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1.  Assessment of serum arginase I as a type 2 diabetes mellitus diagnosis biomarker in patients 
Previous studies have reported that levels of serum arginase I are increased in certain diseases. However, the exact association between arginase I and diabetes mellitus (DM) has yet to be determined. The aim of the present study was to investigate the correlation between arginase I activity and DM to determine whether arginase I activity may be used as a diagnostic biomarker for DM. DM was induced by a streptozotocin injection, while the arginase inhibitor, citrulline, was administered daily. Serum levels of glucose, reactive oxygen species (ROS) and arginase I activity were analyzed, and quantitative polymerase chain reaction and western blot analysis were performed to detect the mRNA and protein expression levels of arginase I, respectively. In addition, western blot analysis was used to determine the protein expression of the Tie 2 receptor. Pearson’s analysis was used to determine the correlation between arginase I activity and Tie 2 expression, while concordance analysis was performed using the Cohen’s test to obtain the Kappa statistic. The results demonstrated that serum arginase I activity levels in the rats with DM were significantly elevated compared with the control group, and positively correlated with the blood glucose levels. In addition, the blood glucose and ROS levels were increased significantly in the rats with DM. Arginase I mRNA and protein expression levels were significantly elevated in the diabetic rats when compared with the control group, and Tie 2 expression levels increased and were shown to correlate with arginase I activity in the diabetic rats. In addition, arginase I activity was shown to correlate with glucose intolerance and post-load glucose values. Good concordance was observed between arginase I activity and the clinical diagnosis for DM (κ=0.876; P<0.001). Therefore, the results indicated that arginase I may function as a diagnostic biomarker for DM rats model.
doi:10.3892/etm.2014.1768
PMCID: PMC4079409  PMID: 25009624
diabetes mellitus; arginase I; biomarker; diagnosis
2.  Treatment Patterns in Patients with Metastatic Melanoma: A Retrospective Analysis 
Journal of Skin Cancer  2014;2014:371326.
Objective. To describe treatment patterns and factors influencing treatment in a real-world setting of US patients with metastatic melanoma (MM). Methods. This was a retrospective claims-based study among patients with MM diagnosed between 2005 and 2010 identified from MarketScan databases. Results. Of 2546 MM patients, 66.8% received surgery, 44.7% received radiation, 38.7% received systemic therapies, and 17.7% received all modalities. Patients with lung, brain, liver, or bone metastases were less likely to undergo surgery (all P < 0.0001); patients with lung (P = 0.04), brain (P < 0.001), or liver metastases (P = 0.03) were more likely to receive systemic therapies; patients with brain (P < 0.0001) or bone metastases (P < 0.0001) were more likely to receive radiation therapy. Oncologists were more likely to recommend systemic therapy (P < 0.0001) or radiation (P < 0.0001), while dermatologists were more likely to recommend surgery (P = 0.002). Monotherapy was the dominant systemic therapy (82.4% patients as first-line). Conclusions. Only 39% of MM patients received systemic therapies, perhaps reflecting efficacy and safety limitations of conventional systemic therapies for MM. Among those receiving systemic therapy, monotherapy was the most common approach. Sites of metastases and physician speciality influenced treatment patterns. This study serves as a baseline against which future treatment pattern studies, following approval of new agents, can be compared.
doi:10.1155/2014/371326
PMCID: PMC4026983  PMID: 24883209
3.  Optical properties of epitaxial BiFeO3 thin film grown on SrRuO3-buffered SrTiO3 substrate 
Nanoscale Research Letters  2014;9(1):188.
The BiFeO3 (BFO) thin film was deposited by pulsed-laser deposition on SrRuO3 (SRO)-buffered (111) SrTiO3 (STO) substrate. X-ray diffraction pattern reveals a well-grown epitaxial BFO thin film. Atomic force microscopy study indicates that the BFO film is rather dense with a smooth surface. The ellipsometric spectra of the STO substrate, the SRO buffer layer, and the BFO thin film were measured, respectively, in the photon energy range 1.55 to 5.40 eV. Following the dielectric functions of STO and SRO, the ones of BFO described by the Lorentz model are received by fitting the spectra data to a five-medium optical model consisting of a semi-infinite STO substrate/SRO layer/BFO film/surface roughness/air ambient structure. The thickness and the optical constants of the BFO film are obtained. Then a direct bandgap is calculated at 2.68 eV, which is believed to be influenced by near-bandgap transitions. Compared to BFO films on other substrates, the dependence of the bandgap for the BFO thin film on in-plane compressive strain from epitaxial structure is received. Moreover, the bandgap and the transition revealed by the Lorentz model also provide a ground for the assessment of the bandgap for BFO single crystals.
