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author:("Wang, honei")
1.  HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade 
PLoS ONE  2014;9(3):e91142.
Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
PMCID: PMC3953338  PMID: 24625757
2.  A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis 
Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).
PMCID: PMC3498587  PMID: 22821242
human papillomavirus; oropharyngeal cancer; prognosis; expression signature
3.  Papillary Squamous Cell Carcinoma of the Head and Neck 
A relationship between human papillomavirus (HPV) infection and papillary squamous cell carcinoma (PSCC) has been suggested. However, to date, no studies have thoroughly and directly evaluated for transcriptional activity of the virus or the clinicopathologic significance of HPV-positive PSCC. Forty-eight cases of PSCC were retrieved from our surgical pathology database and were reviewed by 4 study pathologists, with tumors defined as SCC with a significant component of papillary growth in the tumor. Immunohistochemical analysis for p16 and p53 was performed. Overexpression of p16 was used as a surrogate marker of transcriptionally active HPV. Transcriptional activity was also directly evaluated using RNA in situ hybridization to detect high-risk HPV E6/E7 mRNA. Clinical follow-up data were obtained by chart review. Seven cases were located in the oral cavity, 19 in the oropharynx, and 22 in the larynx. Two morphologic types of PSCC were identified: keratinizing type, in which the epithelial cells showed a maturation trend with minimal surface parakeratin, and nonkeratinizing type, in which the papillae were completely covered by immature basaloid cells. Transcriptionally active HPV was present in 23 of 43 (53.4%) tumors. The majority of tumors harboring transcriptionally active HPV arose in the oropharynx, showed nonkeratinizing morphology, were p16 positive, and p53 negative. Transcriptionally active HPV was also present in many laryngeal and oral cavity PSCCs. Overall survival, disease-specific survival, and disease-free survival were favorable and did not significantly differ by anatomic subsite. However, HPV-related tumors showed a trend toward better survival.
PMCID: PMC3903508  PMID: 23797720
papillary squamous cell carcinoma; head and neck; human papillomavirus; p16; RNA in situ hybridization
4.  A Test of Rensch’s Rule in Greater Horseshoe Bat (Rhinolophus ferrumequinum) with Female-Biased Sexual Size Dimorphism 
PLoS ONE  2014;9(1):e86085.
Sexual size dimorphism (SSD) is widespread within the animal kingdom. Rensch’s rule describes a relationship between SSD and body size: SSD increases with body size when males are the larger sex, and decreases with body size when females are the larger sex. Rensch’s rule is well supported for taxa that exhibit male-biased SSD but patterns of allometry among taxa with female-biased size dimorphism are mixed, there is evidence both for and against the rule. Furthermore, most studies have investigated Rensch’s rule across a variety of taxa; but among-population studies supporting Rensch’s rule are lacking, especially in taxa that display only slight SSD. Here, we tested whether patterns of intraspecific variation in SSD in greater horseshoe bats conform to Rensch’s rule, and evaluated the contribution of latitude to Rensch’s rule. Our results showed SSD was consistently female-biased in greater horseshoe bats, although female body size was only slightly larger than male body size. The slope of major axis regression of log10 (male) on log10 (female) was significantly different from 1. Forearm length for both sexes of greater horseshoe bats was significantly negatively correlated with latitude, and males displayed a slightly but nonsignificant steeper latitudinal cline in body size than females. We suggest that variation in patterns of SSD among greater horseshoe bat populations is consistent with Rensch’s rule indicating that males were the more variable sex. Males did not have a steeper body size–latitude relationship than females suggesting that sex-specific latitudinal variation in body size may not be an important contributing factor to Rensch’s rule. Future research on greater horseshoe bats might best focus on more comprehensive mechanisms driving the pattern of female-biased SSD variation.
PMCID: PMC3896443  PMID: 24465886
5.  Grateloupia tenuis Wang et Luan sp. nov. (Halymeniaceae, Rhodophyta): A New Species from South China Sea Based on Morphological Observation and rbcL Gene Sequences Analysis 
BioMed Research International  2013;2013:560163.
