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1.  Differential expression of circulating miRNAs in maternal plasma in pregnancies with fetal macrosomia 
Macrosomia is associated with problems at birth and has life-long health implications for the infant. The aim of this study was to profile the plasma microRNAs (miRNAs or miRs) and evaluate the potential of circulating miRNAs to predict fetal macrosomia. The expression levels of miRNAs in plasma samples obtained from pregnant women with fetal macrosomia and from women with normal pregnancies (controls) were analyzed using TaqMan Low-Density Arrays (TLDAs) followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) validation and analysis. The TLDA data revealed that 143 miRNAs were differentially expressed in the plasma samples from pregnant women with fetal macrosomia compared with the controls (43 upregulated and 100 downregulated miRNAs). Twelve of these miRNAs were selected for RT-qPCR analysis. Receiver operational characteristic (ROC) curve analysis indicated that several miRNAs (e.g., miR-141-3p and miR-200c-3p) were clearly distinguished between pregnancies with fetal macrosomia and other types of abnormal pregnancy and healthy pregnancies with high sensitivity and specificity (AUC >0.9). The expression of miRNA clusters also showed a similar trend in pregnancies with fetal macrosomia. This study provides a platform for profiling circulating miRNAs in maternal plasma. Our data also suggest that altered levels of maternal plasma miRNAs have great potential to serve as non-invasive biomarkers and as a mechanistic indicator of abnormal pregnancies.
PMCID: PMC4249743  PMID: 25370776
circulating microRNAs; low-density array; fetal macrosomia; abnormal pregnancy
2.  Genome-wide mapping of DNase I hypersensitive sites and association analysis with gene expression in MSB1 cells 
Frontiers in Genetics  2014;5:308.
DNase I hypersensitive sites (DHSs) mark diverse classes of cis-regulatory regions, such as promoters and enhancers. MSB-1 derived from chicken Marek's disease (MD) lymphomas is an MDV-transformed CD4+ T-cell line for MD study. Previously, DNase I HS sites were studied mainly in human cell types for mammalian. To capture the regulatory elements specific to MSB1 cells and explore the molecular mechanisms of T-cell transformation caused by MDV in MD, we generated high-quality of DHSs map and gene expression profile for functional analysis in MSB1 cell line. The total of 21,724 significant peaks of DHSs was identified from around 40 million short reads. DHSs distribution varied between chromosomes and they preferred to enrich in the gene-rich chromosomes. More interesting, DHSs enrichments appeared to be scarce on regions abundant in CpG islands. Besides, we integrated DHSs into the gene expression data and found that DHSs tended to enrich on high expressed genes throughout whole gene regions while DHSs did not show significant changes for low and silent expressed genes. Furthermore, the correlation of DHSs with lincRNAs expression was also calculated and it implied that enhancer-associated lincRNAs probably originated from enhancer-like regions of DHSs. Together, our results indicated that DNase I HS sites highly correlate with active genes expression in MSB1 cells, suggesting DHSs can be considered as markers to identify the cis-regulatory elements associated with chicken Marek's disease.
PMCID: PMC4195362  PMID: 25352859
DNase I; DHS; intergenic DHSs; MSB1; CpG islands; gene expressions; long non-coding RNAs; Marek's disease (MD)
3.  Fabrication of highly fluorescent graphene quantum dots using L-glutamic acid for in vitro/in vivo imaging and sensing 
A facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under the irradiation of ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range of 800–850 nm with the excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing significant advantage for sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as quantum yields, fluorescence life time, and photostability, were measured and the fluorescence quantum yield was as high as 54.5 %. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs is chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to the graphene sheets and carbon nanotubes. Coupled with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 μM.
PMCID: PMC3755467  PMID: 23997934
4.  Nrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-κB-mediated inflammatory response 
Kidney international  2013;85(2):333-343.