doi:10.1186/1556-276X-9-188
PMCID: PMC4002908  PMID: 24791162
BiFeO3 thin film; Optical properties; Spectroscopic ellipsometry; Lorentz model; Dielectric function; 78.67.-n; 78.20.-e; 07.60.Fs
4.  Th9/IL-9 Profile in Human Echinococcosis: Their Involvement in Immune Response during Infection by Echinococcus granulosus 
Mediators of Inflammation  2014;2014:781649.
Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group), the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group), as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF-β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group) compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF-β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.
doi:10.1155/2014/781649
PMCID: PMC3985320  PMID: 24799769
5.  The Regulation of Autophagy by Influenza A Virus 
BioMed Research International  2014;2014:498083.
Influenza A virus is a dreadful pathogen of animals and humans, causing widespread infection and severe morbidity and mortality. It is essential to characterize the influenza A virus-host interaction and develop efficient counter measures against the viral infection. Autophagy is known as a catabolic process for the recycling of the cytoplasmic macromolecules. Recently, it has been shown that autophagy is a critical mechanism underlying the interaction between influenza A virus and its host. Autophagy can be induced by the infection with influenza A virus, which is considered as a necessary process for the viral proliferation, including the accumulation of viral elements during the replication of influenza A virus. On the other hand, influenza A virus can inhibit the autophagic formation via interaction with the autophagy-related genes (Atg) and signaling pathways. In addition, autophagy is involved in the influenza virus-regulated cell deaths, leading to significant changes in host apoptosis. Interestingly, the high pathogenic strains of influenza A virus, such as H5N1, stimulate autophagic cell death and appear to interplay with the autophagy in distinct ways as compared with low pathogenic strains. This review discusses the regulation of autophagy, an influenza A virus driven process.
doi:10.1155/2014/498083
PMCID: PMC3980786  PMID: 24779013
6.  Exploration of microRNAs in porcine milk exosomes 
BMC Genomics  2014;15:100.
Background
Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18–25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets.
Results
In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3’UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network.
Conclusions
Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.
doi:10.1186/1471-2164-15-100
PMCID: PMC4008308  PMID: 24499489
Porcine milk exosomes; Solexa sequencing; miRNA
7.  Embryonic Trophoblasts Induce Decidual Regulatory T Cell Differentiation and Maternal–Fetal Tolerance through Thymic Stromal Lymphopoietin Instructing Dendritic Cells 
Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c+ dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4+CD25− T cells into CD4+CD25+FOXP3+ regulatory T cells (Tregs) through TGF-β1. TSLP-activated dDC–induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4+CD25+FOXP3+ Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56brightCD16− NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal–fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy.
doi:10.4049/jimmunol.1203425
PMCID: PMC3918863  PMID: 24453244
8.  Diversity of Rotavirus Strains Causing Diarrhea in <5 Years Old Chinese Children: A Systematic Review 
PLoS ONE  2014;9(1):e84699.
Background
We conducted a systematic review of the diversity and fluctuation of group A rotavirus strains circulating in China.
Methods and Findings
Studies of rotavirus-based diarrhea among children less than 5 years, published in English or Chinese between 1994 and 2012, were searched in PubMed, SinoMed, and CNKI and reviewed by applying standardized algorithms. The temporal and spatial trends of genotyping and serotyping were analyzed using a random-effects model. Ninety-three studies met the inclusion/exclusion criteria and were included in the meta-analysis. Overall, 22,112 and 10,660 rotavirus samples had been examined for G and P types, respectively. The most common G types were G1 (39·5%), G3 (35·6%), G2 (1·3%), and G9 (0·1%). Among P types, P[8] (54·6%) was the predominant type, followed by P[4] (11·1%) and P6 (0·1%). The most common G-P combinations were G3P[8] (32·1%) and G1P[8] (24·5%), followed by G2P[6] (13·2%) and G2P[4] (10·1%). Before 2000, serotype G1 was the predominant strain and accounted for 74·3% of all rotavirus infections; however, since 2000, G3 (45·2%) has been the predominant strain. Rotavirus P types showed little variation over the study period.
