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1.  Expression of Derlin-1 and its effect on expression of autophagy marker genes under endoplasmic reticulum stress in lung cancer cells 
Background
Recent findings indicated that Derlin-1 has an important function in tumour progression. In this study, we aimed to determine whether Derlin-1 has an oncogene function as a cross-talk molecule with autophagy.
Methods
Cancer cells were treated with tunicamycin (TM) for 8 and 24 h. The expression of Derlin-1 and autophagy-related genes was determined by western blot. Autophagy was analysed by fluorescence microscopy after staining the cancer cells with monodansylcadaverine. The interaction between Derlin-1 and other proteins was identified using co-immunoprecipitation assay.
Results
Our study demonstrated high Derlin-1 expression levels in most non-small lung cancer cell lines. Derlin-1 expression was enhanced under endoplasmic reticulum (ER) stress. Previous studies revealed that TM triggers the initiation of autophagy by activating Beclin 1, converting LC3I to LC3II and degrading p62. Knockdown of Derlin-1 did not affect Beclin 1 and LC3II expression but disrupted the degradation of p62 under ER stress, which resulted in the blockage of autophagy flux. Furthermore, Derlin-1 and p62 were observed to interact under ER stress.
Conclusion
This study is the first report about the interaction between Derlin-1 and p62. Derlin-1 may function in tumour progression partially by interacting with p62.
doi:10.1186/1475-2867-14-50
PMCID: PMC4061450  PMID: 24944523
Derlin-1; Autophagy; ER stress; p62
2.  Down-regulated γ-catenin expression is associated with tumor aggressiveness in esophageal cancer 
AIM: To evaluate the significance of γ-catenin in clinical pathology, cellular function and signaling mechanism in esophageal squamous cell carcinoma (ESCC).
METHODS: The mRNA expression of γ-catenin was detected by real-time quantitative reverse transcription-polymerase chain reaction in 95 tissue specimens and evaluated for association with the clinicopathologic characteristics and survival time of patients with ESCC. siRNAs against human γ-catenin were used to inhibit γ-catenin expression. Hanging drop aggregation assay and dispase-based dissociation assay were performed to detect the effect of γ-catenin on ESCC cell-cell adhesion. Transwell assay was performed to determine cell migration. Luciferase-based transcriptional reporter assay (TOPflash) was used to measure β-catenin-dependent transcription in cells with reduced γ-catenin expression. The expression and subcellular localizations of β-catenin and E-cadherin were examined using Western blot and immunofluorescence analysis.
RESULTS: γ-catenin mRNA expression was significantly associated with tumor histological grade (P = 0.017) in ESCC. Kaplan-Meier survival analysis showed that γ-catenin expression levels had an impact on the survival curve, with low γ-catenin indicating worse survival (P = 0.003). The multivariate Cox regression analysis demonstrated that γ-catenin was an independent prognostic factor for survival. Experimentally, silencing γ-catenin caused defects in cell-cell adhesion and a concomitant increase in cell migration in both KYSE150 and TE3 ESCC cells. Analysis of Wnt signaling revealed no activation event associated with γ-catenin expression. Total β-catenin and Triton X-100-insoluble β-catenin were significantly reduced in the γ-catenin-specific siRNA-transfected KYSE150 and TE3 cells, whereas Triton X-100-soluble β-catenin was not altered. Moreover, knocking down γ-catenin expression resulted in a significant decrease of E-cadherin and Triton X-100-insoluble desmocollin-2, along with reduced β-catenin and E-cadherin membrane localization in ESCC cells.
CONCLUSION: γ-catenin is a tumor suppressor in ESCC and may serve as a prognostic marker. Dysregulated expression of γ-catenin may play important roles in ESCC progression.
doi:10.3748/wjg.v20.i19.5839
PMCID: PMC4024793  PMID: 24914344
γ-catenin; Esophageal squamous cell carcinoma; Independent prognostic factor; Cell-cell adhesion; Cell migration
3.  Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis 
BMC Genomics  2014;15(1):363.