The generation of reactive oxygen species plays a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. Since the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2−/− mice was established using pristine injection. In this model, Nrf2−/− mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2−/− mice had higher expression of TGFβ1, fibronectin and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the NF-κB pathway and increased the level of reactive oxygen species, iNOS, TGFβ1 and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways.
PMCID: PMC3992978  PMID: 24025640
lupus nephritis; Nrf2; ROS; NF-κB; TGFβ1; iNOS
5.  Comparison of Next-Generation Sequencing and Clone-Based Sequencing in Analysis of Hepatitis B Virus Reverse Transcriptase Quasispecies Heterogeneity 
Journal of Clinical Microbiology  2013;51(12):4087-4094.
We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = −Σi(pilnpi)/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.
PMCID: PMC3838070  PMID: 24088859
6.  DNMT gene expression and methylome in Marek’s disease resistant and susceptible chickens prior to and following infection by MDV 
Epigenetics  2013;8(4):431-444.
Marek’s disease (MD) is characterized as a T cell lymphoma induced by a cell-associated α-herpesvirus, Marek’s disease virus type 1 (MDV1). As with many viral infectious diseases, DNA methylation variations were observed in the progression of MD; these variations are thought to play an important role in host-virus interactions. We observed that DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) were differentially expressed in chicken MD-resistant line 63 and MD-susceptible line 72 at 21 d after MDV infection. To better understand the role of methylation variation induced by MDV infection in both chicken lines, we mapped the genome-wide DNA methylation profiles in each line using Methyl-MAPS (methylation mapping analysis by paired-end sequencing). Collectively, the data sets collected in this study provide a more comprehensive picture of the chicken methylome. Overall, methylation levels were reduced in chickens from the resistant line 63 after MDV infection. We identified 11,512 infection-induced differential methylation regions (iDMRs). The number of iDMRs was larger in line 72 than in line 63, and most of iDMRs found in line 63 were overlapped with the iDMRs found in line 72. We further showed that in vitro methylation levels were associated with MDV replication, and found that MDV propagation in the infected cells was restricted by pharmacological inhibition of DNA methylation. Our results suggest that DNA methylation in the host may be associated with disease resistance or susceptibility. The methylation variations induced by viral infection may consequentially change the host transcriptome and result in diverse disease outcomes.
PMCID: PMC3674052  PMID: 23538681
DNA methylation; Marek’s disease; chicken; epigenetics; tumor; viral infection
7.  MAGEC2, an epithelial-mesenchymal transition inducer, is associated with breast cancer metastasis 
MAGEC2 is a member of melanoma antigen (MAGE) family of cancer-testis antigens and associated with tumor relapse and metastasis. Here, we investigated the expression of MAGEC2 in patients with breast cancer and its clinical effects with underlying mechanisms. The expression levels of MAGEC2 were compared between 420 invasive ductal carcinoma (IDC) and 120 ductal carcinoma in situ of the breast. Correlations between MAGEC2 expression and clinico-pathologic factors or survival of patients with IDC were analyzed. In addition, MAGEC2 expression levels in tumor tissues dissected from the primary focus and matched tumor-invaded axillary lymph nodes were analyzed in 8 breast cancer patients. The functional effects of MAGEC2 overexpression were assessed in vitro using scratch assay and transwell chamber assay. MAGEC2 expression was increased in metastatic breast cancer in comparison to the non-metastatic. MAGEC2 expression was significantly associated with ER negative expression (P = 0.037), high tumor grade (P = 0.014) and stage (P = 0.002), high incidence of axillary lymph node metastasis (P = 0.013), and distant metastasis (P = 0.004). Patients with tumor with MAGEC2 positive expression have a worse prognosis and a shorter metastasis free interval. Multivariate analyses showed that MAGEC2 expression was an independent risk factor for patient overall survival and metastasis-free survival. Breast cancer cells that overexpressed MAGEC2 had stronger migratory and invasive potential than control-treated cells. Epithelial markers (E-cadherin and cytokeratin) were down-regulated in MAGEC2-overexpressing cells compared to controls, whereas mesenchymal markers (vimentin and fibronectin) were upregulated. Our results indicate that MAGEC2 has a role in breast cancer metastasis through inducing epithelial-mesenchymal transition. In addition, MAGEC2 is a novel independent poor prognostic factor in patients with IDC. Thus, targeting MAGEC2 may provide a novel therapeutic strategy for breast cancer treatment.
