
2.  Overexpression of SLC38A1 is associated with poorer prognosis in Chinese patients with gastric cancer 
BMC Gastroenterology  2014;14:70.
Background
Current literature has demonstrated that host glutamine depletion facilitates tumorigenesis. Likewise, the glutamine transporter SLC38A1 is putatively associated with malignant transformation and tumor progression. Taken together, this forms the premise for undertaking the current study. The twofold aim of this study was to provide insight into whether or not a variance in the expression of SLC38A1 exists between human gastric cancer and healthy human tissues, and to determine how silencing the SLC38A1 gene could affect the proliferation, viability, migration, and invasion of gastric cancer cells.
Methods
Immunohistochemical staining was used to analyze the expression of SLC38A1 in gastric cancer tissues and adjacent healthy mucosa in 896 patients with pathologically confirmed gastric cancer who had underwent R0 resection. SH-10-TC cells (a gastric cancer cell line) were used to examine whether silencing SLC38A1 with siRNA could affect cell viability, migration and invasion.
Results
The SLC38A1 protein was very low or undetectable in healthy gastric mucosa. In contrast, strong staining of SLC38A1 protein was found in the cytoplasm in 495 out of the 896 gastric cancer samples. More pronounced SLC38A1 expression in gastric cancer tissues was significantly associated with age, differentiation status, lymph node metastasis, TNM stage and PCNA (proliferating cell nuclear antigen) expression. Upon univariate survival analysis, SLC38A1 expression was correlated with poor survival. Multivariate survival analysis revealed that SLC38A1 was an independent prognostic factor.
Conclusion
SLC38A1 is overexpressed in gastric cancer, which suggests that it is contributory to tumor progression. These results encourage the exploration of SLC38A1 as a target for intervention in gastric cancer.
doi:10.1186/1471-230X-14-70
PMCID: PMC3984425  PMID: 24712400
Gastric cancer; Tissue microarray; Immunohistochemistry; SLC38A1; Prognostic factor
3.  A combination of flaxseed oil and astaxanthin alleviates atherosclerosis risk factors in high fat diet fed rats 
Background
Atherosclerosis is the most common pathologic process underlying cardiovascular disease. Both flaxseed oil (FO) and astaxanthin (ASX) are believed to benefit cardiovascular system. The combined effect of FO and ASX on the atherosclerosis risk factors in rats fed a high-fat diet was investigated.
Methods
Astaxanthin was dissolved in flaxseed oil to a final concentration of 1g/kg (FO + ASX). Male Sprague–Dawley rats were fed a rodent diet contained 20% fat whose source was lard (HFD) or 75% lard and 25% FO + ASX (50 mg ASX/kg diet) or 50% lard and 50% FO + ASX (100 mg ASX/kg diet) or FO + ASX (200 mg ASX/kg diet) for 10 weeks.
Results
The combination of FO and ASX significantly increased the antioxidant defense capacity and decreased lipid peroxidation in plasma. Evident decreases in the levels TG, TC and LDL-C contents, as well as IL-6 and CRP were also observed in plasma of FO and ASX fed rats.
Conclusion
The combination of FO and ASX can improve oxidative stress, lipid abnormalities and inflammation, providing evidence that the combination of FO and ASX could be a promising functional food in cardiovascular health promotion.
doi:10.1186/1476-511X-13-63
PMCID: PMC3994197  PMID: 24708887
Flaxseed oil; Astaxanthin; Atherosclerosis; Oxidant stress; Plasma lipids; Inflammation
4.  The Association between TGF-β1 Polymorphisms and Radiation Pneumonia in Lung Cancer Patients Treated with Definitive Radiotherapy: A Meta-Analysis 
PLoS ONE  2014;9(3):e91100.
Background
Previous studies investigating the association between TGF-β1 polymorphisms and Radiation Pneumonia (RP) risk have provided inconsistent results. The aim of our study was to assess the association between the TGF-β1 genes C509T, G915C and T869C polymorphisms and risk of RP in lung cancer patients treated with definitive radiotherapy.
Methods
Two investigators independently searched the Medline, Embase, CNKI, and Chinese Biomedicine Databases for studies published before September 2013. Summary odds ratios (ORs) and 95% confidence intervals (CIs) for TGF-β1 polymorphisms and RP were calculated in a fixed-effects model or a random-effects model when appropriate.