Grateloupia tenuis Wang et Luan sp. nov. is a new species described from Lingshui, Hainan Province, South China Sea. Based on the external form and internal structure, combined with rbcL gene sequence analysis, Grateloupia tenuis is distinct from other Grateloupia species as follows: (1) thalli is slippery and cartilaginous in texture; possess fewer branches, relatively slight main axes, and two or three dichotomous branches; (2) cortex is 5-6 layers; medulla is solid when young, but hollow in old branches; reproductive structures are dispersed in main axes of thalli and lower portions of branchlets; exhibits Grateloupia-type auxiliary cell ampullae; (3) the four studied G. tenuis sequences were positioned in a large Grateloupia clade of Halymeniaceae, which included sister group generitype G. filicina with 68 bp differences; G. tenuis was determined to be a sister taxon to the G. catenata, G. ramosissima, G. orientalis, and G. filiformis subclade. The pairwise distances between G. tenuis and these species were 39 to 50 bp. The sequences of G. tenuis differed by 81–108 bp from the sequences of other samples in Grateloupia; there are 114–133 bp changes between G. tenuis and other genera of Halymeniaceae. In final analysis, we considered Grateloupia tenuis Wang et Luan sp. nov. to be a new species of genus Grateloupia.
PMCID: PMC3885149  PMID: 24455703
6.  Tumor Evolution and Intratumor Heterogeneity of an Oropharyngeal Squamous Cell Carcinoma Revealed by Whole-Genome Sequencing12 
Neoplasia (New York, N.Y.)  2013;15(12):1371-1378.
Head and neck squamous cell carcinoma (HNSCC) is characterized by significant genomic instability that could lead to clonal diversity. Intratumor clonal heterogeneity has been proposed as a major attribute underlying tumor evolution, progression, and resistance to chemotherapy and radiation. Understanding genetic heterogeneity could lead to treatments specific to resistant and metastatic tumor cells. To characterize the degree of intratumor genetic heterogeneity within a single tumor, we performed whole-genome sequencing on three separate regions of an human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma and two separate regions from one corresponding cervical lymph node metastasis. This approach achieved coverage of approximately 97.9% of the genome across all samples. In total, 5701 somatic point mutations (SPMs) and 4347 small somatic insertions and deletions (indels)were detected in at least one sample. Ninety-two percent of SPMs and 77% of indels were validated in a second set of samples adjacent to the discovery set. All five tumor samples shared 41% of SPMs, 57% of the 1805 genes with SPMs, and 34 of 55 cancer genes. The distribution of SPMs allowed phylogenetic reconstruction of this tumor's evolutionary pathway and showed that the metastatic samples arose as a late event. The degree of intratumor heterogeneity showed that a single biopsy may not represent the entire mutational landscape of HNSCC tumors. This approach may be used to further characterize intratumor heterogeneity in more patients, and their sample-to-sample variations could reveal the evolutionary process of cancer cells, facilitate our understanding of tumorigenesis, and enable the development of novel targeted therapies.
PMCID: PMC3884528  PMID: 24403859
7.  Astragaloside IV Attenuates Experimental Autoimmune Encephalomyelitis of Mice by Counteracting Oxidative Stress at Multiple Levels 
PLoS ONE  2013;8(10):e76495.
Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI), a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE) in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS) as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.
PMCID: PMC3790693  PMID: 24124567
8.  Genome-Wide Analysis of ZmDREB Genes and Their Association with Natural Variation in Drought Tolerance at Seedling Stage of Zea mays L 
PLoS Genetics  2013;9(9):e1003790.