Conclusion
Despite the variation of serotypes observed in China, the G1, G2, G3, and G4 serotypes accounted for most rotavirus strains in recent decades. These results suggest that Chinese children will be adequately protected with currently available or forthcoming rotavirus vaccines.
doi:10.1371/journal.pone.0084699
PMCID: PMC3885581  PMID: 24416267
9.  Suppression of Interferon Lambda Signaling by SOCS-1 Results in Their Excessive Production during Influenza Virus Infection 
PLoS Pathogens  2014;10(1):e1003845.
Innate cytokine response provides the first line of defense against influenza virus infection. However, excessive production of cytokines appears to be critical in the pathogenesis of influenza virus. Interferon lambdas (IFN-λ) have been shown to be overproduced during influenza virus infection, but the precise pathogenic processes of IFN-λ production have yet to be characterized. In this report, we observed that influenza virus induced robust expression of IFN-λ in alveolar epithelial cells (A549) mainly through a RIG-I-dependent pathway, but IFN-λ-induced phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was dramatically inhibited in the infected cells. Remarkably, influenza virus infection induced robust expression of suppressor of cytokine signaling-1 (SOCS-1), leading to inhibition of STAT1 activation. Interestingly, the virus-induced SOCS-1 expression was cytokine-independent at early stage of infection both in vitro and in vivo. Using transgenic mouse model and distinct approaches altering the expression of SOCS-1 or activation of STAT signaling, we demonstrated that disruption of the SOCS-1 expression or expression of constitutively active STAT1 significantly reduced the production of IFN-λ during influenza virus infection. Furthermore, we revealed that disruption of IFN-λ signaling pathway by increased SOCS-1 protein resulted in the activation of NF-κB and thereby enhanced the IFN-λ expression. Together, these data imply that suppression of IFN-λ signaling by virus-induced SOCS-1 causes an adaptive increase in IFN-λ expression by host to protect cells against the viral infection, as a consequence, leading to excessive production of IFN-λ with impaired antiviral response.
Author Summary
Influenza virus infection triggers innate immune responses. However, aberrant host immune responses such as excessive production of cytokines contribute to the pathogenesis of influenza virus. Type III interferons (IFN-λ) constitute the major innate immune response to influenza virus infection, but the precise pathogenic processes of IFN-λ production and mechanistic underpinnings are not well understood. In this study, we report that influenza virus induces robust IFN-λ expression mainly through a RIG-I-dependent pathway, but signaling activated by IFN-λ was dramatically inhibited by virus-induced SOCS-1. Importantly, we found that disruption of the SOCS-1 expression or forced activation of STAT1 significantly reduced the expression of IFN-λ in vitro and in vivo, suggesting that suppression of IFN-λ signaling by SOCS-1 results in their excessive production during influenza virus infection. Furthermore, our experiments revealed that disruption of IFN-λ signaling pathway resulted in the activation of NF-κB that governs the IFN-λ expression. Together these findings, we propose that impaired antiviral response of IFN-λ due to the inhibitory effect of SOCS-1 causes an adaptive increase in IFN-λ expression by host to protect cells against the viral infection. This is a novel mechanism that may be critical in the pathogenesis of the influenza virus strains that induce hypercytokinemia.
doi:10.1371/journal.ppat.1003845
PMCID: PMC3879354  PMID: 24391501
10.  Hyaluronan-CD44 Interaction Promotes Growth of Decidual Stromal Cells in Human First-Trimester Pregnancy 
PLoS ONE  2013;8(9):e74812.
Hyaluronan (HA) and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs) continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS) 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA), but not medium molecular weight (MMW-HA) or low molecular weight (LMW-HA), promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy.
doi:10.1371/journal.pone.0074812
PMCID: PMC3777984  PMID: 24069351
11.  Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways 
Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.
PMCID: PMC3796221  PMID: 24133577
Cyclosporine A; trophoblast; PCNA; migration; signal transduction pathway
12.  Enhancement of Antiviral Activity of Human Alpha-Defensin 5 against Herpes Simplex Virus 2 by Arginine Mutagenesis at Adaptive Evolution Sites 
Journal of Virology  2013;87(5):2835-2845.
