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1.  Evolutionary analysis of rubella viruses in mainland China during 2010–2012: endemic circulation of genotype 1E and introductions of genotype 2B 
Scientific Reports  2015;5:7999.
Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010–2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010–2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.
doi:10.1038/srep07999
PMCID: PMC4303870  PMID: 25613734
2.  Gastrointestinal symptom due to lupus peritonitis: a rare form of onset of SLE 
Serositis is commonly seen in systemic lupus erythematosus (SLE). Approximately 16% of patients with SLE have pleural or pericardial involvement. However, peritoneal involvement is extremely rare, and SLE with ascites as the first manifestation is an even rarer condition. This is the case report of a 19-year old male with discoid lupus who evolved with gastrointestinal symptoms as the first manifestation of the disease, characterized by significant abdominal distension and pain, asthenia, vomiting, and signs of ascites. An abdominal CT scan demonstrated ascites and marked edematous thickening of the bowel wall, which appeared as “target sign”, and “double-track sign”. Laboratory tests showed that his serum complement levels decreased and that he was positive for anti-nRNP/Sm antibodies, anti-Sm antibodies, anti-SS-A antibody, and anti-nuclear antibodies. The patient was treated with prednisone and chloroquine, with substantial improvement of his condition.
PMCID: PMC4307580  PMID: 25664133
Systemic lupus erythematosus; serositis; ascites; lupus peritonitis; CT
3.  Outbreak of Epidemic Keratoconjunctivitis Caused by Human Adenovirus Type 56, China, 2012 
PLoS ONE  2014;9(10):e110781.
HAdV-56 is a new recombinant type isolated from epidemic keratoconjunctivitis (EKC) patients and has been sporadically isolated in Japan several times. Here, an outbreak of EKC in the city of Dalian, China involving a large number of workers in two factories was reported; this was the first outbreak of EKC associated with HAdV-56 worldwide.
doi:10.1371/journal.pone.0110781
PMCID: PMC4208770  PMID: 25343525
4.  Effect of ECRG2 in combination with cisplatin on the proliferation and apoptosis of EC9706 cells 
The aim of the present study was to explore the effect of esophageal cancer-related gene 2 (ECRG2) protein in combination with cisplatin (DDP) on the proliferation and apoptosis of esophageal cancer cells. A 3-(4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the effects of ECRG2 alone and ECRG2 in combination with DDP on the proliferation of EC9706 esophageal cancer cells. Hoechst 33258 staining was performed to analyze the effects of ECRG2 alone and ECRG2 in combination with DDP on apoptosis in the EC9706 cells. The expression levels of Bcl-2-associated X protein (Bax) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results from the MTT assay revealed that ECRG2 inhibited the proliferation of EC9706 cells and that ECRG2 in combination with DDP had a greater inhibitory effect on cell proliferation. The antiproliferative effects were time- and concentration-dependent, within a certain range of concentrations. The Hoechst 33258 staining results demonstrated that the number of apoptotic cells following treatment with ECRG2 in combination with DDP for 24 h was higher than that following treatment with ECRG2 alone for the same duration. Western blot analysis and RT-PCR results revealed that the expression levels of Bax mRNA and protein were upregulated in cells treated with ECRG2 in combination with DDP compared with those in cells treated with ECRG2 alone. Thus, ECRG2 in combination with DDP had an enhanced inhibitory effect on EC9706 cell proliferation compared with that of ECRG2 alone, and an increased inductive effect on EC9706 cell apoptosis, possibly due to the upregulation of the expression of Bax.
doi:10.3892/etm.2014.1972
PMCID: PMC4186322  PMID: 25289046
esophageal cancer related-gene 2; bcl-2-associated X protein; cisplatin; proliferation; apoptosis
5.  The Rise of China in the International Trade Network: A Community Core Detection Approach 
PLoS ONE  2014;9(8):e105496.