PMCID: PMC3984411  PMID: 24687377
MAGEC2; Breast cancer; Metastasis; Clinical outcome; Epithelial-to-mesenchymal transition
8.  Immunologic cross-reactivity between Muscovy duck parvovirus and goose parvovirus on the basis of epitope prediction 
Brazilian Journal of Microbiology  2013;44(2):519-521.
Through bioinformatic prediction, between Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV), there were one epitope AA503–509 (RANEPKE) on non-structural protein and three epitopes AA426–430 (SQDLD), 540–544 (DPYRS), 685–691 (KENSKRW) on structural protein might cross-react with each other. Furthermore, the four epitops were expressed in Escherichia coli. All the four recombinant proteins could react with GPV-antisera and MDPV-antisera in Western blot.
PMCID: PMC3833156  PMID: 24294250
MDPV; GPV; cross-reactivity; prediction; epitope
9.  Recompression in new levels after percutaneous vertebroplasty and kyphoplasty compared with conservative treatment 
Study design
A prospective clinical study assessing new vertebral compression fracture after previous treatment.
The purpose of this study was to investigate the incidence and associated risk factors of new symptomatic osteoporotic vertebral compression fractures (OVCFs) in patients treated by percutaneous vertebroplasty (PVP) and kyphoplasty (PKP) versus conservative treatment, and to elucidate our findings.
Summary of background data
There are a lot of reports concerning the feasibility and efficacy of this minimally invasive procedure compared with conservative treatment, especially in pain soothing. However, it is still unclear whether the risk of subsequent fracture has increased among operative treatment patients in the long term.
From November 2005 to July 2009, 290 consecutive patients with 363 OVCFs were randomly selected for PVP/PKP or conservative treatment and evaluated with a mean follow-up of 49.4 months (36–80 months). Some parameters were characterized and statistically compared in this study. Telephone questionnaires, clinical reexamine, and plain radiographs were performed in the follow-up.
Thirty-one of 290 (10.7 %) patients had experienced 42 newly developed symptomatic secondary OVCFs. Among 169 operation (53.3 % vertebroplasty, 46.7 % kyphoplasty) and 121 comparison patients, there is no significant statistical difference of new OVCFs incidence between the two groups calculated by patient proportion. However, in separate, the rate of secondary adjacent fractures calculated by vertebral refracture number is significantly higher than non-adjacent levels in PVP/PKP group but no significant statistical difference was observed in conservative group. The time interval of recompression after operative procedure was much shorter than that for comparison group (9.7 ± 17.8 versus 22.4 ± 7.99 months, p = 0.017). In addition, older age, gender, fracture times, location of original fracture segment, the amount of cement, cement leakage, operation modality (PVP or PKP),and initial number of OVCFs were documented, but these were not the influencing factors in this study (p > 0.05).
Patients who had experienced PVP/PKP were not associated with an increased risk of recompression in new levels. However, recompression in new levels of PVP/PKP group occurred much sooner than that of conservative group in the follow-up period. The incidence of new vertebral fractures observed at adjacent levels was substantially higher but no sooner than at distant levels in PVP/PKP group. No major risk factors involving new OVCFs have been found in this study and  augmentation for sandwich situation is not necessary.
PMCID: PMC3889698  PMID: 24287674
Percutaneous vertebroplasty; Kyphoplasty; Adjacent fracture; Conservative treatment
10.  Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats 
Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.
PMCID: PMC3821587  PMID: 24084726
Achyranthes bidentata polypeptides; oxidative stress; myocardial ischemia/reperfusion; apoptosis
11.  Alpha-Linolenic Acid Exerts an Endothelial Protective Effect against High Glucose Injury via PI3K/Akt Pathway 
PLoS ONE  2013;8(7):e68489.
