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1.  Synergism between upregulation of Rab7 and inhibition of autophagic degradation caused by mycoplasma facilitates intracellular mycoplasma infection 
Molecular Medicine Reports  2014;9(3):793-800.
Following fusion of a mycoplasma with a host cell membrane, the inserted components of mycoplasma may then be transported through the endocytic pathway. However, the effects of mycoplasmas on the host cell endomembrane system are largely unknown. In this study, mycoplasma-induced changes in the dynamics of endocytic and autophagic systems were investigated. Endocytosis and autophagy are two major processes involved in the survival of intracellular prokaryotic pathogens. It was found that, immediately following infection, mycoplasmas induce endocytosis in the host cell; however, in the long term the mycoplasmas suppress turnover of the components of the endocytic pathway. Immunofluorescence microscopy revealed that Rab7 and LC3-II are recruited to the intracellular mycoplasma-containing compartments. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) showed that mycoplasmas increase expression of Rab7 by upregulating transcription, but increase levels of LC3-II and p62 by post-translational regulation. Furthermore, it was demonstrated that mycoplasma infection causes inhibition of autophagic degradation of LC3-II and p62. In addition, it was found that upregulation of Rab7 and inhibition of autophagic degradation synergistically contributes to intracellular mycoplasma accumulation. In conclusion, these findings suggest that mycoplasmas may manipulate host cell endosomal and autophagic systems in order to facilitate intracellular infection.
doi:10.3892/mmr.2014.1907
PMCID: PMC3926517  PMID: 24452847
mycoplasma; endocytic pathway; autophagy; Rab7; LC3; p62
2.  Dietary Glutamate: Interactions With the Enteric Nervous System 
Background/Aims
Digestion of dietary protein elevates intraluminal concentrations of glutamate in the small intestine, some of which gain access to the enteric nervous system (ENS). Glutamate, in the central nervous system (CNS), is an excitatory neurotransmitter. A dogma that glutamatergic neurophysiology in the ENS recapitulates CNS glutamatergic function persists. We reassessed the premise that glutamatergic signaling in the ENS recapitulates its neurotransmitter role in the CNS.
Methods
Pharmacological analysis of actions of receptor agonists and antagonists in concert with immunohistochemical localization of glutamate transporters and receptors was used. Analysis focused on intracellularly-recorded electrical and synaptic behavior of ENS neurons, on stimulation of mucosal secretion by secretomotor neurons in the submucosal plexus and on muscle contractile behavior mediated by musculomotor neurons in the myenteric plexus.
Results
Immunoreactivity for glutamate was expressed in ENS neurons. ENS neurons expressed immunoreactivity for the EAAC-1 glutamate transporter. Neither L-glutamate nor glutamatergic receptor agonists had excitatory actions on ENS neurons. Metabotropic glutamatergic receptor agonists did not directly stimulate neurogenic mucosal chloride secretion. Neither L-glutamate nor the metabotropic glutamatergic receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), changed the mean amplitude of spontaneously occurring contractions in circular or longitudinal strips of intestinal wall from either guinea pig or human small intestinal preparations.
Conclusions
Early discoveries, for excitatory glutamatergic neurotransmission in the CNS, inspired enthusiasm that investigation in the ENS would yield discoveries recapitulating the CNS glutamatergic story. We found this not to be the case.
doi:10.5056/jnm.2014.20.1.41
PMCID: PMC3895608  PMID: 24466444
Intestines; Motility; Proteolysis; Receptors, glutamate; Secretion
3.  Activation of IL-27 signalling promotes development of postinfluenza pneumococcal pneumonia 
EMBO Molecular Medicine  2013;6(1):120-140.
Postinfluenza pneumococcal pneumonia is a common cause of death in humans. However, the role of IL-27 in the pathogenesis of secondary pneumococcal pneumonia after influenza is unknown. We now report that influenza infection induced pulmonary IL-27 production in a type I IFN-α/β receptor (IFNAR) signalling-dependent manner, which sensitized mice to secondary pneumococcal infection downstream of IFNAR pathway. Mice deficient in IL-27 receptor were resistant to secondary pneumococcal infection and generated more IL-17A-producing γδ T cells but not αβ T cells, thereby leading to enhanced neutrophil response during the early phase of host defence. IL-27 treatment could suppress the development of IL-17A-producing γδ T cells activated by Streptococcus pneumoniae and dendritic cells. This suppressive activity of IL-27 on γδ T cells was dependent on transcription factor STAT1. Finally, neutralization of IL-27 or administration of IL-17A restored the role of γδ T cells in combating secondary pneumococcal infection. Our study defines what we believe to be a novel role of IL-27 in impairing host innate immunity against pneumococcal infection.