Theory of complex networks proved successful in the description of a variety of complex systems ranging from biology to computer science and to economics and finance. Here we use network models to describe the evolution of a particular economic system, namely the International Trade Network (ITN). Previous studies often assume that globalization and regionalization in international trade are contradictory to each other. We re-examine the relationship between globalization and regionalization by viewing the international trade system as an interdependent complex network. We use the modularity optimization method to detect communities and community cores in the ITN during the years 1995–2011. We find rich dynamics over time both inter- and intra-communities. In particular, the Asia-Oceania community disappeared and reemerged over time along with a switch in leadership from Japan to China. We provide a multilevel description of the evolution of the network where the global dynamics (i.e., communities disappear or reemerge) and the regional dynamics (i.e., community core changes between community members) are related. Moreover, simulation results show that the global dynamics can be generated by a simple dynamic-edge-weight mechanism.
doi:10.1371/journal.pone.0105496
PMCID: PMC4138169  PMID: 25136895
6.  Small RNAs as important regulators for the hybrid vigour of super-hybrid rice 
Journal of Experimental Botany  2014;65(20):5989-6002.
Summary
Differentially expressed miRNAs and ta-siRNAs between hybrid rice and its parents play important roles in hybrid vigour of super-hybrid rice by regulating their target genes controlling the auxin-mediated signalling pathway.
Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis.
doi:10.1093/jxb/eru337
PMCID: PMC4203131  PMID: 25129133
Auxin signalling pathway; differentially expressed gene; heterosis; QTL mapping;  rice; small RNA; transcriptional profile.
7.  Phylogenetic evidence for multiple intertypic recombinations in enterovirus B81 strains isolated in Tibet, China 
Scientific Reports  2014;4:6035.
Enterovirus B81 (EV-B81) is a newly identified serotype within the species enterovirus B (EV-B). To date, only eight nucleotide sequences of EV-B81 have been published and only one full-length genome sequence (the prototype strain) has been made available in the GenBank database. Here, we report the full-length genome sequences of two EV-B81 strains isolated in the Tibet Autonomous Region of China during acute flaccid paralysis surveillance activities, and we also conducted an antibody seroprevalence study in two prefectures of Tibet. The sequence comparison and phylogenetic dendrogram analysis revealed high variability among the global EV-B81 strains and frequent intertypic recombination in the non-structural protein region of EV-B serotypes, suggesting high genetic diversity of EV-B81. However, low positive rates and low titers of neutralizing antibodies against EV-B81 were detected. Nearly 68% of children under the age of five had no neutralizing antibodies against EV-B81. Hence, the extent of transmission and the exposure of the population to this EV type are very limited. Although little is known about the biological and pathogenic properties of EV-B81 because of few research in this field owing to the limited number of isolates, our study provides basic information for further studies of EV-B81.
doi:10.1038/srep06035
PMCID: PMC4129410  PMID: 25112835
8.  Rapid Development of Microsatellite Markers for Callosobruchus chinensis Using Illumina Paired-End Sequencing 
PLoS ONE  2014;9(5):e95458.
Background
The adzuki bean weevil, Callosobruchus chinensis L., is one of the most destructive pests of stored legume seeds such as mungbean, cowpea, and adzuki bean, which usually cause considerable loss in the quantity and quality of stored seeds during transportation and storage. However, a lack of genetic information of this pest results in a series of genetic questions remain largely unknown, including population genetic structure, kinship, biotype abundance, and so on. Co-dominant microsatellite markers offer a great resolving power to determine these events. Here, we report rapid microsatellite isolation from C. chinensis via high-throughput sequencing.
Principal Findings
In this study, 94,560,852 quality-filtered and trimmed reads were obtained for the assembly of genome using Illumina paired-end sequencing technology. In total, the genome with total length of 497,124,785 bp, comprising 403,113 high quality contigs was generated with de novo assembly. More than 6800 SSR loci were detected and a suit of 6303 primer pair sequences were designed and 500 of them were randomly selected for validation. Of these, 196 pair of primers, i.e. 39.2%, produced reproducible amplicons that were polymorphic among 8 C. chinensis genotypes collected from different geographical regions. Twenty out of 196 polymorphic SSR markers were used to analyze the genetic diversity of 18 C. chinensis populations. The results showed the twenty SSR loci were highly polymorphic among these populations.