Herpes simplex virus 2 (HSV-2) infection is still one of the common causes of sexually transmitted diseases worldwide. The prevalence of HSV strains resistant to traditional nucleoside antiviral agents has led to the development of novel antiviral drugs. Human alpha-defensin 5 (HD5), a kind of endogenous antimicrobial peptide expressed in the epithelia of the small intestine and urogenital tract, displays natural antiviral activity. Based on arginine-rich features and adaptive evolution characteristics of vertebrate defensins, we conducted a screen for HD5 derivatives with enhanced anti-HSV-2 activity by a single arginine substitution at the adaptive evolution sites. Cell protection assay and temporal antiviral studies showed that HD5 and its mutants displayed affirmatory but differential anti-HSV-2 effects in vitro by inhibiting viral adhesion and entry. Inspiringly, the E21R-HD5 mutant had significantly higher antiviral activity than natural HD5, which is possibly attributed to the stronger binding affinity of the E21R-HD5 mutant with HSV-2 capsid protein gD, indicating that E21R mutation can increase the anti-HSV-2 potency of HD5. In a mouse model of lethal HSV-2 infection, prophylactic and/or therapeutic treatment with E21R-HD5 via intravaginal instillation remarkably alleviated the symptoms and delayed disease progress and resulted in about a 1.5-fold-higher survival rate than in the HD5 group. Furthermore, the E21R variant exhibited a 2-fold-higher antiviral potency against HIV-1 over parental HD5 in vitro. This study demonstrates that arginine mutagenesis at appropriate evolution sites may significantly enhance the antiviral activity of HD5, which also paves a facile way to search for potent antiviral drugs based on natural antimicrobial peptides.
doi:10.1128/JVI.02209-12
PMCID: PMC3571410  PMID: 23269800
13.  Hepatitis E Virus Genotype 4, Nanjing, China, 2001–2011 
Emerging Infectious Diseases  2013;19(9):1528-1530.
During 2001–2011, hepatitis E virus (HEV) was found in the blood of patients in Nanjing, China. All HEV-positive patients had virus genotype 4; subgenotype 4a was predominant. The effective population of HEV in Nanjing increased in ≈1980 and continued until ≈2003 when it plateaued.
doi:10.3201/eid1909.130013
PMCID: PMC3810912  PMID: 23965731
hepatitis E virus; viruses; hepatitis E; acute infection; evolution; genotype 4; Nanjing; China
14.  Subcutaneous administration of rhIGF-I post irradiation exposure enhances hematopoietic recovery and survival in BALB/c mice 
Journal of radiation research  2012;53(4):581-587.
It is unclear how to effectively mitigate against irradiation injury. In this study, we studied the capacity of recombinant human insulin-like growth factor-I (rhIGF-I) on hematologic recovery in irradiated BALB/c mice and its possible mechanism. BALB/c mice were injected with rhIGF-I subcutaneously at a dose of 100 μg/kg twice daily for 7 days after total body irradiation. Compared with a saline control group, treatment with rhIGF-I significantly improved the survival of mice after lethal irradiation (7.5 Gy). It was found that treatment with rhIGF-I not only could increase the frequency of Sca-1+ cells in bone marrow harvested at Day 14 after irradiation, but also it could decrease the apoptosis of mononuclear cells induced by irradiation as measured by flow cytometry, suggesting that rhIGF-I may mediate its effects primarily through promoting hematopoietic stem cell/progenitor survival and protecting mononuclear cells from apoptosis after irradiation exposure. Moreover, we have found that rhIGF-I might facilitate thrombopoiesis in an indirect way. Our data demonstrated that rhIGF-I could promote overall hematopoietic recovery after ionizing radiation and reduce the mortality when administered immediately post lethal irradiation exposure.
doi:10.1093/jrr/rrs029
PMCID: PMC3393355  PMID: 22843623
insulin-like growth factor-I; irradiation injury; hematopoietic recovery; apoptosis
15.  Occurrence and Distribution of Microcystins in Lake Taihu, China 
The Scientific World Journal  2013;2013:838176.
