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1.  A Comparison of Loop-Mediated Isothermal Amplification (LAMP) with Other Surveillance Tools for Echinococcus granulosus Diagnosis in Canine Definitive Hosts 
PLoS ONE  2014;9(7):e100877.
Background
Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of Echinococcus infections in canine definitive hosts.
Methods
The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation.
Results
The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China.
Conclusions
We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.
doi:10.1371/journal.pone.0100877
PMCID: PMC4089910  PMID: 25007051
2.  Interactions between pork consumption, CagA status and IL-1B-31 genotypes in gastric cancer 
AIM: To explore potential interactions among Helicobacter pylori (H. pylori), CagA status, interleukin (IL)-1B-31 genotypes, and non-cardiac gastric cancer (GC) risk.
METHODS: A case-control study of non-cardia GC was performed at 3 hospitals located in Xi’an, China, between September 2008 and July 2010. We included 171 patients with histologically diagnosed primary non-cardia GC and 367 population based controls (matched by sex, age and city of residence). A standardized questionnaire was used to obtain information regarding potential risk factors, including pork consumption. H. pylori CagA status was assessed by enzyme-linked immunosorbent assay, and IL-1B-31 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Multivariate unconditional logistic regression was used to explore potential interactions among the factors.
RESULTS: The CagA appeared to confer an increased risk of GC (OR = 1.81, 95%CI: 1.25-2.61). The main associations with IL-1B-31C allele here were 0.98 (95%CI: 0.59-1.63) for CC vs TT and 0.99 (95%CI: 0.64-1.51) for C Carriers vs TT. However, no associations were observed for CagA or IL-1B-31 genotype status among subjects who reported low pork consumption (P for interaction = 0.11). In contrast, high pork consumption and IL-1B-31C genotypes appeared to synergistically increase GC risk (P for interaction = 0.048) after adjusting for confounding factors, particularly among subjects with CagA (OR = 3.07, 95%CI: 1.17-10.79). We did not observe effect modification of pork consumption by H. pylori CagA status, or between H. pylori CagA status and IL-1B-31 genotypes after adjustment for pork consumption and other factors.
CONCLUSION: These interaction relationships among CagA, IL-1B-31 and pork consumption may have implications for development of the preventive strategies for the early detection of non-cardiac GC.
doi:10.3748/wjg.v20.i25.8151
PMCID: PMC4081686  PMID: 25009387
Gastric cancer; Pork; CagA; interleukin-1B; Interaction; Helicobacter pylori
3.  DNA methyltransferase3a expression is an independent poor prognostic indicator in gastric cancer 
AIM: To explore the alteration of DNA methyltransferase expression in gastric cancer and to assess its prognostic value.
METHODS: From April 2000 to December 2010, 227 men and 73 women with gastric cancer were enrolled in the study. The expression of DNA methyltransferases (DNMTs), including DNMT1, DNMT3a and DNMT3b, in the 300 cases of gastric carcinoma, of which 85 had paired adjacent normal gastric mucus samples, was evaluated by immunohistochemistry using a tissue microarray. Serum anti-Helicobacter pylori (H. pylori) IgG was detected by enzyme-linked immunosorbent assay (ELISA). The relationships between the above results and the clinicopathological characteristics were analyzed. Their prognostic value was evaluated using the Cox proportional hazards model.
RESULTS: In gastric cancer, expression of DNMTs was mainly seen in the nucleus. Weak staining was also observed in the cytoplasm. Expression of DNMT1, DNMT3a and DNMT3b in gastric cancer was significantly higher compared to that in the paired control samples (60.0% vs 37.6%, 61.2% vs 4.7%, and 94.1% vs 71.8%, P < 0.01). The overall survival rate was significantly higher in the DNMT3a negative group than in the DNMT3a positive group in gastric cancer patients (Log-rank test, P = 0.032). No significant correlation was observed between DNMT1 and DNMT3b expression and the overall survival time (Log-rank test, P = 0.289, P = 0.347). Multivariate regression analysis indicated that DNMT3a expression (P = 0.025) and TNM stage (P < 0.001), but not DNMT1 (P = 0.54) or DNMT3b (P = 0.62), were independent prognostic factors in gastric cancer. H. pylori infection did not induce protein expression of DNMTs.