Results
Ultimately, each 7 studies were found to be eligible for meta-analyses of C509T, G915C and T869C, respectively. Our analysis suggested that the variant genotypes of T869C were associated with a significantly increased RP risk in dominant model (OR = 0.59, 95% CI = 0.45–0.79) and CT vs. TT model (OR = 0.47, 95% CI = 0.32–0.69). In the subgroup analyses by ethnicity/country, a significantly increased risk was observed among Caucasians. For C509T and G915C polymorphism, no obvious associations were found for all genetic models.
Conclusion
This meta-analysis suggests that T869C polymorphism of TGF-β1 may be associated with RP risk only in Caucasians, and there may be no association between C509T and G915C polymorphism and RP risk.
doi:10.1371/journal.pone.0091100
PMCID: PMC3958356  PMID: 24642488
5.  Differential expressed genes of change in glucose metabolism in the liver of GCK knockout mice 
Gene  2013;516(2):248-254.
To investigate the role of liver-specific expression of GCK in the pathogenesis of hyperglycemia and identify candidate genes involved in the mechanisms for onset and progression of MODY2, we examined the differentially expression of genes in the liver of liver-specific glucokinase (GCK) knockout mice (gckw/−) model at 2 and 26 weeks by suppression subtractive hybridization (SSH). The expression levels of the ACAT2 and PEPCK genes identified by SSH, glycogen Synthase (GS) and glycogen phosphorylase (GP) genes were further examined at different ages by Real time PCR. In addition, we also characterized the expression of mouse GCK mRNA at different ages as well as the fasting blood glucose, serum insulin, GCK activity, total cholesterol (TC), triglyceride(TG) and glycogen content. The results show that, except for 2-week-old gckw/− mice, the fasting blood glucose levels are significantly higher for gckw/− mice (P<0.01)and the GCK activity of gckw/− mice lower about 50% than for GCK wild type(gckw/w )mice(P<0.05). The glycogen content of 4-week old and 40-week old gckw/− mice was lower than that of 4-week old and 40-week old gckw/w mice. The GP mRNA levels decrease at 40-week old gckw/− mice compared to age-matched gckw/w mice. PEPCK mRNA decreases at 2-week old gckw/− mice, but increases at 4-week old gckw/− mice(P<0.05). Changes in the expression of PEPCK genes, delayed development of GCK and impaired hepatic glycogen synthesis in liver potentially lead to onset and progression of maturity onset diabetes of the young, type 2 (MODY-2).
doi:10.1016/j.gene.2012.12.036
PMCID: PMC3642073  PMID: 23291412 CAMSID: cams2937
glucokinase; MODY2; differentially expressed genes
6.  Cardiotoxic and Cardioprotective Features of Chronic β-adrenergic Signaling 
Circulation research  2012;112(3):498-509.
Rationale
In the failing heart, persistent β-adrenergic receptor (βAR) activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II (CaMKII). β-Adrenergic signaling pathways are also capable of activating cardioprotective mechanisms.
Objective
This study used a novel PKA inhibitor peptide (PKI) to inhibit PKA activity to test the hypothesis that βAR signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways.
Methods and Results
In PKI transgenic mice, chronic isoproterenol (ISO) failed to induce cardiac hypertrophy, fibrosis, myocyte apoptosis and depressed cardiac function. In cultured adult feline ventricular myocytes (AFVMs), PKA inhibition protected myocytes from death induced by β1-AR agonists by preventing cytosolic and SR Ca2+ overload and CaMKII activation. PKA inhibition revealed a cardioprotective role of β-adrenergic signaling via cAMP/EPAC /Rap1/Rac/ERK pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction (MI) that was superior to β-blocker therapy.
Conclusion
These results suggest that selective block of PKA could be a novel heart failure therapy.
doi:10.1161/CIRCRESAHA.112.273896
PMCID: PMC3562387  PMID: 23104882
Apoptosis; Ca2+/dependent protein kinase II; ERK2; EPAC
7.  High Serum miR-19a Levels Are Associated with Inflammatory Breast Cancer and Are Predictive of Favorable Clinical Outcome in Patients with Metastatic HER2+ Inflammatory Breast Cancer 
PLoS ONE  2014;9(1):e83113.