Conclusions
This study presents a first report of genome sequencing and de novo assembly for C. chinensis and demonstrates the feasibility of generating a large scale of sequence information and SSR loci isolation by Illumina paired-end sequencing. Our results provide a valuable resource for C. chinensis research. These novel markers are valuable for future genetic mapping, trait association, genetic structure and kinship among C. chinensis.
doi:10.1371/journal.pone.0095458
PMCID: PMC4023940  PMID: 24835431
9.  Squalene Synthase As a Target for Chagas Disease Therapeutics 
PLoS Pathogens  2014;10(5):e1004114.
Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.
Author Summary
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects eight million individuals, primarily in Latin America. Currently there is no cure for chronic T. cruzi infections. Unlike humans, this parasite use a variety of sterols (e.g. ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta ol, and its 22-dihydro analogs), rather than cholesterol in their cell membranes, so inhibiting endogenous sterol biosynthesis is an important therapeutic target. Here, we report the first structure of the parasite's squalene synthase, which catalyzes the first committed step in sterol biosynthesis, as well as the structures of a broad range of squalene synthase inhibitors active against the clinically relevant intracellular stages, opening the way to new approaches to treating this neglected tropical disease.
doi:10.1371/journal.ppat.1004114
PMCID: PMC4006925  PMID: 24789335
10.  Comparative Transcriptional Profiling of Three Super-Hybrid Rice Combinations 
Utilization of heterosis has significantly increased rice yields. However, its mechanism remains unclear. In this study, comparative transcriptional profiles of three super-hybrid rice combinations, LY2163, LY2186 and LYP9, at the flowering and filling stages, were created using rice whole-genome oligonucleotide microarray. The LY2163, LY2186 and LYP9 hybrids yielded 1193, 1630 and 1046 differentially expressed genes (DGs), accounting for 3.2%, 4.4% and 2.8% of the total number of genes (36,926), respectively, after using the z-test (p < 0.01). Functional category analysis showed that the DGs in each hybrid combination were mainly classified into the carbohydrate metabolism and energy metabolism categories. Further analysis of the metabolic pathways showed that DGs were significantly enriched in the carbon fixation pathway (p < 0.01) for all three combinations. Over 80% of the DGs were located in rice quantitative trait loci (QTLs) of the Gramene database, of which more than 90% were located in the yield related QTLs in all three combinations, which suggested that there was a correlation between DGs and rice heterosis. Pathway Studio analysis showed the presence of DGs in the circadian regulatory network of all three hybrid combinations, which suggested that the circadian clock had a role in rice heterosis. Our results provide information that can help to elucidate the molecular mechanism underlying rice heterosis.
doi:10.3390/ijms15033799
PMCID: PMC3975368  PMID: 24595241
rice; transcriptional profiling; heterosis; photosynthesis; circadian clock
11.  Effects of the antihypertensive drug benidipine on osteoblast function in vitro 
The dihydropyridine-type calcium channel blocker, benidipine (BD) has been widely used in hypertension therapy. Previous studies have demonstrated that BD has a positive effect on bone metabolism. Inspired by this promoting phenomenon, the present study investigated the effects of BD on osteoblasts in vitro. Experiments were designed and performed, including an MTT assay, reverse transcription-polymerase chain reaction, western blot analysis, alkaline phosphatase activity measurements and alizarin red S staining. The results demonstrated that BD promoted osteoblast proliferation and osteogenic differentiation at concentrations from 1×10−6 to 1×10−9 M by upregulating Runx2, BMP2 and OCN gene expression levels. Overall, BD at appropriate concentrations has been demonstrated to have positive effects on osteoblast function in addition to its conventional clinical usage.