The occurrence and distribution of microcystins were investigated in Lake Taihu, the third largest lake in China. An extensive survey, larger and broader in scale than previous studies, was conducted in summer 2010. The highest microcystin concentration was found at southern part of Taihu, which was newly included in this survey. In northern coastal areas, total cellular concentrations of 20 to 44 μg/L were observed. In northern offshore waters, levels were up to 4.8 μg/L. Microcystin occurrence was highly correlated with chemical oxygen demand, turbidity, and chlorophyll-a. Extracellular/total cellular microcystin (E/T) ratios were calculated and compared to other water quality parameters. A higher correlation was found using E/T ratios than original microcystin values. These results show that algal blooms are having a severe impact on Lake Taihu, and further and extensive monitoring and research are required to suppress blooms effectively.
doi:10.1155/2013/838176
PMCID: PMC3703407  PMID: 23853542
16.  Tumor suppressor gene RBM5 delivered by attenuated Salmonella inhibits lung adenocarcinoma through diverse apoptotic signaling pathways 
Background
RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 has been demonstrated to be a tumor suppressor. Current researches in vitro confirm that RBM5 can suppress the growth of lung adenocarcinoma cells by inducing apoptosis. There is still no effective model in vivo, however, that thoroughly investigates the effect and molecular mechanism of RBM5 on lung adenocarcinoma.
Method
We established the transplanted tumor model on BALB/c nude mice using the A549 cell line. The mice were treated with the recombinant plasmids carried by attenuated Salmonella to induce the overexpression of RBM5 in tumor tissues. RBM5 overexpression was confirmed by immunohistochemistry staining. H&E staining was performed to observe the histological performance on plasmids-treated A549 xenografts. Apoptosis was assessed by TUNEL staining with a TUNEL detection kit. Apoptosis-regulated genes were detected by Western blot.
Results
We successful established the lung adenocarcinoma animal model in vivo. The growth of tumor xenografts was significantly retarded on the mice treated with pcDNA3.1-RBM5 carried by attenuated Salmonella compared to that on mice treated with pcDNA3.1. Overexpression of RBM5 enhanced the apoptosis in tumor xenografts. Furthermore, the expression of Bcl-2 protein was decreased significantly, while the expression of BAX, TNF-α, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and cleaved PARP proteins was significantly increased in the pcDNA3.1-RBM5-treated mice as compared to that in the control mice.
Conclusions
In this study, we established a novel animal model to determine RBM5 function in vivo, and concluded that RBM5 inhibited tumor growth in mice by inducing apoptosis. The study suggests that although RBM5’s involvement in the death receptor-mediated apoptotic pathway is still to be investigated, RBM5-mediated growth suppression, at least in part, employs regulation of the mitochondrial apoptotic pathways.
doi:10.1186/1477-7819-11-123
PMCID: PMC3673837  PMID: 23721095
RBM5; Lung adenocarcinoma; Apoptosis; A549; Xenograft mice model; Attenuated Salmonella
17.  Glycyrrhizic acid nanoparticles inhibit LPS-induced inflammatory mediators in 264.7 mouse macrophages compared with unprocessed glycyrrhizic acid 
Glycyrrhizic acid (GA), the main component of radix glycyrrhizae, has a variety of pharmacological activities. In the present study, suspensions of GA nanoparticles with the average particle size about 200nm were prepared by a supercritical antisolvent (SAS) process. Comparative studies were undertaken using lipopolysaccardide(LPS)-stimulated mouse macrophages RAW 264.7 as in vitro inflammatory model. Several important inflammation mediators such as NO, PGE2, TNF-α and IL-6 were examined. These markers were highly stimulated by LPS and were inhibited both by nano-GA and unprocessed GA in a dose-dependent manner, especially PGE2 and TNF-α. However nano-GA and unprocessed GA inhibited NO only at a high concentration. In general, we found that GA nanoparticle suspensions exhibited much better anti-inflammatory activities compared to unprocessed GA.