Introduction
Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2+] and inflammatory breast cancer [IBC]).
Experimental Design
In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls.
Results
Patients with non-metastatic HER2+ breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2− disease (p = 0.044); whereas patients with metastatic HER2+ breast cancer had higher serum miR-10b median levels than patients with metastatic HER2− disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2+ and HER2− groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2+ IBC.
Conclusion
High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2+ breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2+ IBC.
doi:10.1371/journal.pone.0083113
PMCID: PMC3885405  PMID: 24416156
8.  aP2-Cre-Mediated Inactivation of Estrogen Receptor Alpha Causes Hydrometra 
PLoS ONE  2014;9(1):e85581.
In this study we describe the reproductive phenotypes of a novel mouse model in which Cre-mediated deletion of ERα is regulated by the aP2 (fatty acid binding protein 4) promoter. ERα-floxed mice were crossed with transgenic mice expressing Cre-recombinase under the control of the aP2 promoter to generate aP2-Cre/ERαflox/flox mice. As expected, ERα mRNA levels were reduced in adipose tissue, but in addition we also detected an 80% reduction of ERα levels in the hypothalamus of aP2-Cre/ERαflox/flox mice. Phenotypic analysis revealed that aP2-Cre/ERαflox/flox female mice were infertile. In line with this, aP2-Cre/ERαflox/flox female mice did not cycle and presented 3.8-fold elevated estrogen levels. That elevated estrogen levels were associated with increased estrogen signaling was evidenced by increased mRNA levels of the estrogen-regulated genes lactoferrin and aquaporin 5 in the uterus. Furthermore, aP2-Cre/ERαflox/flox female mice showed an accumulation of intra-uterine fluid, hydrometra, without overt indications for causative anatomical anomalies. However, the vagina and cervix displayed advanced keratosis with abnormal quantities of accumulating squamous epithelial cells suggesting functional obstruction by keratin plugs. Importantly, treatment of aP2-Cre/ERαflox/flox mice with the aromatase inhibitor Letrozole caused regression of the hydrometra phenotype linking increased estrogen levels to the observed phenotype. We propose that in aP2-Cre/ERαflox/flox mice, increased serum estrogen levels cause over-stimulation in the uterus and genital tracts resulting in hydrometra and vaginal obstruction.
doi:10.1371/journal.pone.0085581
PMCID: PMC3885723  PMID: 24416430
9.  CD11b/CD18 (Mac-1) Is a Novel Surface Receptor for Extracellular Double-stranded RNA to Mediate Cellular Inflammatory Responses 
During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C, a synthetic dsRNA) in mouse sera and livers and in cultured peritoneal macrophages. DsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. Firstly, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of interferon regulatory factor 3 (IRF3) in macrophages. Secondly, poly I:C induced activation of phagocyte NADPH oxidase (NOX2) in a TLR3-independent, but Mac-1 dependent manner. Subsequently, NOX2-derived intracellular reactive oxygen species activated MAPK and NFκB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates Mac-1 as a potential therapeutic target for virus-related inflammatory diseases.
doi:10.4049/jimmunol.1202136
PMCID: PMC3529770  PMID: 23209319
Extracellular dsRNA; Innate immunity; Pathogen recognition receptor (PRR); Integrin; Mac-1(CD11b/CD18); Macrophage; Internalization; NADPH oxidase; Polyinosinic:polycytidylic acid (poly I:C); Reactive oxygen species (ROS); TLR3
10.  Activity of sputum p38 MAPK is correlated with airway inflammation and reduced FEV1 in COPD patients 
Background
Inflammation and remodeling of the small airways are major determinants of the progression and severity of COPD. The present study explored the correlation between sputum p38 mitogen-activated protein kinase (MAPK) activity and airway inflammation and reduction of lung function in the patients with chronic obstructive pulmonary disease (COPD).
Material/Methods
Sputum samples were collected from 48 COPD patients and 12 healthy persons. Sputum p38 MAPK activity was measured by Western blotting and sputum levels of CXCL8 and neutrophil, and lung function was measured. The correlation between p38MAPK activity and airway inflammation and reduction of lung function was analyzed.