doi:10.3892/etm.2014.1475
PMCID: PMC3919856  PMID: 24520261
benidipine; calcium channel blockers; osteoporosis; hypertension
12.  Association of Polymorphisms of Exon 2 of the Growth Hormone Gene with Production Performance in Huoyan Goose 
Primers based on the cDNA sequence of the goose growth hormone (GH) gene in GenBank were designed to amplify exon 2 of the GH gene in Huoyan goose. A total of 552 individuals were brooded in one batch and raised in Liaoning and Jiangsu Provinces, China. Single nucleotide polymorphisms (SNPs) of exon 2 in the GH gene were detected by the polymerase chain reaction (single strand conformation polymorphism method). Homozygotes were subsequently cloned, sequenced and analyzed. Two SNP mutations were detected, and 10 genotypes (referred to as AA, BB, CC, DD, AB, AC, AD, BC, BD and CD) were obtained. Allele D was predominant, and the frequencies of the 10 genotypes fit the Hardy-Weinberg equilibrium in the male, female and whole populations according to the chi-square test. Based on SNP types, the 10 genotypes were combined into three main genotypes. Multiple comparisons were carried out between different genotypes and production traits when the geese were 10 weeks old. Some indices of production performance were significantly (p < 0.05) associated with the genotype. Particularly, geese with genotype AB or BB were highly productive. Thus, these genotypes may serve as selection markers for production traits in Huoyan geese.
doi:10.3390/ijms15010670
PMCID: PMC3907831  PMID: 24402125
multiple comparisons; growth hormone gene; Huoyan goose; production performance; single strand conformation polymorphism
13.  Increase of ADAM10 Level in Coronary Artery In-Stent Restenosis Segments in Diabetic Minipigs: High ADAM10 Expression Promoting Growth and Migration in Human Vascular Smooth Muscle Cells via Notch 1 and 3 
PLoS ONE  2013;8(12):e83853.
Background
This study aimed to identify major proteins in the pathogenesis of coronary artery in-stent restenosis (ISR) in diabetic minipigs with sirolimus-eluting stenting, and to investigate the roles of key candidate molecules, particularly ADAM10, in human arterial smooth muscle cells (HASMCs).
Methods and Results
The stents were implanted in the coronary arteries of 15 diabetic and 26 non-diabetic minipigs, and angiography was repeated at six months. The intima of one vascular segment with significant ISR and one with non-ISR in diabetic minipigs were isolated and cultured in conditioned medium (CM). The CM was analyzed by LC-MS/MS to uncover proteins whose levels were significantly increased (≥1.5-fold) in ISR than in non-ISR tissues. After literature searching, we focused on the identified proteins, whose biological functions were most potentially related to ISR pathophysiology. Among them, ADAM10 was significantly increased in diabetic and non-diabetic ISR tissues as compared with non-ISR controls. In cell experiments, retrovirus-mediated overexpression of ADAM10 promoted growth and migration of HASMCs. The effects of ADAM10 were more remarkable in high-glucose culture than in low-glucose culture. Using shRNA and an inhibitor of γ-secretase (GSI), we found that the influences of ADAM10 were in part mediated by Notch1 and notch 3 pathway, which up-regulated Notch downstream genes and enhanced nuclear translocation of the small intracellular component of Notch1 and Notch3.
Conclusions
This study has identified significantly increased expression of ADAM10 in the ISR versus non-ISR segment in diabetic minipigs and implicates ADAM10 in the enhanced neointimal formation observed in diabetes after vascular injury.
doi:10.1371/journal.pone.0083853
PMCID: PMC3873985  PMID: 24386293
14.  Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro 
Acta Pharmacologica Sinica  2013;34(11):1457-1466.
Aim:
To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro.
Methods:
Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-κB ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNFα was analyzed with ELISA.
Results:
Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 μmol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group (96 h).
Conclusion:
Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.
doi:10.1038/aps.2013.99
PMCID: PMC4006473  PMID: 24056707
wear particle; bone marrow macrophage; osteoclast; TNF-α; calcineurin; nuclear factor of activated T cells (NFAT); 11R-VIVIT peptide; arthroplasty; periprosthetic osteolysis; aseptic loosening
15.  Comprehensive profiling of Epstein-Barr virus-encoded miRNA species associated with specific latency types in tumor cells 
Virology Journal  2013;10:314.