doi:10.1002/emmm.201302890
PMCID: PMC3936494  PMID: 24408967
influenza virus; IL-17; IL-27; Streptococcus pneumoniae; γδ T cells
4.  Correction: High Weight Loss during Radiation Treatment Changes the Prognosis in Under-/Normal Weight Nasopharyngeal Carcinoma Patients for the Worse: A Retrospective Analysis of 2433 Cases 
PLoS ONE  2013;8(12):10.1371/annotation/3dc37158-1f60-436e-9bd5-2a822aa2c9cb.
doi:10.1371/annotation/3dc37158-1f60-436e-9bd5-2a822aa2c9cb
PMCID: PMC3865324
5.  High Weight Loss during Radiation Treatment Changes the Prognosis in Under-/Normal Weight Nasopharyngeal Carcinoma Patients for the Worse: A Retrospective Analysis of 2433 Cases 
PLoS ONE  2013;8(7):e68660.
Background
Although weight loss is common in nasopharyngeal carcinoma (NPC) patients receiving radiotherapy, the prognostic influence of weight loss and its impact modified by body mass index (BMI) are still unclear.
Methods
2433 NPC patients receiving radical radiotherapy at Sun Yat-sen University Cancer Center from November, 2000 to December, 2004 were enrolled. Weight change during radiation treatment was categorized into high weight loss (HWL) and low weight loss (LWL). The associations of HWL with overall survival (OS) and disease-specific survival (DSS) were analyzed by Cox regression.
Results
Among underweight patients, HWL was independently associated with poor OS (hazard ratio [HR], 2.06; 95% CI 1.36–3.11) and DSS (HR, 2.27; 95% CI 1.38–3.73), as compared with LWL, after adjusting for covariates. In normal weight patients, the impact of HWL on OS (HR, 1.47; 95% CI 1.19–1.80) and DSS (HR, 1.59; 95% CI 1.24–2.03) was moderate. Among overweight/obese patients, no significant association between HWL and OS (HR, 1.22; 95% CI 0.95–1.55), or DSS (HR, 1.23; 95% CI 0.93–1.64) was found.
Conclusion
Except for overweight/obese patients, high weight loss during radiation treatment was independently associated with poor survival in NPC. This impact was more prominent in the underweight patient group.
doi:10.1371/journal.pone.0068660
PMCID: PMC3711826  PMID: 23869226
6.  Conversion of human fibroblasts into angioblast-like multipotent progenitor cells 
Nature methods  2012;10(1):77-83.
Lineage conversion of one somatic cell type into another constitutes an attractive approach for research and clinical use. Lineage conversion can proceed in a direct manner, in the absence of proliferation and multipotent progenitor generation, or in an indirect manner, by the generation of expandable multipotent progenitor states. Here we report on the development of a combined reprogramming methodology that, transitioning through a plastic intermediate state, allows for the generation of human mesodermal progenitor cells while circumventing the traditional hallmarks of pluripotency. Converted mesodermal progenitor cells demonstrated bi-potent differentiation potential and were able to generate endothelial and smooth muscle lineages. Importantly, human fibroblasts can be converted into angioblast-like progenitor cells by non-integrative approaches. Differentiated angioblast-like cells exhibit neo-angiogenesis and anastomosis in vivo. The methodology for indirect lineage conversion to angioblast-like cells described here adds to the armamentarium of reprogramming approaches aimed at the clinical treatment of ischemic pathologies.