Background
Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.
Results
The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.
Conclusions
This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-363) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-363
PMCID: PMC4048454  PMID: 24884615
Amycolatopsis orientalis; Complete genome sequencing; Molecular taxonomic characteristics; Vancomycin biosynthesis
4.  Vitamin D-Binding Protein Levels in Plasma and Gingival Crevicular Fluid of Patients with Generalized Aggressive Periodontitis 
Vitamin D-binding protein (DBP) is the main transport protein of vitamin D and plays an important role in the immune system and host defenses. The purpose of this study was to measure DBP levels in plasma and gingival crevicular fluid (GCF) of patients with generalized aggressive periodontitis (GAgP), in comparison to healthy controls, with the goal of elucidating the relationship between DBP and GAgP. Fifty-nine GAgP patients and 58 healthy controls were recruited for the study; clinical parameters of probing depths (PD), bleeding index, and attachment loss (AL) were recorded. DBP levels were measured by enzyme-linked immunosorbent assay. From the results, GAgP patients had higher plasma DBP concentrations (P < 0.001) but lower GCF DBP concentrations (P < 0.001) than healthy controls. In GAgP group, after controlling the potential confounders of age, gender, smoking status, and BMI index, GCF DBP concentrations correlated negatively with PD (P < 0.001) and AL (P = 0.009). Within the limits of the study, we concluded that decreased GCF DBP level and increased plasma DBP level are associated with periodontitis.
doi:10.1155/2014/783575
PMCID: PMC4036603  PMID: 24899892
5.  Complete Genome Sequence of Neisseria meningitidis Serogroup A Strain NMA510612, Isolated from a Patient with Bacterial Meningitis in China 
Genome Announcements  2014;2(3):e00360-14.
Serogroup A meningococcal strains have been involved in several pandemics and a series of epidemics worldwide in the past. Determination of the genome sequence of the prevalent genotype strain will help us understand the genetic background of the evolutionary and epidemiological properties of these bacteria. We sequenced the complete genome of Neisseria meningitidis NMA510612, a clinical isolate from a patient with meningococcal meningitis.
doi:10.1128/genomeA.00360-14
PMCID: PMC4014685  PMID: 24812217
6.  Synthesis, Characterization, In Vitro Cytotoxicity, and Apoptosis-Inducing Properties of Ruthenium(II) Complexes 
PLoS ONE  2014;9(5):e96082.
Two new Ru(II) complexes, [Ru(bpy)2(FAMP)](ClO4)2 1 and 2, are synthesized and characterized by elemental analysis, electrospray mass spectrometry, and 1H nuclear magnetic resonance. The in vitro cytotoxicities and apoptosis-inducing properties of these complexes are extensively studied. Complexes 1 and 2 exhibit potent antiproliferative activities against a panel of human cancer cell lines. The cell cycle analysis shows that complexes 1 and 2 exhibit effective cell growth inhibition by triggering G0/G1 phase arrest and inducing apoptosis by mitochondrial dysfunction. The in vitro DNA binding properties of the two complexes are investigated by different spectrophotometric methods and viscosity measurements.
doi:10.1371/journal.pone.0096082
PMCID: PMC4013043  PMID: 24804832
7.  Identification and characterization of microRNAs in the ovaries of multiple and uniparous goats (Capra hircus) during follicular phase 
BMC Genomics  2014;15(1):339.
Background
Superior kidding rate is an important economic trait in production of meat goat, and ovulation rate is the precondition of kidding rate. MicroRNAs (miRNAs) play critical roles in almost all ovarian biological processes, including folliculogenesis, follicle development, follicle atresia, luteal development and regression. To find out the different ovarian activity and follicle recruitment with miRNA-mediated posttranscriptional regulation, the small RNAs expressed pattern in the ovarian tissues of multiple and uniparous Anhui White goats during follicular phase was analyzed using Solexa sequencing data.