CONCLUSION: The results suggest that expression of DNMT3a is an independent poor prognostic indicator in gastric cancer. DNMT3a might play an important role in gastric carcinogenesis.
doi:10.3748/wjg.v20.i25.8201
PMCID: PMC4081693  PMID: 25009393
DNA methyltransferase; Prognosis; Gastric cancer; Expression; Helicobacter pylori
4.  Androgens Increase lws Opsin Expression and Red Sensitivity in Male Three-Spined Sticklebacks 
PLoS ONE  2014;9(6):e100330.
Optomotor studies have shown that three-spined sticklebacks (Gasterosteus aculeatus) are more sensitive to red during summer than winter, which may be related to the need to detect the red breeding colour of males. This study aimed to determine whether this change of red light sensitivity is specifically related to reproductive physiology. The mRNA levels of opsin genes were examined in the retinae of sexually mature and immature fish, as well as in sham-operated males, castrated control males, or castrated males implanted with androgen 11-ketoandrostenedione (11 KA), maintained under stimulatory (L16:D8) or inhibitory (L8:D16) photoperiods. In both sexes, red-sensitive opsin gene (lws) mRNA levels were higher in sexually mature than in immature fish. Under L16:D8, lws mRNA levels were higher in intact than in castrated males, and were up-regulated by 11 KA treatment in castrated males. Moreover, electroretinogram data confirmed that sexual maturation resulted in higher relative red spectral sensitivity. Mature males under L16:D8 were more sensitive to red light than males under L8:D16. Red light sensitivity under L16:D8 was diminished by castration, but increased by 11 KA treatment. Thus, in sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression. This is the first study to demonstrate that sex hormones can regulate spectral vision sensitivity.
doi:10.1371/journal.pone.0100330
PMCID: PMC4070989  PMID: 24963891
5.  Metallothionein III (MT3) is a putative tumor suppressor gene that is frequently inactivated in pediatric acute myeloid leukemia by promoter hypermethylation 
Background
Acute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear.
Methods
Eleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis.
Results
The MT3 promoter was hypermethylated in leukemia cell lines. More CpG’s methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P < 0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P = 0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P < 0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1.
Conclusion
MT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.
doi:10.1186/1479-5876-12-182
PMCID: PMC4082423  PMID: 24962166
Metallothionein III; Pediatric acute myeloid leukemia; Methylation; Tumor suppressor
6.  A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates 
Brazilian Journal of Microbiology  2014;45(1):163-173.
Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.
PMCID: PMC4059291  PMID: 24948927
bacterial diversity; endophytes; moss; molecular method; cultivated isolates
7.  Detecting overlapping protein complexes based on a generative model with functional and topological properties 
BMC Bioinformatics  2014;15:186.
Background
Identification of protein complexes can help us get a better understanding of cellular mechanism. With the increasing availability of large-scale protein-protein interaction (PPI) data, numerous computational approaches have been proposed to detect complexes from the PPI networks. However, most of the current approaches do not consider overlaps among complexes or functional annotation information of individual proteins. Therefore, they might not be able to reflect the biological reality faithfully or make full use of the available domain-specific knowledge.
Results
In this paper, we develop a Generative Model with Functional and Topological Properties (GMFTP) to describe the generative processes of the PPI network and the functional profile. The model provides a working mechanism for capturing the interaction structures and the functional patterns of proteins. By combining the functional and topological properties, we formulate the problem of identifying protein complexes as that of detecting a group of proteins which frequently interact with each other in the PPI network and have similar annotation patterns in the functional profile. Using the idea of link communities, our method naturally deals with overlaps among complexes. The benefits brought by the functional properties are demonstrated by real data analysis. The results evaluated using four criteria with respect to two gold standards show that GMFTP has a competitive performance over the state-of-the-art approaches. The effectiveness of detecting overlapping complexes is also demonstrated by analyzing the topological and functional features of multi- and mono-group proteins.