doi:10.1038/nmeth.2255
PMCID: PMC3531579  PMID: 23202434
7.  In Vitro Cytotoxicity of Fluorescent Silica Nanoparticles Hybridized with Aggregation-Induced Emission Luminogens for Living Cell Imaging 
Fluorescent silica nanoparticles (FSNPs) can provide high-intensity and photostable fluorescent signals as a probe for biomedical analysis. In this study, FSNPs hybridized with aggregation-induced emission (AIE) luminogens (namely FSNP-SD) were successfully fabricated by a surfactant-free sol-gel method. The FSNP-SD were spherical, monodisperse and uniform in size, with an average diameter of approximately 100 nm, and emitted strong fluorescence at the peak of 490 nm. The FSNP-SD selectively stained the cytoplasmic regions and were distributed in the cytoplasm. Moreover, they can stay inside cells, enabling the tacking of cells over a long period of time. The intracellular vesicles and multinucleated cells were increase gradually with the rise of FSNP-SD concentration. Both cell viability and survival only lost less than 20% when the cells were exposed to the high concentration of 100 μg/mL FSNP-SD. Additionally, the cell apoptosis and intracellular ROS assay indicated that FSNP-SD had no significant toxic effects at the maximum working concentration of 80 μg/mL. This study demonstrated that the FSNP-SD are promising biocompatible fluorescent probes for living cell imaging.
doi:10.3390/ijms14011080
PMCID: PMC3565308  PMID: 23296280
aggregation-induced emission; fluorescent silica nanoparticles; cytotoxicity; cell imaging; silole
8.  Influence of masticatory fatigue on the fracture resistance of the pulpless teeth restored with quartz-fiber post–core and crown 
To investigate whether masticatory fatigue affects the fracture resistance and pattern of lower premolars restored with quartz-fiber post–core and full crown, 44 single rooted lower premolars recently extracted from orthodontic patients were divided into two groups of 22 each. The crowns of all teeth were removed and endodontically treated and then restored with quartz-fiber post–core and full crown. Twenty-two teeth in one group were selected randomly and circularly loaded at 45° to the long axis of the teeth of 127.4 N at a 6 Hz frequency, and the other group was not delivered to cyclic loading and considered as control. Subsequently, all teeth in two groups were continually loaded to fail at 45° to the long axis of the teeth at a crosshead speed of 1 mm⋅min−1. The mean destructive force values were (733.88±254.99) and (869.14±280.26) N for the experimental and the control group, respectively, and no statistically significant differences were found between two groups (P>0.05). Bevel fracture and horizontal fracture in the neck of root were the major fracture mode of the specimens. Under the circumstances of this study, it seems that cyclic loading does not affect the fracture strength and pattern of the quartz-fiber post–core–crown complex.
doi:10.1038/ijos.2012.78
PMCID: PMC3633776  PMID: 23306858
fracture resistance; masticatory fatigue; pulpless teeth; quartz-fiber
9.  A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura, Dicroglossidae) 
PLoS ONE  2012;7(10):e46163.
Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.
doi:10.1371/journal.pone.0046163
PMCID: PMC3463521  PMID: 23056254
10.  CaMKII Is Essential for the Function of the Enteric Nervous System 
PLoS ONE  2012;7(8):e44426.
Background
Ca2+/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species.
Methodology/Principal Findings
CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue.
Conclusion
ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.
doi:10.1371/journal.pone.0044426
PMCID: PMC3432132  PMID: 22952977
11.  Characterization of Carbapenemase Genes in Enterobacteriaceae Species Exhibiting Decreased Susceptibility to Carbapenems in a University Hospital in Chongqing, China 
Annals of Laboratory Medicine  2012;32(4):270-275.
Background
Our study was to investigate the prevalence of carbapenemase genes in strains of Enterobacteriaceae species exhibiting decreased susceptibility to carbapenems in our hospital.
Methods
The carbapenemase producing Enterobacteriaceae species were confirmed by modified Hodge test (MHT) and EDTA-disc synergy test which indicating the production of class B carbapenemases. PCR and sequencing analysis were used to identify the drug-resistant genes. DNA fingerprinting based on enterobacterial repetitive intergenic consensus (ERIC)-PCR was applied to investigate the homology of Enterobacteriaceae species.
Results
From a collection of 1,472 Enterobacteriaceae species, 18 isolates with decreased susceptibility to carbapenem treatment were identified and 9 of which were positive by MHT, and 6 of which produced class B carbapenemases. PCR and sequencing analysis of the 18 isolates revealed 4 different carbapenemase genes (blaIMP-8, blaoxa-1, blaIMP-26, and blaoxa-47) in 10 isolates, with the blaIMP-8 and blaoxa-1 genes being the most common (60-70% prevalence). ERIC-PCR showed 5, 2, and 2 unique genotypes for Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae, respectively. Three E. coli strains isolated from different patients from the urologic surgery department exhibited the same DNA banding pattern, suggesting a possible clonal dissemination. Majority (17/18) of the carbapenem-unsusceptible Enterobacteriaceae species isolates was obtained from the surgery department of our hospital.