Results
1008 miRNAs co-expressed, 309 and 433 miRNAs specifically expressed in the ovaries of multiple and uniparous goats during follicular phase were identified. The 10 most highly expressed miRNAs in the multiple library were also the highest expressed in the uniparous library, and there were no significantly different between each other. The highest specific expressed miRNA in the multiple library was miR-29c, and the one in the uniparous library was miR-6406. 35 novel miRNAs were predicted in total. GO annotation and KEGG Pathway analyses were implemented on target genes of all miRNA in two libraries. RT-PCR was applied to detect the expression level of 5 randomly selected miRNAs in multiple and uniparous hircine ovaries, and the results were consistent with the Solexa sequencing data.
Conclusions
In the present study, the different expression of miRNAs in the ovaries of multiple and uniparous goats during follicular phase were characterized and investigated using deep sequencing technology. The result will help to further understand the role of miRNAs in kidding rate regulation and also may help to identify miRNAs which could be potentially used to increase hircine ovulation rate and kidding rate in the future.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-339) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-339
PMCID: PMC4035069  PMID: 24886377
MicroRNA; Kidding rate; Solexa sequencing; Ovary; Follicular phase; Goat
8.  Significance of MDM2 and P14ARF polymorphisms in susceptibility to differentiated thyroid carcinoma 
Surgery  2012;153(5):711-717.
Background
Murine double minute 2 (MDM2) oncoprotein and p14ARF tumor suppressor play pivotal roles in regulating p53 and function in the MAPK pathway, which is frequently mutated in differentiated thyroid carcinoma (DTC). We hypothesized that functional polymorphisms in the promoters of MDM2 and p14ARF contribute to the inter-individual difference in predisposition to DTC.
Methods
MDM2-rs2279744, MDM2-rs937283, p14ARF-rs3731217, and p14ARF-rs3088440 were genotyped in 303 patients with DTC and 511 cancer-free controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs).
Results
MDM2-rs2279744 and p14ARF-rs3731217 were associated with a significantly increased risk of DTC (MDM2-rs2279744: TT vs. TG/GG, OR = 1.5, 95% CI, 1.1–2.0; p14ARF-rs3731217: TG/GG vs. TT, OR = 1.7, 95% CI, 1.2–2.3). No association was found for MDM2-rs937283 or p14ARF-rs3088440. Individuals carrying 3–4 risk genotypes of MDM2 and p14ARF had 2.2 times (95% CI, 1.4–3.5) the DTC risk of individuals carrying 0–1 risk genotypes (Ptrend = 0.021). The combined effect of MDM2 and p14ARF on DTC risk was confined to young subjects (≤45 years), non-smokers, non-drinkers, and subjects with a first-degree family history of cancer. These associations were quite similar in strength when cases were restricted to those with papillary thyroid cancer.
Conclusion
Our results suggest that polymorphisms of MDM2 and p14ARF contribute to the inter-individual difference in susceptibility to DTC, either alone or more likely jointly. The observed associations warrant further confirmation in independent studies.
doi:10.1016/j.surg.2012.11.009
PMCID: PMC3610784  PMID: 23218882
papillary thyroid carcinoma; p53 pathway; case-control study
9.  Long-term survival of patients with hepatocellular carcinoma with inferior vena cava tumor thrombus treated with sorafenib combined with transarterial chemoembolization: report of two cases and literature review 
Chinese Journal of Cancer  2014;33(5):259-264.