Conclusions
Based on the results obtained in this study, GMFTP presents to be a powerful approach for the identification of overlapping protein complexes using both the PPI network and the functional profile. The software can be downloaded from http://mail.sysu.edu.cn/home/stsddq@mail.sysu.edu.cn/dai/others/GMFTP.zip.
doi:10.1186/1471-2105-15-186
PMCID: PMC4073817  PMID: 24928559
Protein complex detection; Protein-protein interaction network; Functional profile; Generative model
8.  B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells 
BMC Cancer  2014;14:418.
Background
B-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients.
Methods
Expression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells.
Results
BCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins.
Conclusions
The current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer.
doi:10.1186/1471-2407-14-418
PMCID: PMC4065600  PMID: 24917186
Breast cancer; BCL6; microRNA
9.  Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium 
BMC Genomics  2014;15(1):428.
Background
Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets.
Results
Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases. Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases.
Conclusion
Numerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-428) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-428
PMCID: PMC4070553  PMID: 24899069
Proteases; Taenia solium; Drug target; Vaccine candidate antigen; Genome-wide analysis; Cysticercosis; Platyhelminth
10.  Oxidative Responses Induced by Pharmacologic Vitreolysis and/or Long-term Hyperoxia Treatment in Rat Lenses 
Current eye research  2013;38(6):639-648.
Purpose
The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo.
Methods
Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors.
Results
No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis.
Conclusions
Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.
doi:10.3109/02713683.2012.760741
PMCID: PMC3740155  PMID: 23534693
Cataract; hyperoxia; oxidative stress; pharmacologic vitreolysis; posterior vitreous detachment
11.  Diethyl (E)-2,3-bis­[(E)-(2-methyl-2-phenyl­hydrazin-1-yl­idene)meth­yl]but-2-enedioate 
The complete mol­ecule of the title compound, C24H28N4O4, is generated by crystallographic inversion symmetry. The ethyl side chain is disordered over two sets of sites in a 0.57 (4):0.43 (4) ratio. The dihedral angles between the methyl­idene group and the phenyl ring and ester side chain (major conformation) are 7.61 (8) and 86.95 (8)°, respectively. In the crystal, mol­ecules are linked via C—H⋯O hydrogen bonds, forming corrugated sheets lying parallel to (010).
doi:10.1107/S1600536814011970
PMCID: PMC4051109  PMID: 24940292
12.  Delayed Increase of Tyrosine Hydroxylase Expression in Rat Nigrostriatal System after Traumatic Brain Injury 
Brain research  2006;1134(1):171-179.
Tyrosine hydroxylase (TH) is the key enzyme for synthesizing dopamine (DA) in dopaminergic neurons and its terminals. Emerging experimental and clinical evidence support the hypothesis of a disturbance in dopamine neurotransmission following traumatic brain injury (TBI). However, the effect of controlled cortical impact (CCI) injury on TH in the nigrostriatal system is currently unknown. To determine if there is an alteration in TH after CCI injury, we examined TH levels at 1 day, 7 days, and 28 days post-injury by utilizing a commercially available antibody specific to TH. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 3.2 mm) or sham surgery. Injured (n=6) and sham animals (n=6) were sacrificed and coronally sectioned (35 μm thick) through the striatum and substantia nigra (SN) for immunohistochemistry. Additionally, semiquantitative measurements of TH protein in striatal and SN homogenates from injured (n=6) and sham (n=6) rats sacrificed at the appropriate time post-surgery were assessed using Western blot analysis. TH protein is bilaterally increased at 28 days post-injury in nigrostriatal system revealed by immunohistochemistry in injured rats compared to sham controls. Western blot analysis confirms the findings of immunohistochemistry in both striatum and SN. We speculate that the increase in TH in the nigrostriatal system may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and a delayed increase in the efficiency of DA neurotransmission after TBI.
doi:10.1016/j.brainres.2006.11.087
PMCID: PMC4017583  PMID: 17196177
Immunohistochemistry; Nigrostriatal; Rat; Traumatic brain injury (TBI); Tyrosine hydroxylase (TH); Western blot
13.  Acculturative Stress and Influential Factors among International Students in China: A Structural Dynamic Perspective 
PLoS ONE  2014;9(4):e96322.