Result
Our results showed the significantly increased expression of phospho-p38 MAPK and CXCL8 in the sputum samples of the COPD patients. The p38 MAPK activity was remarkably correlated with the CXCL8 level and neutrophils infiltration in the airway, and the decline of lung function in the COPD patients.
Conclusions
These findings suggest the pivotal role of p38 MAPK in the airway inflammation of COPD patients. We propose p38 MAPK as a potential target for the treatment of COPD.
doi:10.12659/MSM.889880
PMCID: PMC3890402  PMID: 24382347
p38 mitogen-activated protein kinase; chronic obstructive pulmonary disease; CXCL8
11.  Phosphoglycerate dehydrogenase is dispensable for breast tumor maintenance and growth 
Oncotarget  2013;4(12):2502-2511.
Cancer cells rely on aerobic glycolysis to maintain cell growth and proliferation via the Warburg effect. Phosphoglycerate dehydrogenase (PHDGH) catalyzes the first step of the serine biosynthetic pathway downstream of glycolysis, which is a metabolic gatekeeper both for macromolecular biosynthesis and serine-dependent DNA synthesis. Here, we report that PHDGH is overexpressed in many ER-negative human breast cancer cell lines. PHGDH knockdown in these cells leads to a reduction of serine synthesis and impairment of cancer cell proliferation. However, PHGDH knockdown does not affect tumor maintenance and growth in established breast cancer xenograft models, suggesting that PHGDH-dependent cancer cell growth may be context-dependent. Our findings suggest that other mechanisms or pathways may bypass exclusive dependence on PHGDH in established human breast cancer xenografts, indicating that PHGDH is dispensable for the growth and maintenance of tumors in vivo.
PMCID: PMC3926844  PMID: 24318446
PHGDH; breast cancer cells; in vivo
12.  Decrease in Prosaposin in the Dystrophic mdx Mouse Brain 
PLoS ONE  2013;8(11):e80032.
Background
Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities.
Methodology/Principal Findings
The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells.
Conclusions/Significance
Low levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.
doi:10.1371/journal.pone.0080032
PMCID: PMC3828254  PMID: 24244600
13.  Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling 
Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs.
doi:10.1016/j.yjmcc.2012.08.005
PMCID: PMC3472041  PMID: 22921230
L-type calcium channel; Hypertrophy; Nuclear factor of activated T cells; Ventricular myocyte; Transient receptor potential channel
14.  Epithelial-Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer 
Molecular cancer therapeutics  2012;11(11):2526-2534.
Currently, there is extensive information about circulating tumor cells (CTCs) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition inducing transcription factors (EMT-TFs) and cancer stem cell features in HER2+ metastatic breast cancer (MBC) patients. Epithelial cells were enriched from peripheral blood mononuclear cells (PBMCs) using antibody-coated anti-CD326 antibody (CD326+) magnetic beads, and the residual CD326− PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326−CD45−). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). Additionally, PBMCs were analyzed using multi-parameter flow cytometry for ALDH activity and cancer stem cells (CSCs) that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326−CD45− cell fraction of 60.7% of patients. The CD326−CD45− fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH+/CD133+ cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P=0.038). Our data indicate that HER2+ MBC patients have EMT-CTCs. Moreover, an enrichment of cancer stem cells was found in CD326−CD45− cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in HER2+ MBC patients treated with trastuzumab-based therapy.
doi:10.1158/1535-7163.MCT-12-0460
PMCID: PMC3500676  PMID: 22973057
circulating tumor cells; epithelial to mesenchymal transition; stem cells; HER2; CD133; metastatic breast cancer
15.  Identifying Influential Nodes in Large-Scale Directed Networks: The Role of Clustering 
PLoS ONE  2013;8(10):e77455.
Identifying influential nodes in very large-scale directed networks is a big challenge relevant to disparate applications, such as accelerating information propagation, controlling rumors and diseases, designing search engines, and understanding hierarchical organization of social and biological networks. Known methods range from node centralities, such as degree, closeness and betweenness, to diffusion-based processes, like PageRank and LeaderRank. Some of these methods already take into account the influences of a node’s neighbors but do not directly make use of the interactions among it’s neighbors. Local clustering is known to have negative impacts on the information spreading. We further show empirically that it also plays a negative role in generating local connections. Inspired by these facts, we propose a local ranking algorithm named ClusterRank, which takes into account not only the number of neighbors and the neighbors’ influences, but also the clustering coefficient. Subject to the susceptible-infected-recovered (SIR) spreading model with constant infectivity, experimental results on two directed networks, a social network extracted from delicious.com and a large-scale short-message communication network, demonstrate that the ClusterRank outperforms some benchmark algorithms such as PageRank and LeaderRank. Furthermore, ClusterRank can also be applied to undirected networks where the superiority of ClusterRank is significant compared with degree centrality and k-core decomposition. In addition, ClusterRank, only making use of local information, is much more efficient than global methods: It takes only 191 seconds for a network with about nodes, more than 15 times faster than PageRank.