doi:10.2147/IJN.S37788
PMCID: PMC3629880  PMID: 23610519
glycyrrhizic acid; nanoparticle; mouse macrophages RAW 264.7; inflammatory cytokines
18.  Roles of Rho GTPases in Intracellular Transport and Cellular Transformation 
Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis. Cdc42 influences trafficking through interaction with Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, leading to changes in actin dynamics. Rac1 mediates endocytic and exocytic vesicle trafficking by interaction with its effectors, PI3kinase, synaptojanin 2, IQGAP1 and phospholipase D1. RhoA participates in the regulation of endocytosis through controlling its downstream target, Rho kinase. Interestingly, these GTPases play important roles at different stages of viral protein and genome transport in infected host cells. Importantly, dysregulation of Cdc42, Rac1 and RhoA leads to numerous disorders, including malignant transformation. In some cases, hyperactivation of Rho GTPases is required for cellular transformation. In this article, we review a number of findings related to Rho GTPase function in intracellular transport and cellular transformation.
doi:10.3390/ijms14047089
PMCID: PMC3645678  PMID: 23538840
Rho GTPases; vesicle trafficking; viral transport; cellular transformation; actin cytoskeleton
19.  Role of a Novel Functional Variant in the PPP2R1A Promoter on the Regulation of PP2A-Aalpha and the Risk of Hepatocellular Carcinoma 
PLoS ONE  2013;8(3):e59574.
Previously, we identified the genetic variant −241 (−/G) (rs11453459) in the PP2A-Aα gene (PPP2R1A) promoter and demonstrated that this variant influences the DNA-binding affinity of nuclear factor-kappa B (NF-κB). In this study, we further confirmed that the transcriptional activity of PPP2R1A may be regulated by NF-κB through the functional genetic variant −241 (−/G). Moreover, we also demonstrated that the methylation status of CpG islands in the promoter of PPP2R1A influences the activity of this gene promoter. Few studies have examined the role of this −241 (−/G) variant in genetic or epigenetic regulation in hepatocellular carcinoma (HCC). To investigate whether this functional variant in the PPP2R1A promoter is associated with the risk of HCC and confirm the function of the −241 (−/G) variant in the HCC population, we conducted a case-control study involving 251 HCC cases and 252 cancer-free controls from a Han population in southern China. Compared with the −241 (−−) homozygote, the heterozygous −241 (−G) genotype (adjusted OR  = 0.32, 95% confidence interval (CI)  = 0.17–0.58, P<0.001) and the −241 (−G)/(GG) genotypes (adjusted OR  = 0.38, 95% CI  = 0.22–0.67, P  = 0.001) were both significantly associated with a reduced risk of HCC. Stratification analysis indicated that the protective role of −241 (−G) was more pronounced in individuals who were ≤ 40 years of age, female and HBV-negative. Our data suggest that the transcriptional activity of PPP2R1A is regulated by NF-κB through the −241 (−/G) variant and by the methylation of the promoter region. Moreover, the functional −241 (−/G) variant in the PPP2R1A promoter contributes to the decreased risk of HCC. These findings contribute novel information regarding the gene transcription of PPP2R1A regulated by the polymorphism and methylation in the promoter region through genetic and epigenetic mechanisms in hepatocarcinogenesis.
doi:10.1371/journal.pone.0059574
PMCID: PMC3612049  PMID: 23555712
20.  An Improbable Monometallic Cluster Entrapped in a Popular Fullerene Cage: YCN@Cs(6)-C82 
Scientific Reports  2013;3:1487.
Since the first proposal that fullerenes are capable of hosting atoms, ions, or clusters by the late Smalley in 1985, tremendous examples of endohedral metallofullerenes (EMFs) have been reported. Breaking the dogma that monometallofullerenes (mono-EMFs) always exist in the form of M@C2n while clusterfullerenes always require multiple (two to four) metal cations to stabilize a cluster that is unstable as a single moiety, here we show an unprecedented monometallic endohedral clusterfullerene entrapping an yttrium cyanide cluster inside a popular C82 cage—YCN@Cs(6)-C82. X-ray crystallography and 13C NMR characterization unambiguously determine the cage symmetry and the endohedal cyanide structure, unexpectedly revealing that the entrapped YCN cluster is triangular. The unprecedented monometallic clusterfullerene structure unveiled by YCN@Cs(6)-C82 opens up a new avenue for stabilizing a cluster by a single metal cation within a carbon cage, and will surely stimulate further studies on the stability and formation mechanism of EMFs.
doi:10.1038/srep01487
PMCID: PMC3601605  PMID: 23512079
21.  MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria 
Applied and Environmental Microbiology  2012;78(17):6113-6120.