Stress represents a prominent aspect of modern life and is associated with numerous negative health consequences. International students are a key force in shaping globalization. However, these students often experience acculturative stress, influencing their health and well-being. The growing number of international students in China emerges as a new global health challenge and presents an opportunity to advance our understanding of acculturative stress. This study aims to investigate the acculturative stress of international students in China, and verify the mechanism and influential factors of acculturative stress. We analyzed survey data from 567 international students attending universities in Wuhan, China. We used a network-based analytical approach to assess the structure of the Acculturative Stress Scale for International Students and used regression analysis to assess the relationships between acculturative stress and theoretically related factors. We found that higher levels of acculturative stress were reported by students from Asia and Africa than from other regions (Europe/America/Oceania). Lower acculturative stress was reported by unmarried students than others and by students well prepared than not well prepared. We verified seven acculturative stress subconstructs: rejection, identity threat, opportunity deprivation, self-confidence, value conflict, cultural competence, and homesickness; and discovered a three-dimensional network structure of these subconstructs. Our results suggest that acculturative stress was more common among international students in China than in developed countries. Acculturative stress was also more common among international students who did not well prepared, married, and belonged to an organized religion. African and Asian students' stress was higher than that for students from other regions. Acculturative stress prevention programs should seek to improve preparedness of the international students for studying abroad and pay extra attention to the high risk subgroups.
doi:10.1371/journal.pone.0096322
PMCID: PMC4005751  PMID: 24788357
14.  Sodium Tanshinone IIA Silate Inhibits High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration through Activation of AMP-Activated Protein Kinase 
PLoS ONE  2014;9(4):e94957.
The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G0/G1 phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.
doi:10.1371/journal.pone.0094957
PMCID: PMC3989257  PMID: 24739942
15.  Pulmonary immune responses to 2009 pandemic influenza A (H1N1) virus in mice 
BMC Infectious Diseases  2014;14:197.
Background
Well-characterized mice models will afford a cheaper, easy-handling opportunity for a more comprehensive understanding of 2009 influenza A (H1N1) virus’s pathogenesis potential. We aimed to provide a robust description of pulmonary immune responses in the mice infected by the virus.
Methods
BALB/c mice were inoculated intranasally with A/Beijing/501/2009(H1N1) (BJ501) and A/PR/8/34(H1N1) (PR8) viruses and compared for survival rate, viral replication, and kinetics of pulmonary immune responses.
Results
BJ501 virus replicated less efficiently in the lungs than PR8, and both caused lethal illness in the mice. The transient increases of pulmonary TNF-α 2 days post infection for BJ501 and of INF-γ and IL-10 at 6 days post infection for PR8 were observed. IL-2+ and IL-4+ secreting cells showed significant increase 12 days post infection, while IFN-γ+, IgG+ and IgA+ secreting cells increased 6 days post infection. The different patterns of pulmonary immunological parameters between two viruses were at most seen in IL-6, IL-17 secretion and IgG1/IgG2a ratio.
Conclusions
The BALB/c mouse is evaluated as a good pathogenic model for studying BJ501 2009 H1N1 virus. The work provided some basic and detailed data, which might be referred when further evaluating innate and adapted pulmonary immune responses and local viral load in mice.
doi:10.1186/1471-2334-14-197
PMCID: PMC4002205  PMID: 24725777
Pandemic influenza; Immunology; BALB/c mice
16.  The Immunoregulation Effect of Alpha 1-Antitrypsin Prolong β-Cell Survival after Transplantation 
PLoS ONE  2014;9(4):e94548.
Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.
doi:10.1371/journal.pone.0094548
PMCID: PMC3983209  PMID: 24722487
17.  Prediction of antiviral efficacy in patients with chronic hepatitis C by changes in forkhead box protein 3 levels 
The aim of the present study was to investigate the distribution of CD4+CD25+ regulatory T cells (Tregs) in the peripheral blood of patients with chronic hepatitis C; in addition to identifying whether the distribution of CD4+CD25+ Tregs predicts the efficacy of antiviral therapy for HCV. The expression of CD4+CD25+ forkhead box protein (FOXP) 3+ Tregs within a CD4+ T cell population was detected in the peripheral blood obtained from patients with chronic hepatitis C and from healthy control subjects using flow cytometry. The hepatitis C virus (HCV)-RNA load was measured using quantitative-fluorescence polymerase chain reaction. CD4+CD25+FOXP3+ Tregs accounted for 14.24±1.33% of the CD4+ T cells in the peripheral blood of patients with chronic hepatitis C, which was higher than that of the healthy control subjects (5.62±1.21%; P<0.001). Furthermore, the frequency of CD4+CD25+FOXP3+ Tregs in CD4+ T cells of the peripheral blood positively correlated with the HCV-RNA load (r=0.73; P=0.032). Therefore, the results of the present study indicated that the expression of CD4+CD25+FOXP3+ Tregs increased in patients that were chronically infected with HCV and positively correlated with the HCV-RNA load.
doi:10.3892/etm.2014.1675
PMCID: PMC4061195  PMID: 24944616
chronic hepatitis C; CD4+CD25+ forkhead box P3+ regulatory T cells; hepatitis C virus-RNA load
18.  Effects of silencing RIP1 with siRNA on the biological behavior of the LoVo human colon cancer cell line 
Oncology Letters  2014;7(6):2065-2072.
The present study aimed to investigate the effects of silencing RIP1 by small interfering RNA (siRNA) on the biological behavior of the LoVo human colorectal carcinoma cell line and to provide evidence for the feasibility of colorectal cancer gene therapy. LoVo cells were divided into the RIP1 siRNA group, the blank control group and the negative control group. Chemically synthesized siRNA targeting RIP1 (RIP1 siRNA) was transfected into LoVo cells. Following transfection of the RIP1-targeted siRNA into the LoVo cells, the expression of the RIP1 gene was effectively inhibited. The results demonstrated that RIP1 effectively regulated the malignant biological behavior of the LoVo colon cancer cell line. Furthermore, the proliferation, motility and invasiveness of LoVo cells were inhibited by siRNA knockdown of RIP1. The results revealed that the RIP1 gene has an important role in the regulation of proliferation and apoptosis in colorectal carcinoma cells.
doi:10.3892/ol.2014.2040
PMCID: PMC4049674  PMID: 24932290
RNA interference; RIP1 gene; colorectal carcinoma
19.  Equity in use of maternal health services in Western Rural China: a survey from Shaanxi province 
Background
The 20th century was marked by a significant improvement in worldwide human health and access to healthcare. However, these improvements were not completely or uniformly distributed among, or even within, nations. This study was designed to assess the use of maternal health services by pregnant women in China, with a focus on the inequity related to family income level.
Methods
Two population-based cross-sectional surveys were carried out in the Zhenan and Lantian counties in March 2007 and from December 2008 to March 2009. A total of 2562 women completed the questionnaires, including 948 who were pregnant in 2006 and 1614 from 2008–2009. The concentration index (CI) was calculated and used to analyze the parameters of maternal health care in the two counties surveyed.