The worldwide production of maize (Zea mays L.) is frequently impacted by water scarcity and as a result, increased drought tolerance is a priority target in maize breeding programs. While DREB transcription factors have been demonstrated to play a central role in desiccation tolerance, whether or not natural sequence variations in these genes are associated with the phenotypic variability of this trait is largely unknown. In the present study, eighteen ZmDREB genes present in the maize B73 genome were cloned and systematically analyzed to determine their phylogenetic relationship, synteny with rice, maize and sorghum genomes; pattern of drought-responsive gene expression, and protein transactivation activity. Importantly, the association between the nucleic acid variation of each ZmDREB gene with drought tolerance was evaluated using a diverse population of maize consisting of 368 varieties from tropical and temperate regions. A significant association between the genetic variation of ZmDREB2.7 and drought tolerance at seedling stage was identified. Further analysis found that the DNA polymorphisms in the promoter region of ZmDREB2.7, but not the protein coding region itself, was associated with different levels of drought tolerance among maize varieties, likely due to distinct patterns of gene expression in response to drought stress. In vitro, protein-DNA binding assay demonstrated that ZmDREB2.7 protein could specifically interact with the target DNA sequences. The transgenic Arabidopsis overexpressing ZmDREB2.7 displayed enhanced tolerance to drought stress. Moreover, a favorable allele of ZmDREB2.7, identified in the drought-tolerant maize varieties, was effective in imparting plant tolerance to drought stress. Based upon these findings, we conclude that natural variation in the promoter of ZmDREB2.7 contributes to maize drought tolerance, and that the gene and its favorable allele may be an important genetic resource for the genetic improvement of drought tolerance in maize.
Author Summary
Water scarcity is one of the most severe threats to maize production worldwide. Although research has demonstrated that DREB-type transcription factors play important roles in plant water stress response, whether the specific genetic variants in DREB genes contribute to plant drought tolerance is largely unknown. Taking advantages of recent technical and methodological advance, we systematically analyzed all the functional DREB genes in maize and examined their associations with the natural variation in drought tolerance of 368 maize varieties collected from tropical and temperate regions. A significant association in the ZmDREB2.7 gene with drought tolerance was detected in that the DNA polymorphisms in the gene promoter region, but not those in the protein coding region, contributed to observed variations in maize drought tolerance, probably due to the distinct gene expression patterns in response to the stress. Overexpressing ZmDREB2.7 in Arabidopsis resulted in enhanced tolerance to drought stress. Moreover, a favorable ZmDREB2.7 allele, identified from drought-tolerant varieties, was effective in improving plant tolerance to drought stress when it was introduced into a drought-sensitive background. ZmDREB2.7 and its favorable allele represent a valuable genetic resource for enhancing maize drought tolerance by marker assisted breeding and transformation technology.
PMCID: PMC3784558  PMID: 24086146
9.  A peptide-mediated targeting gene delivery system for malignant glioma cells 
Glioblastoma multiforme (GBM) is the most common and malignant glioma. Although there has been considerable progress in treatment strategies, the prognosis of many patients with GBM remains poor. In this work, polyethylenimine (PEI) and the VTWTPQAWFQWV (VTW) peptide were modified and synthesized into GBM-targeting nanoparticles. The transfection efficiency of U-87 (human glioblastoma) cells was evaluated using fluorescence microscopy and flow cytometry. Cell internalization was investigated to verify the nanoparticle delivery into the cytoplasm. Results showed that the methods of polymer conjugation and the amount of VTW peptide were important factors to polymer synthesis and transfection. The PEI-VTW20 nanoparticles increased the transfection efficiency significantly. This report describes the use of VTW peptide-based PEI nanoparticles for intracellular gene delivery in a GBM cell-specific manner.
PMCID: PMC3790891  PMID: 24101872
glioblastoma; polyethylenimine; nanoparticles; drug-delivery systems; gene transfer techniques
10.  Correction: Epigenetic Regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA Methylation and Histone Modification 
PLoS ONE  2013;8(9):10.1371/annotation/1b1eb36e-60a4-4d28-bb3a-ee5a12a3d5d8.
PMCID: PMC3779267  PMID: 24086196
11.  Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in Subtype I-C/Dvulg CRISPR-Cas system 
Structure (London, England : 1993)  2012;20(9):1574-1584.
Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several Type I CRISPR-Cas systems. Here we report the molecular function of Subtype I-C/Dvulg Cas5d from B. halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3′ single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116 and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-subunit interference complex similar to E. coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among Type I CRISPR subtypes.
PMCID: PMC3479641  PMID: 22841292
12.  The Human Cathelicidin LL-37 Has Antiviral Activity against Respiratory Syncytial Virus 
PLoS ONE  2013;8(8):e73659.
Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.
PMCID: PMC3758310  PMID: 24023689
13.  Functional Comparison between Genes Dysregulated in Ulcerative Colitis and Colorectal Carcinoma 
PLoS ONE  2013;8(8):e71989.
Patients with ulcerative colitis (UC) are predisposed to colitis-associated colorectal cancer (CAC). However, the transcriptional mechanism of the transformation from UC to CAC is not fully understood.
Firstly, we showed that CAC and non-UC-associated CRC were very similar in gene expression. Secondly, based on multiple datasets for UC and CRC, we extracted differentially expressed (DE) genes in UC and CRC versus normal controls, respectively. Thirdly, we compared the dysregulation directions (upregulation or downregulation) between DE genes of UC and CRC in CRC-related functions overrepresented with the DE genes of CRC, and proposed a regulatory model to explain the CRC-like dysregulation of genes in UC. A case study for “positive regulation of immune system process” was done to reveal the functional implication of DE genes with reversal dysregulations in these two diseases.
Principal Findings
In all the 44 detected CRC-related functions except for “viral transcription”, the dysregulation directions of DE genes in UC were significantly similar with their counterparts in CRC, and such CRC-like dysregulation in UC could be regulated by transcription factors affected by pro-inflammatory stimuli for colitis. A small portion of genes in each CRC-related function were dysregulated in opposite directions in the two diseases. The case study showed that genes related to humoral immunity specifically expressed in B cells tended to be upregulated in UC but downregulated in CRC.
The CRC-like dysregulation of genes in CRC-related functions in UC patients provides hints for understanding the transcriptional basis for UC to CRC transition. A small portion of genes with distinct dysregulation directions in each of the CRC-related functions in the two diseases implicate that their reversal dysregulations might be critical for UC to CRC transition. The cases study indicates that the humoral immune response might be inhibited during the transformation from UC to CRC.
PMCID: PMC3750042  PMID: 23991021
14.  In vitro evaluation of 5-aminolevulinic acid (ALA) loaded PLGA nanoparticles 
5-Aminolevulinic acid (ALA) is a prodrug for topical photodynamic therapy. The effectiveness of topical ALA can be limited by its bioavailability. The aim of this study was to develop a novel ALA delivery approach using poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs).
A modified double emulsion solvent evaporation method was used to prepare ALA loaded PLGA NPs (ALA PLGA NPs). The characteristics, uptake, protoporphyrin IX fluorescence kinetics, and cytotoxicity of ALA PLGA NPs toward a human skin squamous cell carcinoma cell line were examined.
The mean particle size of spherical ALA PLGA NPs was 65.6 nm ± 26 nm with a polydispersity index of 0.62. The encapsulation efficiency was 65.8% ± 7.2% and ALA loading capacity was 0.62% ± 0.27%. When ALA was dispersed in PLGA NPs, it turned into an amorphous phase. ALA PLGA NPs could be taken up by squamous cell carcinoma cells and localized in the cytoplasm. The protoporphyrin IX fluorescence kinetics and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay showed that ALA PLGA NPs were more effective than free ALA of the same concentration.
PLGA NPs provide a promising ALA delivery strategy for topical ALA-photodynamic therapy of skin squamous cell carcinoma.
PMCID: PMC3728265  PMID: 23926429
5-Aminolevulinic acid (ALA); nanoparticles; poly(lactic-co-glycolic acid) (PLGA); skin squamous cell carcinoma; photodynamic therapy (PDT)
15.  A non-synonymous SNP within the isopentenyl transferase 2 locus is associated with kernel weight in Chinese maize inbreds (Zea mays L.) 