Conclusions
The main carbapenemase genes of Enterobacteriaceae species in our hospital were blaIMP-8 and blaoxa-1. Prevalence of carbapenem resistance may be existed in surgery department and infection control should be taken for preventing further dissemination of drug-resistant strains.
doi:10.3343/alm.2012.32.4.270
PMCID: PMC3384808  PMID: 22779068
Enterobacteriaceae species; Carbapenemases; Carbapenems
12.  Challenges in the modification of the M1 stage of the TNM staging system for nasopharyngeal carcinoma: A study of 1027 cases and review of the literature 
A series of modifications have been introduced to the TNM staging system over time for nasopharyngeal carcinoma (NPC), mainly focused on the T (primary tumor) and N (local node) components of the system. The M1 stage is a ‘catch all’ classification, covering a group of patients whose outlook ranges from potentially curable to incurable. Since the current M1 stage does not allow clinicians to stratify patients according to prognosis or guide therapeutic decision-making and allow comparison of results of radical and non-radical treatments, we aimed to subdivide the M1 stage according to a retrospective study of 1027 metastatic NPC patients and to review the relevant literature. Between 1995 and 2007, 1027 inpatients with distant metastasis from NPC were retrospectively analyzed. Various possible subdivisions of the M1 stage were considered, looking at different metastatic sites, the number of metastatic organs and the number of metastases. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test. The most frequently involved metastatic sites were the bone, lung and liver. The incidence rates of solitary metastatic lesions and pulmonary metastasis were 16.2 and 41.3%. Despite the poor survival of these patients with a median survival of 30.8 months, patients in the metachronous metastatic group with metastases to the lung and/or solitary lesions, were defined as M1a, and were significantly associated with favorable median survival of 41.5 and 49.1 months in the univariate and multivariate analysis, respectively. Patients in the metachronous metastatic group with metastasis to the lung and/or solitary lesions (M1a) have a more favorable prognosis compared with those patients with multiple metastases located in other anatomic sites (M1b). These data, in one of the largest reported metastatic NPC cohorts, are the first to show the prognostic impact of metastatic status in NPC. As a powerful predictor, the potential clinical value of a modified M1 of the TNM system for NPC will facilitate patient counseling and individualize management.
doi:10.3892/etm.2012.584
PMCID: PMC3460246  PMID: 23139721
distant metastasis; M1 stage; nasopharyngeal carcinoma; metastatic survival; prognostic factors
13.  Increased pretreatment levels of serum LDH and ALP as poor prognostic factors for nasopharyngeal carcinoma 
Chinese Journal of Cancer  2012;31(4):197-206.
Serum enzymes that play potential roles in tumor growth have recently been reported to have prognostic relevance in a diverse array of tumors. However, prognosis-related serum enzymes are rarely reported for nasopharyngeal carcinoma (NPC). To clarify whether the level of serum enzymes is linked to the prognosis of NPC, we reviewed the pretreatment data of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and glutamyl transferase (GGT) in 533 newly diagnosed NPC patients who underwent radical radiotherapy between May 2002 and October 2003 at Sun Yat-sen University Cancer Center. Patients were grouped according to the upper limit of normal values of LDH, ALP, and GGT. The Kaplan-Meier method and log-rank test were used for selecting prognostic factors from clinical characteristics and serum enzymes, and the Chi-square test was applied to analyze the relationships of clinical characteristics and serum enzymes. Finally, a Cox proportional hazards model was used to identify the independent prognostic factors. We found that increased levels of LDH had poor effects on both overall survival and distant metastasis-free survival (P = 0.009 and 0.035, respectively), and increased pretreatment level of serum ALP had poor effects on both overall survival and local recurrence-free survival (P = 0.037 and 0.039, respectively). In multivariate analysis, increased LDH level was identified as an independent prognostic factor for overall survival. Therefore, we conclude that increased pretreatment serum LDH and ALP levels are poor prognostic factors for NPC.
doi:10.5732/cjc.011.10283
PMCID: PMC3777475  PMID: 22237040
Nasopharyngeal carcinoma; LDH; ALP; GGT; prognosis
14.  Aurora-A is an efficient marker for predicting poor prognosis in human nasopharyngeal carcinoma with aggressive local invasion: 208 cases with a 10-year follow-up from a single institution 
Oncology Letters  2012;3(6):1237-1244.