Results
The responses in both 2006 and 2008–2009 indicated a bias towards higher (rich) economic statuses for the use of maternal and child health services. The CI of ‘delivery at health facility’ was 0.0206 (95% confidence interval between 0.0114 and 0.0299) for 2006 and 0.0053 (95% confidence interval between 0.0015 and 0.0091) for 2008, which represented a statistically significant inequity for women of lower (poor) economic statuses. Similar CI was observed in ‘receiving antenatal care within 12 weeks’ for 2006 (CI2006 = 0.0956, 95% confidence interval between 0.0516 and 0.1396). The CIs of ‘postnatal visit’ and ‘postnatal visit >3-times’ was positive (except for 2006), indicating that the poor used postnatal care less than the non-poor. In 2008, poor women had C-sections more often than non-poor women (CI2008 = −0.0629, 95% confidence interval between-0.1165 and −0.0093), but such a difference was not observed in 2006.
Conclusions
In 2006 and 2008, the use of maternal health services in western rural China was significantly unequal between pregnant women of poor and non-poor economic statuses. Financial support that enables poorer pregnant women to use health services will be beneficial. Utilization of maternal healthcare services can be improved if out-of-pocket expenses can be minimized.
doi:10.1186/1472-6963-14-155
PMCID: PMC3985545  PMID: 24708641
Equity; Maternal and child health service; Economic situation
20.  An attempt to stabilize tanshinone IIA solid dispersion by the use of ternary systems with nano-CaCO3 and poloxamer 188 
Pharmacognosy Magazine  2014;10(Suppl 2):S311-S317.
Background:
Tanshinone IIA (TSIIA) on solid dispersions (SDs) has thermodynamical instability of amorphous drug. Ternary solid dispersions (tSDs) can extend the stability of the amorphous form of drug. Poloxamer 188 was used as a SD carrier. Nano-CaCO3 played an important role in adsorption of biomolecules and is being developed for a host of biotechnological applications.
Objective:
The aim of the present study was to investigate the dissolution behavior and accelerated stability of TSIIA on solid dispersions (SDs) by the use of ternary systems with nano-CaCO3 and poloxamer 188.
Materials and Methods:
The TSIIA tSDs were prepared by a spray-drying method. First, the effect of combination of poloxamer 188 and nano-CaCO3 on TSIIA dissolution was studied. Subsequently, a set of complementary techniques (DSC, XRPD, SEM and FTIR) was used to monitor the physical changes of TSIIA in the SDs. Finally, stability test was carried out under the conditions 40°C/75% RH for 6 months.
Results:
The characterization of tSDs by differential scanning calorimetry analysis (DSC) and X-ray powder diffraction (XRPD) showed that TSIIA was present in its amorphous form. Fourier transforms infrared spectroscopy (FTIR) suggested the presence of interactions between TSIIA and carriers in tSDs. Improvement in the dissolution rate was observed for all SDs. The stability study conducted on SDs with nano-CaCO3 showed stable drug content and dissolution behavior, over the period of 6 months as compared with freshly prepared SDs.
Conclusion:
SDs preparation with nano-CaCO3 and poloxamer 188 may be a promising approach to enhance the dissolution and stability of TSIIA.
doi:10.4103/0973-1296.133286
PMCID: PMC4078353  PMID: 24991109
Combination carriers; in vitro dissolution; stability; ternary solid dispersions
21.  Field comparison of circulating antibody assays versus circulating antigen assays for the detection of schistosomiasis japonica in endemic areas of China 
Parasites & Vectors  2014;7:138.
Background
Schistosomiasis remains a serious public health problem in affected countries, and routine, highly sensitive and cost-effective diagnostic methods are lacking. We evaluated two immunodiagnostic techniques for the detection of Schistosoma japonicum infections: circulating antibody and circulating antigen assays.
Methods
A total of 1864 individuals (between 6 and 72 years old) residing in five administrative villages in Hubei province were screened by serum examination with an indirect hemagglutination assay (IHA). The positive individuals (titer ≥20 in IHA) were reconfirmed by stool examination with the Kato-Katz method (three slides from a single stool specimen). Samples of good serum quality and a volume above 0.5 ml were selected for further testing with two immunodiagnostic antibody (DDIA and ELISA) and two antigen (ELISA) assays.