BMC Plant Biology  2013;13:98.
Kernel weight, controlled by quantitative trait loci (QTL), is an important component of grain yield in maize. Cytokinins (CKs) participate in determining grain morphology and final grain yield in crops. ZmIPT2, which is expressed mainly in the basal transfer cell layer, endosperm, and embryo during maize kernel development, encodes an isopentenyl transferase (IPT) that is involved in CK biosynthesis.
The coding region of ZmIPT2 was sequenced across a panel of 175 maize inbred lines that are currently used in Chinese maize breeding programs. Only 16 single nucleotide polymorphisms (SNPs) and seven haplotypes were detected among these inbred lines. Nucleotide diversity (π) within the ZmIPT2 window and coding region were 0.347 and 0.0047, respectively, and they were significantly lower than the mean nucleotide diversity value of 0.372 for maize Chromosome 2 (P < 0.01). Association mapping revealed that a single nucleotide change from cytosine (C) to thymine (T) in the ZmIPT2 coding region, which converted a proline residue into a serine residue, was significantly associated with hundred kernel weight (HKW) in three environments (P <0.05), and explained 4.76% of the total phenotypic variation. In vitro characterization suggests that the dimethylallyl diphospate (DMAPP) IPT activity of ZmIPT2-T is higher than that of ZmIPT2-C, as the amounts of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) consumed by ZmIPT2-T were 5.48-, 2.70-, and 1.87-fold, respectively, greater than those consumed by ZmIPT2-C. The effects of artificial selection on the ZmIPT2 coding region were evaluated using Tajima’s D tests across six subgroups of Chinese maize germplasm, with the most frequent favorable allele identified in subgroup PB (Partner B).
These results showed that ZmIPT2, which is associated with kernel weight, was subjected to artificial selection during the maize breeding process. ZmIPT2-T had higher IPT activity than ZmIPT2-C, and this favorable allele for kernel weight could be used in molecular marker-assisted selection for improvement of grain yield components in Chinese maize breeding programs.
PMCID: PMC3704264  PMID: 23826856
Maize; Isopentenyl transferase 2; Association mapping; Artificial selection
16.  The Correlation Analysis of Two Common Polymorphisms in STAT6 Gene and the Risk of Asthma: A Meta-Analysis 
PLoS ONE  2013;8(7):e67657.
Several studies have reported that the GT dinucleotide repeat length polymorphism and the G2964A polymorphism in signal transducer and activator of transcriptional factor 6 gene are associated with asthma susceptibility, but others have conflicting results. Our meta-analysis aimed to elucidate the emerging paradigms.
We searched PUBMED, EMBASE, ISI web of knowledge, Chinese National Knowledge Infrastructure, and Wanfang databases. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to evaluate the strength of association. We applied Bonferroni step-down and Benjamini-Hochberg step-up methods to adjust the values for multiple comparisons.
A total of 12 individual studies in 11 articles were included in the meta-analysis. For GT repeat polymorphism, the S allele had approximately 45% increased risk of asthma (S vs. L: OR = 1.45, 95% CI = 1.22–1.71, PUNCORRECTED <0.001, PBon <0.001, PFDR <0.001). Further analysis indicated that GT13 and GT14 contributed to asthma risk, whereas GT15 and GT16 were protective (GT13 vs. GT15: OR = 1.38, 95% CI = 1.16–1.65, PUNCORRECTED = 0.001, PBon = 0.005, PFDR = 0.002). Similar results were obtained in the subgroup analysis of Asian population. G2964A polymorphism analysis showed that the AA genotype moderately increased the risk of asthma by 47% compared with the GG genotype (OR = 1.47, p = 0.068) in Chinese population, whereas the 2964A allele moderately increased the risk of asthma in Chinese population by 18% (2964A vs. 2964G: OR = 1.18, p = 0.08). However, none of the associations reached statistically significant levels particularly after correction for multiple testing.