doi:10.1371/journal.pone.0077455
PMCID: PMC3814409  PMID: 24204833
16.  Expression of activator protein-1 (AP-1) family members in breast cancer 
BMC Cancer  2013;13:441.
Background
The activator protein-1 (AP-1) transcription factor is believed to be important in tumorigenesis and altered AP-1 activity was associated with cell transformation. We aimed to assess the potential role of AP-1 family members as novel biomarkers in breast cancer.
Methods
We studied the expression of AP-1 members at the mRNA level in 72 primary breast tumors and 37 adjacent non-tumor tissues and evaluated its correlation with clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and HER2/neu status. Expression levels of Ubiquitin C (UBC) were used for normalization. Protein expression of AP-1 members was assessed using Western blot analysis in a subset of tumors. We used student’s t-test, one-way ANOVA, logistic regression and Pearson’s correlation coefficient for statistical analyses.
Results
We found significant differences in the expression of AP-1 family members between tumor and adjacent non-tumor tissues for all AP-1 family members except Fos B. Fra-1, Fra-2, Jun-B and Jun-D mRNA levels were significantly higher in tumors compared to adjacent non-tumor tissues (p < 0.001), whilst c-Fos and c-Jun mRNA levels were significantly lower in tumors compared with adjacent non-tumor tissues (p < 0.001). In addition, Jun-B overexpression had outstanding discrimination ability to differentiate tumor tissues from adjacent non-tumor tissues as determined by ROC curve analysis. Moreover, Fra-1 was significantly overexpressed in the tumors biochemically classified as ERα negative (p = 0.012) and PR negative (p = 0.037). Interestingly, Fra-1 expression was significantly higher in triple-negative tumors compared with luminal carcinomas (p = 0.01).
Conclusions
Expression levels of Fra-1 and Jun-B might be possible biomarkers for prognosis of breast cancer.
doi:10.1186/1471-2407-13-441
PMCID: PMC3849565  PMID: 24073962
AP-1 family members; Breast cancer; Estrogen receptor; Progesterone receptor
17.  Increased MicroRNA-146a Levels in Plasma of Patients with Newly Diagnosed Type 2 Diabetes Mellitus 
PLoS ONE  2013;8(9):e73272.
Background
MicroRNAs (miRNAs), a class of small non-coding RNAs, are thought to serve as crucial regulators of gene expression. Dysregulated expression of miRNAs has been described in various diseases and may contribute to related pathologic processes. Our aim was to examine circulating miRNA-146a levels in newly diagnosed type 2 diabetes mellitus (new-T2DM) patients from a Chinese Han population.
Methodology/Principal Findings
Circulating miRNA-146a was extracted from plasma samples of 90 new-T2DM patients and 90 age- and sex-matched controls. Quantitative PCR assessment revealed that circulating miRNA-146a levels were significantly elevated in new-T2DM patients compared with controls. Participants in the highest tertile of circulating miRNA-146a levels showed a notably higher risk for new-T2DM (crude OR 4.333, 95% CI, 1.935 to 9.705, P = 0.001) than persons in the lowest tertile. Controlling for known risk factors and some biochemical indicators did not attenuate the aforementioned association. In addition, receiver operating characteristic (ROC) curves generated for miRNA-146a revealed an area under the curve (AUC) of 0.725 (95% CI, 0.651 to 0.799, P < 0.001). Moreover, higher circulating miRNA-146a levels were significantly associated with higher plasma heme oxygenase-1 (HO-1) concentrations (β coefficient = 0.131, P < 0.001) and lower HOMA-beta (β coefficient = -0.153, P = 0.015).