Results
The average antibody positive rate in the five villages was 12.7%, while the average parasitological prevalence was 1.50%; 25 of the 28 egg-positive samples were also circulating antigen-positive. Significant differences were observed between the prevalence according to the Kato-Katz method and all three immunodiagnostic antibody assays (P-value <0.0001). Similar differences were observed between the Kato-Katz method and the two immunodiagnostic antigen assays (P-value <0.0001) and between the antigen and antibody assays (P-value <0.0001).
Conclusion
Both circulating antibody and circulating antigen assays had acceptable performance characteristics. Immunodiagnostic techniques to detect circulating antigens have potential to be deployed for schistosomiasis japonica screening in the endemic areas.
doi:10.1186/1756-3305-7-138
PMCID: PMC3978087  PMID: 24684924
Schistosoma japonicum; Circulating antibody; Circulating antigen; China
22.  Maintenance of Stemness in Oxaliplatin-Resistant Hepatocellular Carcinoma Is Associated with Increased Autocrine of IGF1 
PLoS ONE  2014;9(3):e89686.
Background
Evidence suggests that many types of cancers are composed of different cell types, including cancer stem cells (CSCs). We have previously shown that the chemotherapeutic agent oxaliplatin induced epithelial-mesenchymal transition, which is thought to be an important mechanism for generating CSCs. In the present study, we investigate whether oxaliplatin-treated cancer tissues possess characteristics of CSCs, and explore oxaliplatin resistance in these tissues.
Methods
Hepatocellular carcinoma cells (MHCC97H cells) were subcutaneously injected into mice to form tumors, and the mice were intravenously treated with either oxaliplatin or glucose. Five weeks later, the tumors were orthotopically xenografted into livers of other mice, and these mice were treated with either oxaliplatin or glucose. Metastatic potential, sensitivity to oxaliplatin, and expression of CSC-related markers in the xenografted tumor tissues were evaluated. DNA microarrays were used to measure changes in gene expression as a result of oxaliplatin treatment. Additionally, an oxaliplatin-resistant cell line (MHCC97H-OXA) was established to assess insulin-like growth factor 1 secretion, cell invasion, cell colony formation, oxaliplatin sensitivity, and expression of CSC-related markers. The effects of an insulin-like growth factor 1 receptor inhibitor were also assessed.
Results
Oxaliplatin treatment inhibited subcutaneous tumor growth. Tumors from oxaliplatin-treated mice that were subsequently xenografted into livers of other mice exhibited that decreasing sensitivity to oxaliplatin and increasing pulmonary metastatic potential. Among the expression of CSC-related proteins, the gene for insulin-like growth factor 1, was up-regulated expecially in these tumor tissues. Additionally, MHCC97H-OXA cells demonstrated that increasing cell invasion, colony formation, and expression of insulin-like growth factor 1 and CSC-related markers, whereas treatment with an inhibitor of the insulin-like growth factor 1 receptor suppressed these effects.
Conclusion
Maintenance of stemness in oxaliplatin-resistant hepatocellular carcinoma cells is associated with increased autocrine of IGF1.
doi:10.1371/journal.pone.0089686
PMCID: PMC3954560  PMID: 24632571
23.  Prevalence and Determinants of Metabolic Syndrome among Adults in a Rural Area of Northwest China 
PLoS ONE  2014;9(3):e91578.
Objectives
To evaluate the prevalence and determinants of metabolic syndrome (MetS) among adults in a rural area of Northwest China.
Methods
A population-based cross-sectional study was conducted in 2010 among adults aged 18 to 80 years in rural areas of Hanzhong, in Northwest China. Interview, physical and clinical examinations, and fasting blood glucose and lipid measurements were completed for 2990 adults. The definitions of MetS proposed by the Third Report of the National Cholesterol Education Program Expert Panel (Adults Treatment Panel III, ATP III) and the International Diabetes Federation (IDF), and the modified ATP III definition for Asian population were used and compared. Proportions were adjusted for age and sex.