This meta-analysis suggests that S allele (GT13 and GT14) of the GT repeat polymorphism confers significant risks to asthma. However, the G2964A polymorphism does not have an association with the susceptibility to asthma.
PMCID: PMC3701693  PMID: 23861779
17.  Characteristics of patients with spinal tuberculosis: seven-year experience of a teaching hospital in Southwest China 
International Orthopaedics  2012;36(7):1429-1434.
The purpose of this study was to investigate the characteristics of patients managed for spinal tuberculosis at the orthopaedics department of a teaching hospital in Chongqing, China, between 2004 and 2010.
The study used a retrospective chart review. The epidemiology, clinical features, laboratory test results, imaging study findings, and treatment methods were recorded.
The annual incidence of spinal tuberculosis was stable throughout the study period. There were 284 patients, 147 women and 137 men, with a mean age of 38.2 years. The majority of the lesions involved the thoracic spine (45.3%), followed by the lumbar spine (45.0%). Multiple level skip lesions were seen in 5.6% of cases. The erythrocyte sedimentation rate was normal in 26.8% of patients. The C-reactive protein (CRP) was normal in 30.2% of patients. Type A and type O were the most common blood types. Neurological involvement was seen in 21.8% of patients. Concomitant tuberculosis of the lung was seen in 73 (25.7%). The patients with middle school education and above account for 60.4% (102/169) in rural patients and 68.7% (79/115) in urban patients. Mean time from symptom onset to diagnosis was 18.0 months (range, three days to 360 months), and there was a significant difference between the rural patients (23.0 months) and the urban patients (10.7 months) (p = 0.001, t = −3.300). Surgical treatment was performed in 233 patients (82.0%). The preferred surgical procedure was radical anterior debridement, bone grafting and internal fixation (132 patients, 46.5%). There were 13 patients (4.2%) with anti-tuberculous chemotherapy drug allergy or toxicity, streptomycin anaphylaxis and toxicity in 12, and isoniazide anaphylaxis and toxicity in one. No mortality was related to spinal TB.
The annual incidence of spinal tuberculosis remained unchanged throughout the study period and most of the patients did not pay much attention to the disease and received timely treatment. Thus, we should strengthen the census and treatment of spinal tuberculosis in Southwest China.
PMCID: PMC3385881  PMID: 22358176
18.  Solid and hollow pedicle screws affect the electrical resistance: A potential source of error with stimulus-evoked electromyography 
Indian Journal of Orthopaedics  2013;47(4):352-356.
Although stimulus evoked electromyography (EMG) is commonly used to confirm the accuracy of pedicle screw placement. There are no studies to differentiate between solid screws and hollow screws to the electrical resistance of pedicle screws. We speculate that the electrical resistance of the solid and hollow pedicle screws may be different and then a potential source of error with stimulus-evoked EMG may happen.
Materials and Methods:
Resistance measurements were obtained from 12 pedicle screw varieties (6 screws of each manufacturer) across the screw shank based on known constant current and measured voltage. The voltage was measured 5 times at each site.
Resistance of all solid screws ranged from 0.084 Ω to 0.151 Ω (mean =0.118 ± 0.024 Ω) and hollow screws ranged from 0.148 Ω to 0.402 Ω (mean = 0.285 ± 0.081 Ω). There was a significant difference of resistance between the solid screws and hollow screws (P < 0.05). The screw with the largest diameter no matter solid screws or hollow screws had lower resistance than screws with other diameters. No matter in solid screws group or hollow screws group, there were significant differences (P < 0.05) between the 5.0 mm screws and 6.0 mm screws, 6.0 mm screws and 7.0 mm screws, 5.0 mm screws and 7.0 mm screws, 4.5 mm screws and 5.5 mm screws, 5.5 mm screws and 6.5 mm screws, 4.5 mm screws and 6.5 mm screws. The resistance of hollow screws was much larger than the solid screws in the same diameter group (P < 0.05).