Conclusions/Significance
We found that circulating miRNA-146a levels were significantly elevated in new-T2DM patients compared with healthy controls. Whether expression of circulating miRNA-146a holds predictive value for T2DM warrants further investigations.
doi:10.1371/journal.pone.0073272
PMCID: PMC3759444  PMID: 24023848
18.  The Association between the Pro12Ala Variant in the PPARγ2 Gene and Type 2 Diabetes Mellitus and Obesity in a Chinese Population 
PLoS ONE  2013;8(8):e71985.
Background
Conflicting results have been reported on the association of the Pro12Ala polymorphism of the PPARγ2 gene with the risk of type 2 diabetes or obesity.
Methods and Findings
A total of 3146 subjects with 1145 cases of type 2 diabetes and 2001 healthy controls were included in the study. Genomic DNA was obtained from blood samples and the screening for the gene polymorphisms was done using an allelic discrimination assay-by-design TaqMan method. Overall, the Ala allele frequency was 5.6% in control subjects and 3.9% in diabetes subjects (P = 0.023). We found a statistically significant association of carriers of the Ala allele with greater homoeostasis model assessment of beta cell function index in all subjects (P = 0.046). After controlling for confounders, carriers of the Ala allele had a decreased risk of diabetes compared with noncarriers [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.49–0.83; P = 0.001]. A beneficial effect of the Ala allele was also observed for obesity (OR 0.64, 95% CI 0.42–0.96; P = 0.030).
Conclusion
Our results suggested that the presence of the Ala allele may contribute to improved insulin secretory capacity and may confer protection from type 2 diabetes and obesity in the Chinese population.
doi:10.1371/journal.pone.0071985
PMCID: PMC3749141  PMID: 23991018
19.  Forest Structure, Stand Composition, and Climate-Growth Response in Montane Forests of Jiuzhaigou National Nature Reserve, China 
PLoS ONE  2013;8(8):e71559.
Montane forests of western China provide an opportunity to establish baseline studies for climate change. The region is being impacted by climate change, air pollution, and significant human impacts from tourism. We analyzed forest stand structure and climate-growth relationships from Jiuzhaigou National Nature Reserve in northwestern Sichuan province, along the eastern edge of the Tibetan plateau. We conducted a survey to characterize forest stand diversity and structure in plots occurring between 2050 and 3350 m in elevation. We also evaluated seedling and sapling recruitment and tree-ring data from four conifer species to assess: 1) whether the forest appears in transition toward increased hardwood composition; 2) if conifers appear stressed by recent climate change relative to hardwoods; and 3) how growth of four dominant species responds to recent climate. Our study is complicated by clear evidence of 20th century timber extraction. Focusing on regions lacking evidence of logging, we found a diverse suite of conifers (Pinus, Abies, Juniperus, Picea, and Larix) strongly dominate the forest overstory. We found population size structures for most conifer tree species to be consistent with self-replacement and not providing evidence of shifting composition toward hardwoods. Climate-growth analyses indicate increased growth with cool temperatures in summer and fall. Warmer temperatures during the growing season could negatively impact conifer growth, indicating possible seasonal climate water deficit as a constraint on growth. In contrast, however, we found little relationship to seasonal precipitation. Projected warming does not yet have a discernible signal on trends in tree growth rates, but slower growth with warmer growing season climates suggests reduced potential future forest growth.
doi:10.1371/journal.pone.0071559
PMCID: PMC3739742  PMID: 23951188
20.  Efficacy and Safety of a Chinese Herbal Formula (Invigorating Kidney and Strengthening Spleen) in Chronic Hepatitis B Virus Carrier: Results from a Multicenter, Randomized, Double-Blind, and Placebo-Controlled Trial 
A Chinese Herbal Formula (CHF) has acquired a certain therapeutic effect on chronic HBV infection. To assess the efficacy and safety of CHF on HBV replication in chronic HBV carriers, we performed a randomized, double-blind, and placebo-controlled trial involving patients from 16 centers. A total of 300 confirmed chronic HBV carriers were randomized at baseline in a ratio of 2 : 1 to receive either CHF or placebo for 52 weeks. The results showed that a greater proportion of CHF than placebo treated patients achieved virological response at week 52; the mean decline of serum HBsAg levels in the CHF group dropped more obviously than that in the control group at all stages of the treatment; however, the rates of HBeAg loss and seroconversion had no difference between the two groups. Meanwhile, were presented significant increases in IFN-γ; IL-2 levels and reductions in IL-4 and IL-10 levels in the treatment group compared to the control group at week 52. There were no drug-related serious adverse events. In conclusion, the treatment with 52-week CHF is safe and effective in inhibiting HBV replication in chronic HBV carriers. The ability of the compound to modulate host immune function probably contributed to this effect.