Aurora-A kinase (Aur-A), a member of a family of mitotic serine/threonine kinases, is known to be amplified in epithelial malignancies. In this study, we focused our investigation on Aur-A expression and its prognostic significance in nasopharyngeal carcinoma (NPC). Immunohistochemical staining for Aur-A was performed on the paraffin sections of 208 patients with NPC. Data were subjected to statistical analysis with respect to clinicopathological variables, overall survival and disease-free survival. An immunohistochemical analysis showed that Aur-A was highly expressed in 132 (63.5%) of the 208 NPC tissues examined. Aur-A expression was significantly correlated with T classification (P=0.012), clinical stage (P=0.003) and skull base invasion (P=0.003). Statistical analysis showed that Aur-A expression was inversely correlated with the 10-year overall and disease-free survival rates of NPC patients. Results of the multivariate analysis revealed that Aur-A expression was an independent prognostic indicator for patient survival. More significantly, Aur-A was found to be a marker for poor survival, which was mainly attributed to its high expression in the subgroup of T4 tumor classification with aggressive local invasion. These results indicated that Aur-A expression is inversely correlated with survival and directly correlated with the malignant status of NPC. Therefore, Aur-A may serve as a potential biological marker for poor prognosis in the T4 subgroup of patients.
doi:10.3892/ol.2012.660
PMCID: PMC3392567  PMID: 22783425
nasopharyngeal carcinoma; Aurora-A; prognosis; T4 subgroup patients
15.  Long-term molecular changes in WHO grade II astrocytomas following radiotherapy 
Chinese Journal of Cancer  2012;31(3):159-165.
Monitoring the long-term radiotherapy-associated molecular changes in low-grade gliomas (LGGs) facilitates the understanding of LGG response to radiotherapy. In this study, we used immunohistochemistry to analyze the expression of Ki-67, tumor protein P53 (TP53), P21, and P27 in 8 paired WHO grade II astrocytoma samples. The interval between radiotherapy (RT) and the second surgery was more than 3 months in all cases. The average Ki-67 labeling index (LI) was 5.3% in pre-RT samples and 11.54% in post-RT samples. Ki-67 LI was higher in the primary tumors that underwent malignant transformation observed at the second surgery after radiation. Post-RT Ki-67 LI decreased in 2 cases with an interval of less than 12 months between RT and the second surgery. TP53 expression was found in 3 out of 4 pre-RT samples with malignant transformation and in 1 out of 4 pre-RT samples without malignant transformation. Post-RT TP53 increased in 2 cases in which increased expression of P21 or P27 was also observed. Our study suggests that radiotherapy can inhibit WHO grade II astrocytoma proliferation as reflected by Ki-67 LI, but the effect attenuates with time. In addition, there is a tendency of malignant transformation for WHO grade II astrocytomas with a high Ki-67 level or TP53 expression in initial samples.
doi:10.5732/cjc.011.10149
PMCID: PMC3777483  PMID: 22313596
Radiotherapy; WHO grade II astrocytoma; malignant transformation; Ki-67; TP53
16.  Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient 
Chinese Journal of Cancer  2012;31(1):36-44.
The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage-independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.
doi:10.5732/cjc.011.10317
PMCID: PMC3777465  PMID: 22176775
Nasopharyngeal carcinoma; Epstein-Barr virus; SUNE2 cell line
17.  Bmi-1 induces radioresistance in MCF-7 mammary carcinoma cells 
Oncology Reports  2011;27(4):1116-1122.