The prognosis of hepatocellular carcinoma (HCC) with tumor thrombus formation in the main vasculature is extremely poor. Sorafenib combined with transarterial chemoembolization is a novel treatment approach for advanced HCC. In this study, we report two HCC patients with inferior vena cava tumor thrombus who underwent the combination treatment. The overall survival times for these two patients were 44 months and 35 months, respectively. Our report suggests that sorafenib combined with transarterial chemoembolization may be a viable choice for patients with advanced HCC even with inferior vena cava tumor thrombus. Further studies are required to verify the efficacy and safety of this combination therapy for patients with advanced HCC with inferior vena cava tumor thrombus.
doi:10.5732/cjc.013.10133
PMCID: PMC4026547  PMID: 24325788
Hepatocellular carcinoma; sorafenib; transarterial chemoembolization; inferior vena cava; tumor thrombus
10.  Heat shock protein 27 regulates human prostate cancer cell motility and metastatic progression 
Oncotarget  2014;5(9):2648-2663.
Prostate cancer (PCa) is the most common form of cancer in American men. Mortality from PCa is caused by the movement of cancer cells from the primary organ to form metastatic tumors at distant sites. Heat shock protein 27 (HSP27) is known to increase human PCa cell invasion and its overexpression is associated with metastatic disease. The role of HSP27 in driving PCa cell movement from the prostate to distant metastatic sites is unknown. Increased HSP27 expression increased metastasis as well as primary tumor mass. In vitro studies further examined the mechanism of HSP27-induced metastatic behavior. HSP27 did not affect cell detachment, adhesion, or migration, but did increase cell invasion. Cell invasion was dependent upon matrix metalloproteinase 2 (MMP-2), whose expression was increased by HSP27. In vivo, HSP27 induced commensurate changes in MMP-2 expression in tumors. These findings demonstrate that HSP27 drives metastatic spread of cancer cells from the prostate to distant sites, does so across a continuum of expression levels, and identifies HSP27-driven increases in MMP-2 expression as functionally relevant. These findings add to prior studies demonstrating that HSP27 increases PCa cell motility, growth and survival. Together, they demonstrate that HSP27 plays an important role in PCa progression.
PMCID: PMC4058034  PMID: 24798191
Prostate Cancer; Metastasis; Heat Shock Protein 27; Matrix Metalloprotease-2; Cell Invasion
11.  Ameliorating Adriamycin-Induced Chronic Kidney Disease in Rats by Orally Administrated Cardiotoxin from Naja naja atra Venom 
Previous studies reported the oral administration of Naja naja atra venom (NNAV) reduced adriamycin-induced chronic kidney damage. This study investigated the effects of intragastric administrated cardiotoxin from Naja naja atra venom on chronic kidney disease in rats. Wistar rats were injected with adriamycin (ADR; 6 mg/kg body weight) via the tail vein to induce chronic kidney disease. The cardiotoxin was administrated daily by intragastric injection at doses of 45, 90, and 180 μg/kg body weight until the end of the protocol. The rats were placed in metabolic cages for 24 hours to collect urine, for determination of proteinuria, once a week. After 6 weeks, the rats were sacrificed to determine serum profiles relevant to chronic kidney disease, including albumin, total cholesterol, phosphorus, blood urea nitrogen, and serum creatinine. Kidney histology was examined with hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining. The levels of kidney podocin were analyzed by Western blot analysis and immunofluorescence. We found that cardiotoxin reduced proteinuria and can improve biological parameters in the adriamycin-induced kidney disease model. Cardiotoxin also reduced adriamycin-induced kidney pathology, suggesting that cardiotoxin is an active component of NNAV for ameliorating adriamycin-induced kidney damage and may have a potential therapeutic value on chronic kidney disease.
doi:10.1155/2014/621756
PMCID: PMC4021839  PMID: 24876873
12.  Specific Capture and Release of Circulating Tumor Cells Using Aptamer Modified Nanosubstrates 
doi:10.1002/adma.201300082
PMCID: PMC3786685  PMID: 23495071
Cancer Diagnosis; Circulating Tumor Cells; Nanostructured materials; Aptamer; Cell Capture; Cell Release
13.  Parallel mRNA and MicroRNA Profiling of HEV71-Infected Human Neuroblastoma Cells Reveal the Up-Regulation of miR-1246 in Association with DLG3 Repression 
PLoS ONE  2014;9(4):e95272.
Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3′-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.
doi:10.1371/journal.pone.0095272
PMCID: PMC3989279  PMID: 24739954
14.  Relationship between tone perception and production in prelingually-deafened children with cochlear implants 
Hypothesis
Performance in tone perception and production are correlated in prelingually-deafened pediatric cochlear implant (CI) users across individuals. Demographic variables, such as age at implantation, contribute to the performance variability.
Background
Poor representation of pitch information in CI devices hinders pitch perception and affects perception of lexical tones in cochlear implant users who speak tonal languages.
Methods
One hundred and ten Mandarin-speaking, prelingually-deafened CI subjects and 125 typically-developing, normal-hearing subjects were recruited from Beijing, China. Lexical tone perception was measured using a computerized tone contrast test. Tone production was judged by native Mandarin-speaking adult listeners and analyzed acoustically and with an artificial neural network. A general linear model analysis was performed to determine factors that accounted for performance variability.
Results
CI subjects scored ~67% correct on the lexical tone perception task. The degree of differentiation of tones produced by the CI group was significantly lower than the control group as revealed by acoustic analysis. Tone production performance assessed by the neural network was highly correlated with that evaluated by human listeners. There was a moderate correlation between the overall tone perception and production performance across CI subjects. Duration of implant use and age at implantation jointly explained ~29% of the variance in the tone perception performance. Age at implantation was the only significant predictor for tone production performance in the CI subjects.
Conclusion
Tone production performance in pediatric CI users is dependent on accurate perception. Early implantation predicts better outcome in lexical tone perception and production.
doi:10.1097/MAO.0b013e318287ca86
PMCID: PMC3600119  PMID: 23442566
15.  Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein 
Antimicrobial Agents and Chemotherapy  2013;57(10):4937-4944.
Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients.
doi:10.1128/AAC.00897-13
PMCID: PMC3811438  PMID: 23877701
16.  Molecular Epidemiology of Clostridium difficile Infection in a Major Chinese Hospital: an Underrecognized Problem in Asia? 
Journal of Clinical Microbiology  2013;51(10):3308-3313.
Clostridium difficile infection is almost unrecognized in mainland China. We have undertaken a study in a large Chinese teaching hospital in Changsha, Hunan, China, to identify cases of C. difficile, record patient characteristics, and define the molecular epidemiology with respect to ribotype distribution and cross-infection. Between April 2009 and February 2010, we examined fecal samples from 70 hospitalized patients with diarrhea who were receiving or had received antibiotics within the previous 6 weeks. Clinical information was collected and the samples were cultured for C. difficile retrospectively. Isolates were ribotyped, and multiple-locus variable-number tandem-repeat assay (MLVA) subtyping was performed on clusters of the same ribotype. The mean age of patients from whom C. difficile was cultured was 58 years, with only 4/21 patients aged >65 years. All patients, with a single exception, had received a third-generation cephalosporin and/or a quinolone antibiotic. Twenty-one isolates of C. difficile were recovered, and seven different ribotypes were identified, the dominant types being 017 (48%), 046 (14%), and 012 (14%). We identified two clusters of cross-infection with indistinguishable isolates of ribotype 017, with evidence of spread both within and between wards. We have identified C. difficile as a possibly significant problem, with cross-infection and a distinct ribotype distribution, in a large Chinese hospital. C. difficile may be underrecognized in China, and further epidemiological studies across the country together with the introduction of routine diagnostic testing are needed to ascertain the size of this potentially significant problem.
doi:10.1128/JCM.00587-13
PMCID: PMC3811633  PMID: 23903542
17.  VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation 
PLoS ONE  2014;9(4):e93547.
Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway.
doi:10.1371/journal.pone.0093547
PMCID: PMC3972149  PMID: 24691407
18.  Primary retroperitoneal extraskeletal mesenchymal chondrosarcoma involving the vena cava: A case report 
Oncology Letters  2014;7(6):1970-1974.