Mounting evidence has indicated that the cardiovascular protective effects of dietary alpha-linolenic acid (ALA), but whether ALA exerts an endothelial protective effect against high glucose injury and the underlying mechanisms remain largely unknown. Streptozocin-induced diabetic rats were randomized treated orally for 4 weeks with vehicle (0.01% alcohol) or ALA (500 µg/kg per day by gavage). Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (28 mmol/L) stimulation for 48 hours. ALA significantly improved concentration-dependent vasorelaxation to ACh in diabetic aortic segments and inhibited endothelial inflammation as evidenced by decreased soluble P-selectin and intercellular adhesion molecule-1 (ICAM-1) in diabetic rats. Furthermore, both P-selectin and ICAM-1 expression were increased significantly in high glucose-induced HUVECs, resulting in enhanced neutrophils adhesion to HUVECs compared with normal glucose group. Treatment with ALA (50 µmol/L) increased Akt phosphorylation, attenuated P-selectin and ICAM-1 expressions and thus inhibited neutrophils adhesion in HUVECs exposed to high glucose, all of which was blocked by the PI3K inhibitors LY294002 and wortmannin. These data indicates that ALA inhibits endothelial inflammation and improved endothelial function in STZ-induced diabetic rats. The anti-adhesive effect of ALA against high glucose injury may partially be mediated by the PI3K/Akt pathway.
PMCID: PMC3702615  PMID: 23861910
12.  Compressed carbon nanotubes: A family of new multifunctional carbon allotropes 
Scientific Reports  2013;3:1331.
The exploration of novel functional carbon polymorphs is an enduring topic of scientific investigations. In this paper, we present simulations demonstrating metastable carbon phases as the result of pressure induced carbon nanotube polymerization. The configuration, bonding, electronic, and mechanical characteristics of carbon polymers strongly depend on the imposed hydrostatic/non-hydrostatic pressure, as well as on the geometry of the raw carbon nanotubes including diameter, chirality, stacking manner, and wall number. Especially, transition processes under hydrostatic/non-hydrostatic pressure are investigated, revealing unexpectedly low transition barriers and demonstrating sp2→sp3 bonding changes as well as peculiar oscillations of electronic property (e.g., semiconducting→metallic→semiconducting transitions). These polymerized nanotubes show versatile and superior physical properties, such as superhardness, high tensile strength and ductility, and tunable electronic properties (semiconducting or metallic).
PMCID: PMC3580323  PMID: 23435585
13.  Long-term effects of a combination of D-penicillamine and zinc salts in the treatment of Wilson’s disease in children 
The aim of this study was to investigate the effectiveness of a high-dose zinc sulfate and low-dose D-penicillamine combination in the treatment of pediatric Wilson’s disease (WD). A retropective chart review of 65 patients with WD was conducted. These patients received D-penicillamine (8–10 mg/kg/day) and zinc sulfate as the primary treatment. The pediatric dose of elemental zinc is 68–85 mg/day until 6 years of age, 85–136 mg/day until 8 years of age, 136–170 mg/day until 10 years of age and then 170 mg/day, in 3 divided doses 1 h before meals. After clinical and biochemical improvement or stabilization, zinc sulfate alone was administered as the maintenance therapy. Under treatment, the majority of patients (89.2%) had a favourable outcome and 3 patients succumbed due to poor therapy compliance. No penicillamine-induced neurological deterioration was noted and side-effects were observed in <11% of patients over the entire follow-up period. Benefical results on the liver and neurological symptoms were reported following extremely long-term treatment with a combination of low-dose D-penicillamine and high-dose zinc sulfate. Therefore, this regimen is an effective and safe treatment for children with WD.