Klebsiella pneumoniae M5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene cluster mhbRTDHIM conferred the ability to grow on 3-hydroxybenzoate to Escherichia coli and Pseudomonas putida PaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H+ symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane. P. putida PaW340 containing mhbRΔTDHIM could not grow on 3-hydroxybenzoate; however, supplying mhbT in trans allowed the bacterium to grow on the substrate. K. pneumoniae M5a1 and P. putida PaW340 containing recombinant MhbT transported 14C-labeled 3-hydroxybenzoate but not 14C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake by P. putida PaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.
doi:10.1128/AEM.01511-12
PMCID: PMC3416623  PMID: 22729544
22.  Differentially Expressed miRNAs after GnRH Treatment and Their Potential Roles in FSH Regulation in Porcine Anterior Pituitary Cell 
PLoS ONE  2013;8(2):e57156.
Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3′-untranslated region (3′-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3′-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.
doi:10.1371/journal.pone.0057156
PMCID: PMC3579806  PMID: 23451171
23.  The Anorexigenic Effect of Serotonin Is Mediated by the Generation of NADPH Oxidase-Dependent ROS 
PLoS ONE  2013;8(1):e53142.
Serotonin (5-HT) is a central inhibitor of food intake in mammals. Thus far, the intracellular mechanisms for the effect of serotonin on appetite regulation remain unclear. It has been recently demonstrated that reactive oxygen species (ROS) in the hypothalamus are a crucial integrative target for the regulation of food intake. To investigate the role of ROS in the serotonin-induced anorexigenic effects, conscious mice were treated with 5-HT alone or combination with Trolox (a ROS scavenger) or Apocynin (an NADPH oxidase inhibitor) by acute intracerebroventricular injection. Both Trolox and Apocynin reversed the anorexigenic action of 5-HT and the 5-HT-induced hypothalamic ROS elevation. The mRNA and protein expression levels of pro-opiomelanocortin (POMC) were dramatically increased after ICV injection with 5-HT. The anorexigenic action of 5-HT was accompanied by markedly elevated hypothalamic MDA levels and GSH-Px activity, while the SOD activity was decreased. Moreover, 5-HT significantly increased the mRNA expression of UCP-2 but reduced the levels of UCP-3. Both Trolox and Apocynin could block the 5-HT-induced changes in UCP-2 and UCP-3 gene expression. Our study demonstrates for the first time that the anorexigenic effect of 5-HT is mediated by the generation of ROS in the hypothalamus through an NADPH oxidase-dependent pathway.
doi:10.1371/journal.pone.0053142
PMCID: PMC3541393  PMID: 23326391
24.  Motor Training Increases the Stability of Activation Patterns in the Primary Motor Cortex 
PLoS ONE  2013;8(1):e53555.
Learning to be skillful is an endowed talent of humans, but neural mechanisms underlying behavioral improvement remain largely unknown. Some studies have reported that the mean magnitude of neural activation is increased after learning, whereas others have instead shown decreased activation. In this study, we used functional magnetic resonance imaging (fMRI) to investigate learning-induced changes in the neural activation in the human brain with a classic motor training task. Specifically, instead of comparing the mean magnitudes of activation before and after training, we analyzed the learning-induced changes in multi-voxel spatial patterns of neural activation. We observed that the stability of the activation patterns, or the similarity of the activation patterns between the even and odd runs of the fMRI scans, was significantly increased in the primary motor cortex (M1) after training. By contrast, the mean magnitude of neural activation remained unchanged. Therefore, our study suggests that learning shapes the brain by increasing the stability of the activation patterns, therefore providing a new perspective in understanding the neural mechanisms underlying learning.
doi:10.1371/journal.pone.0053555
PMCID: PMC3538534  PMID: 23308252
25.  Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation 
PLoS ONE  2012;7(12):e51616.
Background
The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.
Methodology/Principal Findings
First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×105 genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶104–1∶105 and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶103–1∶104 and were protected from hepatitis, but two out of the five rabbits showed virus shedding.
Conclusions/Significance
Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.
doi:10.1371/journal.pone.0051616
PMCID: PMC3521758  PMID: 23272124

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