Hollow pedicle screws have the potential for high electrical resistance compared to the solid pedicle screws and therefore may affect the EMG response during stimulus-evoked EMG testing in pedicle screw fixation especially in minimally invasive percutaneous pedical screw fixation surgery.
PMCID: PMC3745688  PMID: 23960278
Electrical resistance; electromyography; pedicle screw; percutaneous pedicle screw
19.  Ginsenoside RK3 Prevents Hypoxia-Reoxygenation Induced Apoptosis in H9c2 Cardiomyocytes via AKT and MAPK Pathway 
Reperfusion therapy is widely utilized for acute myocardial infarction (AMI), but further injury induced by rapidly initiating reperfusion of the heart is often encountered in clinical practice. Ginsenoside RK3 (RK3) is reportedly present in the processed Radix notoginseng that is often used as a major ingredient of the compound preparation for ischemic heart diseases. This study aimed to investigate the possible protective effect of RK3 against hypoxia-reoxygenation (H/R) induced H9c2 cardiomyocytes damage and its underlying mechanisms. Our results showed that RK3 pretreatment caused increased cell viability and decreased levels of LDH leakage compared with the H/R group. Moreover, RK3 pretreatment inhibited cell apoptosis, as evidenced by decreased caspase-3 activity, TUNEL-positive cells, and Bax expression, as well as increased Bcl-2 level. Further mechanism investigation revealed that RK3 prevented H9c2 cardiomyocytes injury and apoptosis induced by H/R via AKT/Nrf-2/HO-1 and MAPK pathways. These observations indicate that RK3 has the potential to exert cardioprotective effects against H/R injury, which might be of great importance to clinical efficacy for AMI treatment.
PMCID: PMC3712237  PMID: 23935671
20.  Detection of Promyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARα) Fusion Gene with Functionalized Graphene Oxide 
An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable.
PMCID: PMC3709817  PMID: 23787474
PML; RARα fusion gene; graphene oxide; fluorescein isothiocyanate (FITC); laser confocal microscopy
21.  Isorhamnetin Protects against Doxorubicin-Induced Cardiotoxicity In Vivo and In Vitro 
PLoS ONE  2013;8(5):e64526.
Doxorubicin (Dox) is an anthracycline antibiotic for cancer therapy with limited usage due to cardiotoxicity. Isorhamnetin is a nature antioxidant with obvious cardiac protective effect. The aim of this study is going to investigate the possible protective effect of isorhamnetin against Dox-induced cardiotoxicity and its underlying mechanisms. In an in vivo investigation, rats were intraperitoneally (i.p.) administered with Dox to duplicate the model of Dox-induced chronic cardiotoxicity. Daily pretreatment with isorhamnetin (5 mg/kg, i.p.) for 7 days was found to reduce Dox-induced myocardial damage significantly, including the decline of cardiac index, decrease in the release of serum cardiac enzymes and amelioration of heart vacuolation. In vitro studies on H9c2 cardiomyocytes, isorhamnetin was effective to reduce Dox-induced cell toxicity. A further mechanism study indicated that isorhamnetin pretreatment can counteract Dox-induced oxidative stress and suppress the activation of mitochondrion apoptotic pathway and mitogen-activated protein kinase pathway. Isorhamnetin also potentiated the anti-cancer activity of Dox in MCF-7, HepG2 and Hep2 cells. These findings indicated that isorhamnetin can be used as an adjuvant therapy for the long-term clinical use of Dox.
PMCID: PMC3665796  PMID: 23724057
22.  Epigenetic Regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA Methylation and Histone Modification 
PLoS ONE  2013;8(4):e61423.
Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone formation through down-regulating BMP2 expression both in vivo and in vitro.