Bmi-1, a member of the polycomb family, it is involved in self renewal of stem cells and functions as an oncogene in many malignant human cancer types. Recent studies have demonstrated that Bmi-1 is a predictive factor for poor patient prognosis. However, the underlying mechanisms of radioresistance mediated by Bmi-1 are poorly understood. In this study, the dose-survival relationship was analyzed using a clonogenic survival assay and combined radiation treatment with Bmi-1 overexpression or silencing. DNA double-strand break (DSB) and repair was assessed by immunofluorescence staining of γH2AX foci. In addition, mitochondrial membrane potential was detected between Bmi-1 knockdown and control MCF-7 cells after irradiation. Apoptosis and cell cycle were evaluated by flow cytometry. We found that exposure of MCF-7 cells overexpressing Bmi-1 to ionizing radiation resulted in dramatically enhanced survival relative to control cells, whereas cells with silenced Bmi-1 showed markedly reduced survival. Bmi-1 inhibition significantly increased DSBs and decreased DSB repair. Furthermore, Bmi-1 knockdown induced loss of mitochondrial membrane potential and enhanced apoptosis by up-regulating p53, p21, Bax expression and down-regulating p-AKT and Bcl-2 expression. These results indicate that Bmi-1 may play an important role in radiosensitivity, and the suppression of its expression might be a potential therapeutic target for breast cancer.
doi:10.3892/or.2011.1615
PMCID: PMC3583403  PMID: 22209830
Bmi-1; radioresistance; mammary carcinoma cells
18.  ENU-Induced Mutagenesis in Grass Carp (Ctenopharyngodon idellus) by Treating Mature Sperm 
PLoS ONE  2011;6(10):e26475.
N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus) were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.
doi:10.1371/journal.pone.0026475
PMCID: PMC3195716  PMID: 22022617
19.  Herbal preparations for uterine fibroids 
Background
Uterine fibroids are the most common non-malignant growths in women of childbearing age. They are associated with heavy menstrual bleeding and subfertility. Herbal preparations are commonly used as alternatives to surgical procedures.
Objectives
To assess the benefits and risks of herbal preparations for uterine fibroids.
Search strategy
Authors searched following electronic databases: the Trials Registers of the Cochrane Menstrual Disorders and Subfertility Group and the Cochrane Complementary Medicine Field, the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2008, Issue 3), MEDLINE, EMBASE, the Chinese Biomedical Database, the Traditional Chinese Medical Literature Analysis and Retrieval System (TCMLARS), AMED, and LILACS. The searches ended on 31st December 2008.
Selection criteria
Randomised controlled trials comparing herbal preparations with no intervention, placebo, medical treatment or surgical procedures in women with uterine fibroids. We also included trials of herbal preparations with or without conventional therapy.
Data collection and analysis
Two review authors collected data independently. We assessed trial risk of bias according to our methodological criteria. We presented dichotomous data as risk ratios (RR) and continuous outcomes as mean difference (MD), both with 95% confidence intervals (CI).
Main results
We included two randomised trials (involved 150 women) with clear description of randomisation methods. The methodological risk of bias of the trials varied. There were variations in the tested herbal preparations, and the treatment duration was six months. The outcomes available were not the primary outcomes selected for this review, such as symptom relief or the need for surgical treatment; trials mainly reported outcomes in terms of shrinkage of the fibroids.
Compared with mifepristone, Huoxue Sanjie decoction showed no significant difference in the disappearance of uterine fibroids, number of patients with shrinking of uterine fibroids or average volume of uterine fibroids, but less effective than mifepristone on reducing average size of uterus (mean difference 23.23 cm3, 95% confidence interval 17.85 to 28.61). There was no significant difference between Nona Roguy herbal product and GnRH agonist in average volume of uterine fibroids or size of uterus. No serious adverse effects from herbal preparations was reported.
Authors’ conclusions
Current evidence does not support or refute the use of herbal preparations for treatment of uterine fibroids due to insufficient studies of large sample and high quality. Further high quality trials evaluating clinically relevant outcomes are warranted.
doi:10.1002/14651858.CD005292.pub2
PMCID: PMC3155698  PMID: 19370619
Drugs; Chinese Herbal [therapeutic use]; Gonadotropin-Releasing Hormone [therapeutic use]; Hormone Antagonists [therapeutic use]; Leiomyoma [*drug therapy]; Mifepristone [therapeutic use]; Phytotherapy [*methods]; Plant Preparations [*therapeutic use]; Randomized Controlled Trials as Topic; Female; Humans
20.  Transcription factor c-Myb promotes the invasion of hepatocellular carcinoma cells via increasing osteopontin expression 
Background
Specific gene expression is tightly regulated by various transcription factors. Osteopontin (OPN) is a phosphoprotein that mediates hepatocellular carcinoma (HCC) progression and metastasis. However, the mechanism of OPN up-regulation in HCC metastasis remains to be clarified.