Background
Epstein-Barr virus (EBV) is an etiological cause of many human lymphocytic and epithelial malignancies. EBV expresses different genes that are associated with three latency types. To date, as many as 44 EBV-encoded miRNA species have been found, but their comprehensive profiles in the three types of latent infection that are associated with various types of tumors are not well documented.
Methods
In the present study, we utilized poly (A)-tailed quantitative real-time RT-PCR in combination with microarray analysis to measure the relative abundances of viral miRNA species in a subset of representative lymphoid and epithelial tumor cells with various EBV latency types.
Results
Our findings showed that the miR-BHRF1 and miR-BART families were expressed differentially in a tissue- and latency type-dependent manner. Specifically, in nasopharyngeal carcinoma (NPC) tissues and the EBV-positive cell line C666-1, the miR-BART family accounted for more than 10% of all detected miRNAs, suggesting that these miRNAs have important roles in maintaining latent EBV infections and in driving NPC tumorigenesis. In addition, EBV miRNA-based clustering analysis clearly distinguished between the three distinct EBV latency types, and our results suggested that a switch from type I to type III latency might occur in the Daudi BL cell line.
Conclusions
Our data provide a comprehensive profiling of the EBV miRNA transcriptome that is associated with specific tumor cells in the three types of latent EBV infection states. EBV miRNA species represent a cluster of non-encoding latency biomarkers that are differentially expressed in tumor cells and may help to distinguish between the different latency types.
doi:10.1186/1743-422X-10-314
PMCID: PMC4231337  PMID: 24161012
Epstein-Barr virus; Viral microRNA; Latency types; Nasopharyngeal carcinoma; Burkitt’s lymphoma
16.  Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L  
PLoS ONE  2013;8(10):e78355.
Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC50 value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.
doi:10.1371/journal.pone.0078355
PMCID: PMC3806812  PMID: 24194922
17.  Oxidative damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease 
Peripheral arterial disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in arteries supplying the legs, affects an estimated 27 million people in Europe and North America. Increased production of reactive oxygen species by dysfunctional mitochondria in leg muscles of PAD patients is viewed as a key mechanism of initiation and progression of the disease. Previous studies demonstrated increased oxidative damage in homogenates of biopsy specimens from PAD gastrocnemius compared to controls, but did not address myofiber-specific damage. In this study, we investigated oxidative damage to myofibers as a possible cause of the myopathy of PAD. To achieve this, we developed and validated fluorescence microscopy procedures for quantitative analysis of carbonyl groups and 4-hydroxy-2-nonenal (HNE) adducts in myofibers of biopsy specimens from human gastrocnemius. PAD and control specimens were evaluated for differences in 1) myofiber content of these two forms of oxidative damage and 2) myofiber cross-sectional area. Furthermore, oxidative damage to PAD myofibers was tested for associations with clinical stage of disease, degree of ischemia in the affected leg, and myofiber cross-sectional area. Carbonyl groups and HNE adducts were increased 30% (p < 0.0001) and 40% (p < 0.0001), respectively, in the myofibers of PAD (N = 34) compared to control (N = 21) patients. Mean cross-sectional area of PAD myofibers was reduced 29.3% compared to controls (p < 0.0003). Both forms of oxidative damage increased with clinical stage of disease, blood flow limitation in the ischemic leg, and reduced myofiber cross-sectional area. The data establish oxidative damage to myofibers as a possible cause of PAD myopathy.
doi:10.1186/1479-5876-11-230
PMCID: PMC3849592  PMID: 24067235
18.  Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009 
PLoS ONE  2013;8(9):e73374.
Background
China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies.
Principal Findings
Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure.
Conclusions
Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.
doi:10.1371/journal.pone.0073374
PMCID: PMC3779233  PMID: 24073194
19.  Overexpression of FXYD-3 Is Involved in the Tumorigenesis and Development of Esophageal Squamous Cell Carcinoma 
Disease markers  2013;35(3):195-202.