PMCID: PMC3631216  PMID: 23620751
23.  Gender-dependent miR-375 promoter methylation and the risk of type 2 diabetes 
Promoter DNA methylation may reflect the interaction between genetic background and environmental factors in the development of metabolic disorders, including type 2 diabetes (T2D). As an epigenetic factor of T2D, miR-375 plays an important role in the functional accommodation of islet cells. In the present study, we investigated the association of promoter DNA methylation of the miR-375 gene with the risk of T2D. Using bisulfite pyrosequencing technology, the DNA methylation levels of eight CpG dinucleotides on the miR-375 promoter were measured in 48 T2D cases and 48 healthy controls. The majority of CpGs (with the exception of CpG7) had significantly higher methylation levels in women compared with those in men (P<0.05). The methylation levels of the eight CpGs were significantly correlated with each other (P<0.001). No significant association between miR-375 gene promoter methylation and the risk of T2D was identified (P=0.417). Similar results were observed in the breakdown analysis by gender (men, P=0.844; women, P=0.234). In addition, although a correlation between the CpG8 methylation level of miR-375 and total triglyceride level was identified in women (P=0.009), DNA methylation of the majority of CpGs in the miR-375 gene promoter was not associated with the clinical metabolic features of the individuals.
PMCID: PMC3702700  PMID: 23837055
type 2 diabetes; miR-375; DNA methylation; promoter
24.  Clinical characteristics and treatment of angiomatous meningiomas: a report of 27 cases 
Angiomatous meningioma (AM) is a rare histological variant of meningioma. Twenty seven patients (14 male and 13 female) with angiomatous meningioma were treated in our institution. Their clinical presentation, neuroimaging studies, treatment and follow-up were investigated. The age of patients ranged from 24 to 72 years with a mean of 51.8 years. The clinical presentation was non-specific and depended on the location of the tumor and was mainly due to the mass effect. On computed tomography (CT) scanning, AMs showed slightly hyperintensity. On magnetic resonance imaging (MRI), AMs demonstrated hypointensity on T1-weighted images (T1WI), hyperintensity on T2-weighted images (T2WI), slight hypointensity on diffusion-weighted images (DWI), enhancement on postcontrast T1WI, peritumoral edema, and rich signal voids of vessels in the tumor. On histology, all tumors exhibited abundant blood vessels with at least focal classic meningothelial differentiation. Thirteen, eight, and six cases were achieved Simpson grade I, II and III-IV resection respectively. Nineteen cases were followed for 8 to 125 months with a mean of 47.9 months. Four patients with residual tumor were treated with postoperative radiation therapy and all of them had stable disease. One patient with Simpson grade II resection was not treated with radiation therapy and developed recurrent tumor in 5 years. In conclusion, angiomatous meningiomas have relative high male to female ratio, more frequent peritumoral edema, and rich blood vessels. Gross total resection is still the treatment of choice. These patients with residual tumor after surgery can benefit from radiation therapy. Overall, the prognosis of AMs are as good as other benign meningiomas.
PMCID: PMC3606859  PMID: 23573316
Angiomatous meningioma; computed tomography (CT); magnetic resonance imaging (MRI); surgery; radiation therapy
25.  The Mechanism of Effective Electroacupuncture on T Cell Response in Rats with Experimental Autoimmune Encephalomyelitis 
PLoS ONE  2013;8(1):e51573.
Previously, we demonstrated that electroacupuncture (EA) decreased lymphocyte infiltration into the spinal cords of rats presenting with experimental autoimmune encephalomyelitis (EAE), a disease model used in the study of multiple sclerosis (MS). The aim of this study was to characterize the effects of EA on the EAE. Female Lewis rats were divided into either CFA, EAE, EA, or injection with naloxone after electroacupuncture (NAL) groups. Electroacupuncture was administered every day for 21 days. To evaluate proliferation and apoptosis, lymphocytes from rats presenting with EAE were collected and cultured with β-endorphin. Immunohistochemisty, flow cytometry and radio-immunity methods were applied to detect the expression of β-endorphin. Results presented in this report demonstrate that the beneficial anti-inflammatory effects of EA on EAE were related to β-endorphin production that balances the Thl/Th2 and Th17/Treg responses. These results suggest that β-endorphin could be an important component in the development of EA-based therapies used for the treatment of EAE.
PMCID: PMC3557272  PMID: 23382807

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