Methods
Oligonucleotide array-based transcription factor assays were applied to compare different activities of transcription factors in two human HCC cell lines with different OPN expression levels. The effects of one selected transcription factor on OPN expression were further evaluated.
Results
Eleven transcription factors were over-expressed in metastatic HCC cell line HCCLM6 cells whereas twelve transcription factors were down-regulated. Electrophoretic mobility shift assays (EMSA) and reporter gene assays showed that one of up-regulated transcription factors c-Myb could bind the OPN promoter and increase its transcription activity. In addition, small interfering RNA targeting c-Myb could inhibit OPN expression and significantly decrease migration and invasion of HCCLM6 cells in vitro.
Conclusion
Our data first demonstrate that c-Myb has a functionally important role in the regulation of OPN expression in HCC cells, suggesting that c-Myb might be a new target to control HCC metastasis.
doi:10.1186/1756-9966-29-172
PMCID: PMC3023683  PMID: 21190594
21.  Prognostic significance and therapeutic implications of centromere protein F expression in human nasopharyngeal carcinoma 
Molecular Cancer  2010;9:237.
Background
Our recent cDNA microarray data showed that centromere protein F (CENP-F) is significantly upregulated in primary cultured nasopharyngeal carcinoma (NPC) tumor cells compared with normal nasopharyngeal epithelial cells. The goal of this study was to further investigate the levels of CENP-F expression in NPC cell lines and tissues to clarify the clinical significance of CENP-F expression in NPC as well as the potential therapeutic implications of CENP-F expression.
Methods
Real-time RT-PCR and western blotting were used to examine CENP-F expression levels in normal primary nasopharyngeal epithelial cells (NPEC), immortalized nasopharyngeal epithelial cells and NPC cell lines. Levels of CENP-F mRNA were determined by real-time RT-PCR in 23 freshly frozen nasopharyngeal biopsy tissues, and CENP-F protein levels were detected by immunohistochemistry in paraffin sections of 202 archival NPC tissues. Statistical analyses were applied to test for prognostic associations. The cytotoxicities of CENP-F potential target chemicals, zoledronic acid (ZOL) and FTI-277 alone, or in combination with cisplatin, in NPC cells were determined by the MTT assay.
Results
The levels of CENP-F mRNA and protein were higher in NPC cell lines than in normal and immortalized NPECs. CENP-F mRNA level was upregulated in nasopharyngeal carcinoma biopsy tissues compared with noncancerous tissues. By immunohistochemical analysis, CENP-F was highly expressed in 98 (48.5%) of 202 NPC tissues. Statistical analysis showed that high expression of CENP-F was positively correlated with T classification (P < 0.001), clinical stage (P < 0.001), skull-base invasion (P < 0.001) and distant metastasis (P = 0.012) inversely correlated with the overall survival time in NPC patients. Multivariate analysis showed that CENP-F expression was an independent prognostic indicator for the survival of the patient. Moreover, we found that ZOL or FTI-277 could significantly enhance the chemotherapeutic sensitivity of NPC cell lines (HONE1 and 6-10B) with high CENP-F expression to cisplatin, although ZOL or FTI-277 alone only exhibited a minor inhibitory effect to NPC cells.
Conclusion
Our data suggest that CENP-F protein is a valuable marker of NPC progression, and CENP-F expression is associated with poor overall survival of patients. In addition, our data indicate a potential benefit of combining ZOL or FTI-277 with cisplatin in NPC suggesting that CENP-F expression may have therapeutic implications.
doi:10.1186/1476-4598-9-237
PMCID: PMC2944187  PMID: 20828406
22.  Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma 
PLoS Pathogens  2010;6(6):e1000940.
It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.
Author Summary
Epstein-Barr virus (EBV) infects about 90% of people worldwide and persists benignly as a latent infection. However, EBV is associated with different types of human cancer. Nasopharyngeal carcinoma (NPC) is the most commonly known EBV associated cancer and expresses a well defined set of latent viral genes, including LMP2A, which has been detected in the majority of NPC samples. Several studies indicated this latent viral protein drove cellular invasion and metastasis. For this study, enforced LMP2A expressing NPC cell lines were generated. We show here that LMP2A induces an Epithelial–Mesenchymal Transition and increases the Stem-like Cancer Cells in NPC. Our results suggest that LMP2A supports tumor initiation and recurrence of the infected nasopharyngeal epithelial cells. For the first time we report a virus protein that functions in the initiation and progression of cancer by inducing the cancer stem-like cells. These findings permit a more detailed understanding of function and contribution to viral pathogenesis and provide a novel therapeutic target for NPC therapy.