Results
The prevalence of MetS was 7.9%, 10.8% and 15.1% according to ATP III, IDF and modified ATP III criteria, respectively. Agreement between ATP III and IDF criteria and that between ATP III and modified ATP III criteria were moderate (Kappa = 0.52 and 0.64, respectively), whereas agreement between IDF and modified ATP III criteria was good (Kappa = 0.83). The prevalence of MetS increased with age, and was higher in women than in men (10.4% versus 5.4%, 13.6% versus 8.1% and 17.4% versus 12.8%, according to ATP III, IDF and modified ATP III criteria, respectively). The most common MetS component was high blood pressure. Having family history of hypertension, lack of physical activity, high economical level, overweight and obesity were positively associated with MetS.
Conclusions
MetS is prevalent among rural adults in Northwest China and high blood pressure is the most common MetS component. Prevention and treatment of hypertension and MetS should be a public health priority to reduce cardiovascular diseases in rural areas of Northwest China. More attention should be given to the elderly, women, people with family history of hypertension and obese people who are at high risk of MetS.
doi:10.1371/journal.pone.0091578
PMCID: PMC3948893  PMID: 24614618
24.  Effect of Naphthalene Acetic Acid on Adventitious Root Development and Associated Physiological Changes in Stem Cutting of Hemarthria compressa 
PLoS ONE  2014;9(3):e90700.
In order to find a way to induce rooting on cuttings of Hemarthria compressa cv. Ya’an under controlled conditions, a project was carried out to study the effect of naphthalene acetic acid (NAA) on rooting in stem cuttings and related physiological changes during the rooting process of Hemarthria compressa. The cuttings were treated with five concentrations of NAA (0, 100, 200 300, 400 mg/l) at three soaking durations (10, 20, 30 minutes), and cuttings without treatment were considered as control. Samples were planted immediately into pots after treatment. IAA-oxidase (IAAO) activity, peroxidase (POD) activity and polyphenol oxidase (PPO) activity were determined after planting. Results showed that NAA had positive effect on rooting at the concentration of 200 mg/l compared to other concentrations at 30 days after planting (DAP). Among the three soaking durations, 20 minutes (min) of 200 mg/l NAA resulted in higher percentages of rooting, larger numbers of adventitious roots and heavier root dry weight per cutting. The lowest IAAO activity was obtained when soaked at 200 mg/l NAA for 20 min soaking duration. This was consistent with the best rooting ability, indicating that the lower IAAO activity, the higher POD activity and PPO activity could be used as an indicator of better rooting ability for whip grass cuttings and might serve as a good marker for rooting ability in cuttings.
doi:10.1371/journal.pone.0090700
PMCID: PMC3942460  PMID: 24595064
25.  Measuring and decomposing the inequality of maternal health services utilization in Western Rural China 
Background
To measure socioeconomic inequalities in maternal health services in rural western China and to analyze the determinants’ contributions of inequalities.
Study design: a cross-sectional study.
Methods
The data utilized in this study were obtained from a cross-sectional study from 10 provinces in rural Western China in 2005. Wealth index of household socioeconomic status was developed by using principle component analysis. Concentration index, concentration curve and decomposition of the concentration index were employed to measure socioeconomic inequality in maternal health services utilization.
Results
For more than four times prenatal visits, the concentration index was 0.0605 (95% CI: 0.0603, 0.0607). The concentration index of hospital delivery was 0.0230 (95% CI: 0.0210, 0.0240) and the concentration index of more than 2 times postnatal visits was 0.0842 (95% CI: 0.0836, 0.0847). Han ethnicity woman, particularly in conjunction with high school education and rich wealth status, was the main contributor to inequality in maternal health services utilization.
Conclusions
There is a strong pro-rich inequality of maternal health services in rural western China. This study suggests that an effective way to reduce the inequality is not only to narrow the gap of income between the rich and poor, but focus education on ethnic minority woman in rural remote areas.
doi:10.1186/1472-6963-14-102
PMCID: PMC3975923  PMID: 24589223
Maternal health care utilization; Rural Western China; Socioeconomic inequality; Decomposition

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