doi:10.1155/2013/961926
PMCID: PMC3722789  PMID: 23935692
21.  Serum Heat Shock Protein 70 Concentration in Relation to Polycystic Ovary Syndrome in a Non-Obese Chinese Population 
PLoS ONE  2013;8(6):e67727.
Background
Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70) concentrations and PCOS in a non-obese Chinese population.
Methods and Results
Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI) levels, as well as Insulin resistance index (HOMA-IR) (P < 0.05). Identically, markers of oxidative stress (malondialdehyde (MDA), 8-Hydroxy-desoxyguanosine (8-OHdG), Nitric oxide (NO)) and inflammation (tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)) were markedly increased when compared to controls (P < 0.05). Elevated serum Hsp70 was positively correlated with IR, oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI). The receiver-operating characteristic curve (ROC) analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946) vs. 0.822 (95% CI 0.744-0.900); P for difference = 0.015).
Conclusions and Significance
Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.
doi:10.1371/journal.pone.0067727
PMCID: PMC3688989  PMID: 23825680
22.  Landscape of transcription in human cells 
Djebali, Sarah | Davis, Carrie A. | Merkel, Angelika | Dobin, Alex | Lassmann, Timo | Mortazavi, Ali M. | Tanzer, Andrea | Lagarde, Julien | Lin, Wei | Schlesinger, Felix | Xue, Chenghai | Marinov, Georgi K. | Khatun, Jainab | Williams, Brian A. | Zaleski, Chris | Rozowsky, Joel | Röder, Maik | Kokocinski, Felix | Abdelhamid, Rehab F. | Alioto, Tyler | Antoshechkin, Igor | Baer, Michael T. | Bar, Nadav S. | Batut, Philippe | Bell, Kimberly | Bell, Ian | Chakrabortty, Sudipto | Chen, Xian | Chrast, Jacqueline | Curado, Joao | Derrien, Thomas | Drenkow, Jorg | Dumais, Erica | Dumais, Jacqueline | Duttagupta, Radha | Falconnet, Emilie | Fastuca, Meagan | Fejes-Toth, Kata | Ferreira, Pedro | Foissac, Sylvain | Fullwood, Melissa J. | Gao, Hui | Gonzalez, David | Gordon, Assaf | Gunawardena, Harsha | Howald, Cedric | Jha, Sonali | Johnson, Rory | Kapranov, Philipp | King, Brandon | Kingswood, Colin | Luo, Oscar J. | Park, Eddie | Persaud, Kimberly | Preall, Jonathan B. | Ribeca, Paolo | Risk, Brian | Robyr, Daniel | Sammeth, Michael | Schaffer, Lorian | See, Lei-Hoon | Shahab, Atif | Skancke, Jorgen | Suzuki, Ana Maria | Takahashi, Hazuki | Tilgner, Hagen | Trout, Diane | Walters, Nathalie | Wang, Huaien | Wrobel, John | Yu, Yanbao | Ruan, Xiaoan | Hayashizaki, Yoshihide | Harrow, Jennifer | Gerstein, Mark | Hubbard, Tim | Reymond, Alexandre | Antonarakis, Stylianos E. | Hannon, Gregory | Giddings, Morgan C. | Ruan, Yijun | Wold, Barbara | Carninci, Piero | Guigó, Roderic | Gingeras, Thomas R.
Nature  2012;489(7414):101-108.
Summary
Eukaryotic cells make many types of primary and processed RNAs that are found either in specific sub-cellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic sub-cellular localizations are also poorly understood. Since RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modifications and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations taken together prompt to a redefinition of the concept of a gene.
doi:10.1038/nature11233
PMCID: PMC3684276  PMID: 22955620
23.  Abdominal aortic calcification is not superior over other vascular calcification in predicting mortality in hemodialysis patients: a retrospective observational study 
BMC Nephrology  2013;14:120.