PMCID: PMC3628594  PMID: 23599735
Wilson’s disease; D-penicillamine; zinc sulfate; child
14.  Ganoderma lucidum polysaccharides counteract inhibition on CD71 and FasL expression by culture supernatant of B16F10 cells upon lymphocyte activation 
Immune responses to tumor-associated antigens are often detectable in tumor-bearing hosts, but they fail to eliminate malignant cells or prevent development of metastases. Tumor cells produce factors such as interleukin-10, transforming growth factor-β1 and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. Culture supernatant of tumor cells may contain these immunosuppressive factors which suppress lymphocyte activation. CD71 and FasL are two important molecules that are expressed upon lymphocyte activation. Counteraction against suppression CD71 and FasL expression upon lymphocyte activation may benefit tumor control. A potential component with this effect is Ganoderma lucidum polysaccharides (Gl-PS). In this study, Gl-PS was used on lymphocytes incubating with culture supernatant of B16F10 melanoma cells (B16F10-CS) in the presence of phytohemagglutinin. Following induction with phytohemagglutinin, B16F10-CS suppressed CD71 expression in lymphocytes (as detected by immunofluorescence and flow cytometry), proliferation in lymphocytes (as detected by MTT assay), and FasL expression in lymphocytes (as detected by immunocytochemistry and western blot analysis), while Gl-PS fully or partially counteracted these suppressions. Gl-PS showed counteractive effects against suppression induced by B16F10-CS on CD71 and FasL expression upon lymphocyte activation, suggesting the potential of Gl-PS to facilitate cancer immunotherapy.
PMCID: PMC3628224  PMID: 23596479
tumor; lymphocyte activation; Ganoderma lucidum; polysaccharides; FasL; CD71
15.  Can MicroRNAs Improve the Management of Lung Cancer Patients? A Clinician's Perspective 
Theranostics  2013;3(12):953-963.
The treatment of patients with lung cancer is increasingly individualised. Rather than treating lung cancer as a single disease, clinicians are often called upon to consider the precise histology and molecular biology of each tumour in addition to the individual characteristics of each patient. Paralleling advances in lung cancer management, advances in the detection of lung cancer are changing practice. Lung cancer screening promises to find disease at a curable stage; however, the high false positive rate in screening trials has clinical and fiscal ramifications which demand attention. Biomarkers able to stratify for the risk of cancer, prognosticate the course of disease, or predict the response to treatment are in increasing demand. This paper summarizes some of the clinical problems faced by those treating lung cancer patients, and examines how knowledge about the role of microRNAs in lung cancer biology may change patient management.
PMCID: PMC3881097  PMID: 24396506
biomarker; microRNA; lung cancer; thoracic oncology; screening; early detection; tumor biology; prognostic marker; predictive marker.
16.  Histone Methylation Analysis and Pathway Predictions in Chickens after MDV Infection 
PLoS ONE  2012;7(7):e41849.
Marek's disease (MD) is a lymphoproliferative disease in chicken induced by Marek's disease virus (MDV). Although studies have focused on the genetic differences between the resistant and susceptible chicken, less is known about the role of epigenetic factors in MD. In this study, genome-wide histone modifications in the non-MHC-associated resistant and susceptible chicken lines were examined. We found that tri-methylation at histone H3 Lys4 (H3K4me3) enrichment is positively correlated with the expression of protein coding genes as well as microRNA (miRNA) genes, whereas tri-methylation at histone H3 Lys27 (H3K27me3) exhibits a negative correlation. By identifying line-specific histone modifications in MDV infection, we found unique H3K4me3 islands in the resistant chicken activated genes, which are related to immune response and cell adhesion. Interestingly, we also found some miRNAs from unique H3K27me3 patterns in the susceptible chickens that targeted genes involved in 5-hydroxytryptamine (5-HT)-receptor and adrenergic receptor pathways. In conclusion, dynamic line-specific histone modifications in response to MDV infection suggested that intrinsic epigenetic mechanisms may play a role in MD-resistance and -susceptibility.