The current study presents a case of extraskeletal mesenchymal chondrosarcoma (ESMC) involving the vena cava that originally occurred in the retroperitoneum of a 61-year-old female. Following excision of the masses, pathological examination confirmed a diagnosis of primary ESMC. Mesenchymal chondrosarcomas are extremely rare in comparison to conventional chondrosarcomas and even more so when arising in an extraskeletal location. In the current report, the major characteristics of ESMC are discussed and a review of the current knowledge regarding this rare disease entity is presented.
doi:10.3892/ol.2014.2012
PMCID: PMC4049765  PMID: 24932271
extraskeletal chondrosarcoma; mesenchymal; X-ray computed tomography; magnetic resonance imaging
19.  L-Serine Treatment May Improve Neurorestoration of Rats after Permanent Focal Cerebral Ischemia Potentially Through Improvement of Neurorepair 
PLoS ONE  2014;9(3):e93405.
The present study was conducted to clarify whether treatment with L-serine can improve the brain repair and neurorestoration of rats after permanent middle cerebral artery occlusion (pMCAO). After pMCAO, the neurological functions, brain lesion volume, and cortical injury were determined. GDNF, NGF, NCAM L1, tenascin-C, and Nogo-A levels were measured. Proliferation and differentiation of the neural stem cells (NSCs) and proliferation of the microvessels in the ischemic boundary zone of the cortex were evaluated. Treatment with L-serine (168 mg/kg body weight, i.p.) began 3 h after pMCAO and was repeated every 12 h for 7 days or until the end of the experiment. L-Serine treatment: 1) reduced the lesion volume and neuronal loss; 2) improved the recovery of neurological functions; 3) elevated the expression of nerve growth-related factors; and 4) facilitated the proliferation of endogenous NSCs and microvessels activated after pMCAO and increased the number of new-born neurons. 5) D-cycloserine, an inhibitor of serine hydroxymethyltransferase, blunted the effects of L-serine on NSC proliferation, differentiation, microvascular proliferation. In conclusions, L-serine treatment in pMCAO rats can reduce brain injury and facilitate neurorestoration which is partly associated with the improvement of proliferation of NSCs and microvessels, reconstruction of neurovascular units and resultant neurorepair. The effects of L-serine on endogenous NSC proliferation and microvascular proliferation are partly mediated by the action of L-serine as a substrate for the production of one-carbon groups used for purine and pyrimidine synthesis and modulation of the expression of some nerve growth-related factors.
doi:10.1371/journal.pone.0093405
PMCID: PMC3966884  PMID: 24671106
20.  Anoctamin 6 Regulates C2C12 Myoblast Proliferation 
PLoS ONE  2014;9(3):e92749.
Anoctamin 6 (Ano6) belongs to a conserved gene family (TMEM16) predicted to code for eight transmembrane proteins with putative Ca2+-activated chloride channel (CaCC) activity. Recent work revealed that disruption of ANO6 leads to a blood coagulation defect and impaired skeletal development. However, its function in skeletal muscle cells remains to be determined. By using a RNA interference mediated (RNAi) loss-of-function approach, we show that Ano6 regulates C2C12 myoblast proliferation. Ano6 is highly expressed in C2C12 myoblasts and its expression decreases upon differentiation. Knocking down Ano6 significantly reduces C2C12 myoblast proliferation but has minimal effect on differentiation. Ano6 deficiency significantly reduces ERK/AKT phosphorylation, which has been shown to be involved in regulation of cancer cell proliferation by another Anoctamin member. Taken together, our data demonstrate for the first time that Ano6 plays an essential role in C2C12 myoblast proliferation, likely via regulating the ERK/AKT signaling pathway.
doi:10.1371/journal.pone.0092749
PMCID: PMC3963950  PMID: 24663380
21.  Neuroprotective Effects of Sevoflurane against Electromagnetic Pulse-Induced Brain Injury through Inhibition of Neuronal Oxidative Stress and Apoptosis 
PLoS ONE  2014;9(3):e91019.