Objective. To investigate the association of FXYD-3 expression with clinicopathological variables and PINCH in patients with ESCC. Patients and Methods. Expression of FXYD-3 protein was immunohistochemically examined in normal esophageal mucous (n = 20) and ESCC (n = 64). Results. Expression of FXYD-3 in the cytoplasm markedly increased from normal esophageal epithelial cells to primary ESCC (P = 0.001). The expression of FXYD-3 was correlated with TNM stages and depth of tumor invasion. Furthermore, the cases with lymph node metastasis tended to show a higher frequency of positive expression than those without metastasis (P = 0.086), and FXYD-3 expression tended to be positively related to the expression of PINCH (P = 0.063). Moreover, the cases positive for both proteins had the highest frequency of lymph node metastasis (P = 0.001). However, FXYD-3 expression was not correlated with patient's gender (P = 0.847), age (P = 0.876), tumor location (P = 0.279), size (P = 0.771), grade of differentiation (P = 0.279), and survival (P = 0.113). Conclusion. Overexpression of FXYD-3 in the cytoplasm may play an important role in the tumorigenesis and development in the human ESCC, particularly in combination with PINCH expression.
doi:10.1155/2013/740201
PMCID: PMC3774971  PMID: 24167366
20.  Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease 
Background
Peripheral arterial disease (PAD) is characterized by myofiber degeneration and loss of function in muscles of the lower limbs. Human enterovirus (HEV) infection has been implicated in the pathogenesis of a number of muscle diseases. However, its association with PAD has not been studied. In this study, we tested the hypothesis that infectious HEV is present in skeletal muscle of patients with PAD and is associated with severity of disease.
Methods and Results
Gastrocnemius biopsies from 37 patients with PAD and 14 controls were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity. HEV RNA was detected in 54% of the biopsies from patients with PAD but was not detected in muscle biopsies from control patients. This difference in prevalence among PAD and control patients was significant at P<0.001. Viral RNA copy numbers were increased significantly at the later stages of disease; Fontaine Stage IV (105.50 copies/mg muscle wet weight, at P<0.005) and Stage III (104.87 copies/mg, at P<0.010) compared to Stage II (102.50 copies/mg). Viral replication was confirmed by the presence of the negative‐strand of viral RNA in all specimens positive for HEV RNA. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein.
Conclusion
Our data identified infectious HEV in the gastrocnemius of PAD patients but not in controls. Viral copy number and prevalence of infection were higher in the later stages of disease. Our data point to the need for further studies to determine the contribution of HEV infection to the pathophysiology of PAD.
doi:10.1161/JAHA.113.000082
PMCID: PMC3828788  PMID: 23920231
coxsackievirus; human enterovirus; peripheral arterial disease; skeletal muscle degeneration
21.  Correction: Viral Etiology of Acute Respiratory Infection in Gansu Province, China, 2011 
PLoS ONE  2013;8(7):10.1371/annotation/91d141f8-549c-475c-891a-5d8b4e5f91fd.
doi:10.1371/annotation/91d141f8-549c-475c-891a-5d8b4e5f91fd
PMCID: PMC3728372
22.  Puerarin Suppress Apoptosis of Human Osteoblasts via ERK Signaling Pathway 
Puerarin, the main isoflavone glycoside extracted from Radix Puerariae, is an isoflavone traditional Chinese herb. Previous studies have demonstrated that puerarin could regulate osteoblast proliferation and differentiation to promote bone formation. However, the effect of puerarin on the process of human osteoblasts (hOBs) apoptosis is still unclear. In this study, we detected the function of puerarin on serum-free-induced cell apoptosis using ELISA and TUNEL arrays and then found that the mortality of hOBs was significantly decreased after exposure to 10−10–10−6 M puerarin and reached the maximal antiapoptotic effect at the concentration of 10−8 M. In addition, compared with the control group, puerarin notably increased the Bcl-2 protein levels while it decreased the Bax protein levels in the hOBs in a dose-dependent way. 10−7 M puerarin decreased the Bax/Bcl-2 ratio with a maximal decrease to 0.08. Moreover, puerarin activated ERK signaling pathways in hOBs, and the antiapoptotic effect induced by puerarin was abolished by incubation of ERK inhibitor PD98059. Similarly, the estrogen receptor antagonist ICI182780 also suppressed the inhibitory effect of puerarin on hOBs apoptosis. In conclusion, puerarin could prevent hOBs apoptosis via ERK signaling pathway, which might be effective in providing protection against bone loss and bone remolding associated with osteoporosis.