doi:10.1371/journal.ppat.1000940
PMCID: PMC2880580  PMID: 20532215
23.  Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System 
The canonical transient receptor potential (TRPC) family of ion channels is implicated in many neuronal processes including calcium homeostasis, membrane excitability, synaptic transmission and axon guidance. TRPC channels are postulated to be important in the functional neurobiology of the enteric nervous system (ENS); nevertheless, details for expression in the ENS are lacking. RT-PCR, Western blotting, and immunohistochemistry were used to study the expression and localization of TRPC channels. We found mRNA transcripts, protein on Western blots and immunoreactivity (IR) for TRPC1/3/4/6 expressed in the small intestinal ENS of adult guinea pigs. TRPC1/3/4/6-IR was localized to distinct subpopulations of enteric neurons and was differentially distributed between the myenteric and submucosal divisions of the ENS. TRPC1-IR was widely distributed and localized to neurons with cholinergic, calretinin, and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to both cholinergic and non-cholinergic secretomotor neurons in the submucosal plexus. TRPC3-IR was found only in the submucosal plexus and was expressed exclusively by neuropeptide Y-IR neurons. TRPC4/6-IR was expressed in only a small population of myenteric neurons, but was abundantly expressed in the submucosal plexus. TRPC4/6-IR was coexpressed with both cholinergic and nitrergic neurochemical codes in the myenteric plexus. In the submucosal plexus, TRPC4/6-IR was expressed exclusively in non-cholinergic secretomotor neurons. No TRPC1/3/4/6-IR was found in calbindin-IR neurons. TRPC3/4/6-IR was widely expressed along varicose nerve fibers and colocalized with synaptophysin-IR at putative neurotransmitter release sites. Our results suggest important roles for TRPC channels in ENS physiology and neuronal regulation of gut function.
doi:10.1002/cne.21874
PMCID: PMC2577062  PMID: 18925632
neurogastroenterology; TRP channels; gastrointestinal tract; myenteric plexus; submucosal plexus
24.  The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells 
The Journal of Clinical Investigation  2009;119(12):3626-3636.
The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1–mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3β signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1–silenced cells, indicating that PTEN might be a major mediator of Bmi-1–induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer.
doi:10.1172/JCI39374
PMCID: PMC2786794  PMID: 19884659
25.  Distribution and Chemical Coding of Corticotropin Releasing Factor-Immunoreactive Neurons in the Guinea-Pig Enteric Nervous System 
Immunofluorescence was used to study immunoreactivity (IR) for corticotropin releasing factor (CRF) in the guinea-pig enteric nervous system. CRF-IR was expressed in both the myenteric and submucosal plexuses of all regions of the large and small intestine and the myenteric plexus of the stomach. CRF-IR-nerve fibers were present in the myenteric and submucosal plexuses, in the circular muscle coat and surrounding submucosal arterioles. Most of the CRF-IR fibers persisted in the myenteric and submucosal plexuses after 7 days in organotypic culture. CRF-IR was not co-expressed with tyrosine hydroxylase-IR or calcitonin gene-related peptide-IR fibers. The proportions of CRF-IR cell bodies in the myenteric plexus increased progressively from the stomach (0.6%) to the distal colon (2.8%). Most of the CRF-IR myenteric neurons (95%) had uniaxonal morphology; the remainder had Dogiel type II multipolar morphology. CRF-IR cell bodies in the myenteric plexus of the ileum expressed IR for choline acetyltransferase (56.9%), substance P (55.0%), and nitric oxide synthase (37.9%). CRF-IR never co-localized with IR for calbindin, calretinin, neuropeptide Y, serotonin or somatostatin in the myenteric plexus. CRF-IR cell bodies were more abundant in the submucosal plexus (29.9-38.0%) than in the myenteric plexus. All CRF-IR neurons in submucosal ganglia expressed vasoactive intestinal peptide-IR and were likely to be secretomotor/vasodilator neurons. CRF-IR neurons did not express IR for the CRF1 receptor. CRF1-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.
doi:10.1002/cne.20781
PMCID: PMC2582187  PMID: 16304680
gastrointestinal tract; myenteric plexus; submucosal plexus; stress

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