Electromagnetic pulse (EMP) causes central nervous system damage and neurobehavioral disorders, and sevoflurane protects the brain from ischemic injury. We investigated the effects of sevoflurane on EMP-induced brain injury. Rats were exposed to EMP and immediately treated with sevoflurane. The protective effects of sevoflurane were assessed by Nissl staining, Fluoro-Jade C staining and electron microscopy. The neurobehavioral effects were assessed using the open-field test and the Morris water maze. Finally, primary cerebral cortical neurons were exposed to EMP and incubated with different concentration of sevoflurane. The cellular viability, lactate dehydrogenase (LDH) release, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were assayed. TUNEL staining was performed, and the expression of apoptotic markers was determined. The cerebral cortexes of EMP-exposed rats presented neuronal abnormalities. Sevoflurane alleviated these effects, as well as the learning and memory deficits caused by EMP exposure. In vitro, cell viability was reduced and LDH release was increased after EMP exposure; treatment with sevoflurane ameliorated these effects. Additionally, sevoflurane increased SOD activity, decreased MDA levels and alleviated neuronal apoptosis by regulating the expression of cleaved caspase-3, Bax and Bcl-2. These findings demonstrate that Sevoflurane conferred neuroprotective effects against EMP radiation-induced brain damage by inhibiting neuronal oxidative stress and apoptosis.
doi:10.1371/journal.pone.0091019
PMCID: PMC3948751  PMID: 24614080
22.  Reduced Intensity Conditioning, Combined Transplantation of Haploidentical Hematopoietic Stem Cells and Mesenchymal Stem Cells in Patients with Severe Aplastic Anemia 
PLoS ONE  2014;9(3):e89666.
We examined if transplantation of combined haploidentical hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) affected graft failure and graft-versus-host disease (GVHD) in patients with severe aplastic anemia (SAA). Patients with SAA-I (N = 17) received haploidentical HSCT plus MSC infusion. Stem cell grafts used a combination of granulocyte colony-stimulating factor (G-CSF)-primed bone marrow and G-CSF-mobilized peripheral blood stem cells of haploidentical donors and the culture-expanded third-party donor-derived umbilical cord MSCs (UC-MSCs), respectively. Reduced intensity conditioning consisted of fludarabine (30 mg/m2·d)+cyclosphamide (500 mg/m2·d)+anti-human thymocyte IgG. Transplant recipients also received cyclosporin A, mycophenolatemofetil, and CD25 monoclonal antibody. A total of 16 patients achieved hematopoietic reconstitution. The median mononuclear cell and CD34 count was 9.3×108/kg and 4.5×106/kg. Median time to ANC was >0.5×109/L and PLT count >20×109/L were 12 and 14 days, respectively. Grade III-IV acute GVHD was seen in 23.5% of the cases, while moderate and severe chronic GVHD were seen in 14.2% of the cases. The 3-month and 6-month survival rates for all patients were 88.2% and 76.5%, respectively; mean survival time was 56.5 months. Combined transplantation of haploidentical HSCs and MSCs on SAA without an HLA-identical sibling donor was safe, effectively reduced the incidence of severe GVHD, and improved patient survival.
doi:10.1371/journal.pone.0089666
PMCID: PMC3940616  PMID: 24594618
23.  Inhibition of Hepatitis C Virus Infection by DNA Aptamer against NS2 Protein 
PLoS ONE  2014;9(2):e90333.
NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies.
doi:10.1371/journal.pone.0090333
PMCID: PMC3938669  PMID: 24587329
24.  Cucurbitacin IIb Exhibits Anti-Inflammatory Activity through Modulating Multiple Cellular Behaviors of Mouse Lymphocytes 
PLoS ONE  2014;9(2):e89751.