doi:10.1155/2013/786574
PMCID: PMC3694486  PMID: 23843790
23.  Expression of huCdc7 in colorectal cancer 
AIM: To detect the expression of huCdc7 in colorectal cancer.
METHODS: The mRNA and protein expression of huCdc7 in 39 colorectal cancer tissue specimens and matched tumor-adjacent normal colorectal tissue specimens was detected by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively.
RESULTS: The relative expression level of huCdc7 mRNA in colorectal cancer was significantly higher than that in tumor-adjacent normal colorectal tissues (0.03675 ± 1.00 vs 0.01199 ± 0.44, P < 0.05). huCdc7-positive cells displayed brown granules in the nucleus. Tumor tissues contained many huCdc7-positive cells, whereas normal colorectal tissues contained very few positive cells.
CONCLUSION: huCdc7 may play an important role in the development and progression of colorectal cancer.
doi:10.3748/wjg.v19.i20.3130
PMCID: PMC3662954  PMID: 23716994
huCdc7; Semiquantitative reverse transcription-polymerase chain reaction; Colorectal cancer
24.  Viral Etiology of Acute Respiratory Infection in Gansu Province, China, 2011 
PLoS ONE  2013;8(5):e64254.
Background
Acute respiratory infections (ARIs) are the leading cause of children and their leading killer. ARIs are responsible for at least six percent of the world's disability and death. Viruses are one of the most common agents causing ARIs. Few studies on the viral etiology and clinical characteristics of ARIs have been performed in the northwest region of China, including Gansu Province.
Methods
Clinical and demographic information and throat swabs were collected from 279 patients from January 1st to December 30st, 2011. Multiplex RT-PCR was performed to detect 16 respiratory viral pathogens.
Results
279 patients were admitted for ARIs. The patients aged from 1 month to 12 years, with the median age of 2 years. Of which, 105 (37.6%) were positive for at least one pathogen. A total of 136 respiratory viral pathogens were identified from the 105 patients. Respiratory syncytial virus (RSV) was the most frequently detected pathogen (26.5%, 36/136), followed by parainfluenza virus (PIV) 1–3 (22.1%, 30/136), human rhinovirus (HRV) (21.3%, 29/136), human coronavirus (CoV) (10.3%, 14/136) and human adenovirus (HAdV) (9.6%, 13/136). Influenza A (Flu A), human metapneumovirus (hMPV) and human bocavirus (BoCA) were found 4.4%, 3.7% and 2.2%, respectively. Influenza B (Flu B) and seasonal influenza A H1N1(sH1N1) were not detected. Single-infections were detected in 30.5% (85/279) of cases. RSV was the most common pathogens in patients under 1 year and showed seasonal variation with peaks during winter and spring.
Conclusions
This paper presents data on the epidemiology of viral pathogens associated with ARIs among children in Gansu Province, China. RSV is most frequently detected in our study. The findings could serve as a reference for local CDC in drawing up further plans to prevent and control ARIs.
doi:10.1371/journal.pone.0064254
PMCID: PMC3653869  PMID: 23691184
25.  The Development and Application of the Two Real-Time RT-PCR Assays to Detect the Pathogen of HFMD 
PLoS ONE  2013;8(4):e61451.
Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.
doi:10.1371/journal.pone.0061451
PMCID: PMC3630163  PMID: 23637836

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