Background
KDIGO (Kidney Disease: Improving Global Outcomes) guidelines recommend that a lateral abdominal radiograph should be performed to assess vascular calcification (VC) in dialysis patients. However, abdominal aortic calcification is a prevalent finding, and it remains unclear whether other anatomical areas of VC can predict mortality more accurately.
Methods
A total of 217 maintenance hemodialysis patients were enrolled at the Sichuan Provincial People’s Hospital between July 2010 and March 2011. Radiographs of the abdomen, pelvis and hands were evaluated by a radiologist to evaluate the presence of VC. The correlation between different areas of VC and all-cause or cardiovascular mortality was analyzed using univariate and multivariate models.
Results
The prevalence of VC was 70.0% (152 patients), and most had abdominal aortic calcification (90.1%). During 26 ± 7 months of follow-up, 37 patients died. The VC score was independently associated with patient mortality. VC observed on abdominal radiographs (abdominal aortic calcification) was associated with all-cause mortality in models adjusted for cardiovascular risk factors (HR, 4.69; 95%CI, 1.60-13.69) and dialysis factors (HR, 3.38; 95%CI, 1.18-9.69). VC in the pelvis or hands was associated with all-cause mortality in the model adjusted for dialysis factors. When three combinations of VC in different radiographs were included in models, the presence of abdominal VC was only significantly associated with all-cause mortality in the integrated model. VC in the abdomen and pelvis was associated with all-cause mortality in the model adjusted for cardiovascular factors and the integrated model, but neither was significantly associated with cardiovascular mortality. VC in all radiographs was significantly associated with a more than 6-fold risk of all-cause mortality and a more than 5-fold risk of cardiovascular mortality compared to patients without VC.
Conclusions
VC in different arteries as shown on radiographs is associated with different levels of risk for mortality. The lateral abdominal radiograph may not be superior to other radiographs for predicting patient outcomes. Further research is needed to elucidate the effects of difference burdens of VC on patient outcomes.
doi:10.1186/1471-2369-14-120
PMCID: PMC3691830  PMID: 23738982
Vascular calcification; Mortality; Hemodialysis; Abdominal aortic calcification
24.  NADPH oxidases: novel therapeutic targets for neurodegenerative diseases 
Oxidative stress is a key pathologic factor in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocytic NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of over-activated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, the suppression of PHOX activity correlates with less neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocytic NADPH oxidases in astroglia and neurons further reinforces the critical role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases.
doi:10.1016/j.tips.2012.03.008
PMCID: PMC3477578  PMID: 22503440
25.  Long-term renal changes in the liver-specific glucokinase knockout mouse: implications for renal disease in MODY2 
To investigate the functional and structural renal changes in a long-term liver-specific glucokinase knockout mouse, model of MODY2. Hemizygous glucokinase knockout mice, gckw/− groups, were compared at 6-, 10- and 14-months to their age-matched normal littermates, gckw/w groups. To examine changes we compared body weight, fasting blood glucose, serum insulin and creatinine levels, as well as 24 h urine samples that were collected for urine volume and protein analysis between the two groups. Renal tissues were collected and stained with hemotoxylin-eosin and periodic-acid Schiff, for light microscopic observation. Expression of renal Transforming Growth Factor β1 (TGF-β1) was determined by Western blot. Our results show that fasting blood glucose levels were significantly higher in gckw/− mice compared to gckw/w mice (P<0.01) for all age groups. Compared to age-matched gckw/w mice, 10-month old gckw/− mice have significantly elevated body weights (P<0.01), and urine volume (P<0.05) and protein concentrations (P<0.01). Renal tubular casts were observed in 10- and 14-month gckw/− mice. Significant increase in mesangial matrix and thickening of the glomerular basement membrane was observed in gckw/− compared to age-matched gckw/w mice at 10 and 14 months. As mice age increases, the levels of renal TGF-β1 are observed in both gckw/− and gckw/w mice. Our results indicate that renal changes occur in the liver-specific glucokinase knockout mouse model of MODY2, and suggest that TGF-β1 may play a key role in pathogenesis of these renal changes.
doi:10.1016/j.trsl.2010.11.003
PMCID: PMC3654931  PMID: 21316027 CAMSID: cams2938
MODY; Diabetic nephropathy; Animal models

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