Cucurbitacin IIb (CuIIb) is one of the major active compounds in Hemsleyadine tablets which have been used for clinical treatment of bacillary dysentery, enteritis and acute tonsilitis. However, its action mechanism has not been completely understood. This study aimed to explore the anti-inflammatory activity of CuIIb and its underlying mechanism in mitogen-activated lymphocytes isolated from mouse mesenteric lymph nodes. The results showed that CuIIb inhibited the proliferation of concanavalin A (Con A)-activated lymphocytes in a time- and dose-dependent manner. CuIIb treatment arrested their cell cycle in S and G2/M phases probably due to the disruption of the actin cytoskeleton and the modulation of p27Kip1 and cyclin levels. Moreover, the surface expression of activation markers CD69 and CD25 on Con A-activated CD3+ T lymphocytes was suppressed by CuIIb treatment. Both Con A- and phorbol ester plus ionomycin-induced expression of TNF-α, IFN-γ and IL-6 proteins was attenuated upon exposure to CuIIb. Mechanistically, CuIIb treatment suppressed the phosphorylation of JNK and Erk1/2 but not p38 in Con A-activated lymphocytes. Although CuIIb unexpectedly enhanced the phosphorylation of IκB and NF-κB (p65), it blocked the nuclear translocation of NF-κB (p65). In support of this, CuIIb significantly decreased the mRNA levels of IκBα and TNF-α, two target genes of NF-κB, in Con A-activated lymphocytes. In addition, CuIIb downregulated Con A-induced STAT3 phosphorylation and increased cell apoptosis. Collectively, these results suggest that CuIIb exhibits its anti-inflammatory activity through modulating multiple cellular behaviors and signaling pathways, leading to the suppression of the adaptive immune response.
doi:10.1371/journal.pone.0089751
PMCID: PMC3934946  PMID: 24587010
25.  Intraocular pressure-lowering efficacy and safety of bimatoprost 0.03% therapy for primary open-angle glaucoma and ocular hypertension patients in China 
BMC Ophthalmology  2014;14:21.
Background
To report the clinical outcomes in Chinese patients with primary open-angle glaucoma and ocular hypertension treated with bimatoprost 0.03% therapy.
Methods
Two hundred sixty-three Chinese patients with primary open-angle glaucoma and ocular hypertension who needed initial or additional intraocular pressure (IOP) lowering were recruited in this prospective, open-label, multicenter clinical study and were treated with bimatoprost 0.03%. Patients received bimatoprost 0.03% as initial, replacement or adjunctive IOP-lowering therapy, and follow-up visits were performed at week 1, and month 1 and 3 of the bimatoprost treatment. The efficacy outcome measure was the post-treatment IOP level. The safety outcome measures included the rate of medication-related symptoms, physical signs, reported adverse events, and the level of conjunctival hyperemia.
Results
Among 240 patients who could be categorized by pre-existing therapies and the bimatoprost therapy regimen in the study, IOP values observed in all medication conditions showed significant IOP reduction at all study visits compared with baseline. At 3 months, 8.0 ± 3.7 mmHg (32.0%) reduction in IOP was observed in treatment-naive patients after bimatoprost monotherapy; in the patients previously on various therapy regimens, 1.9 ± 2.8 mmHg (9.5%) to 6.4 ± 6.1 mmHg (24.8%) additional IOP lowering was achieved after switching to bimatoprost monotherapy or bimatoprost combination therapy. The most common adverse event was conjunctival hyperemia, mainly of trace and mild intensity.
Conclusions
Our results show that bimatoprost 0.03% was effective in lowering IOP with favorable safety in Chinese primary open-angle glaucoma and ocular hypertension patients.
doi:10.1186/1471-2415-14-21
PMCID: PMC3943806  PMID: 24568617
Bimatoprost; Glaucoma; Ocular hypertension; Intraocular pressure; Conjunctival hyperemia

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