PMCID: PMC3406056  PMID: 22848633
17.  miRNA-dysregulation associated with tenderness variation induced by acute stress in Angus cattle 
miRNAs are a class of small, single-stranded, non-coding RNAs that perform post-transcriptional repression of target genes by binding to 3’ untranslated regions. Research has found that miRNAs involved in the regulation of many metabolic processes. Here we uncovered that the beef quality of Angus cattle sharply diversified after acute stress. By performing miRNA microarray analysis, 13 miRNAs were significantly differentially expressed in stressed group compared to control group. Using a bioinformatics method, 135 protein-coding genes were predicted as the targets of significant differentially expressed miRNAs. Gene Ontology (GO) term and Ingenuity Pathway Analysis (IPA) mined that these target genes involved in some important pathways, which may have impact on meat quality and beef tenderness.
PMCID: PMC3436641  PMID: 22958451
miRNA; Bovine; Beef tenderness; Stress
18.  Functional Genomic Analysis of Variation on Beef Tenderness Induced by Acute Stress in Angus Cattle 
Beef is one of the leading sources of protein, B vitamins, iron, and zinc in human food. Beef palatability is based on three general criteria: tenderness, juiciness, and flavor, of which tenderness is thought to be the most important factor. In this study, we found that beef tenderness, measured by the Warner-Bratzler shear force (WBSF), was dramatically increased by acute stress. Microarray analysis and qPCR identified a variety of genes that were differentially expressed. Pathway analysis showed that these genes were involved in immune response and regulation of metabolism process as activators or repressors. Further analysis identified that these changes may be related with CpG methylation of several genes. Therefore, the results from this study provide an enhanced understanding of the mechanisms that genetic and epigenetic regulations control meat quality and beef tenderness.
PMCID: PMC3332163  PMID: 22566754
19.  DNA Methylation Fluctuation Induced by Virus Infection Differs between MD-resistant and -susceptible Chickens 
Marek’s disease (MD) is a lymphoproliferative disease induced by Marek’s disease virus (MDV) infection. To augment vaccination measures in MD control, host genetic resistant to MD becomes obviously more and more important. To elucidate the mechanism of MD-resistance, most of researches were focused on the genetic differences between resistant and susceptible chickens. However, epigenetic features between MD resistant and susceptible chickens are poorly characterized. Using bisulfite pyrosequencing method, we found some candidate genes have higher promoter methylation in the MD-susceptible (L72) chickens than in the MD-resistant (L63) chickens. The hypermethylated genes, involved in cellular component organization, responding to stimulus, cell adhesion, and immune system process, may play important role in susceptibility to disease by deregulation of these genes. MDV infection induced the expression changes of all three methyltransferases genes (DNMT1, DNMT3a, and DNMT3b) in both lines of chickens. The DNMT1 was up-regulated in L72, whereas the DNMT3b was down-regulated in L63 at 21 dpi. Interestingly, a dynamic change of promoter methylation was observed during MDV life cycle. Some genes, including HDAC9, GH, STAT1, CIITA, FABP3, LATS2, and H2Ac, showed differential methylation behaviors between the two lines of chickens. In summary, the findings from this study suggested that DNA methylation heterogeneity and MDV infection induced methylation alterations differences existed between the two lines of chickens. Therefore, it is suggested that epigenetic mechanisms may be involved in modulating the resistance and/or susceptibility to MD in chickens.
PMCID: PMC3281210  PMID: 22363343
chicken; Marek’s disease; MD-resistance; MD-susceptibility; DNA methylation
20.  Temporal transcriptome changes induced by MDV in marek's disease-resistant and -susceptible inbred chickens 
BMC Genomics  2011;12:501.
Marek's disease (MD) is a lymphoproliferative disease in chickens caused by Marek's disease virus (MDV) and characterized by T cell lymphoma and infiltration of lymphoid cells into various organs such as liver, spleen, peripheral nerves and muscle. Resistance to MD and disease risk have long been thought to be influenced both by genetic and environmental factors, the combination of which contributes to the observed outcome in an individual. We hypothesize that after MDV infection, genes related to MD-resistance or -susceptibility may exhibit different trends in transcriptional activity in chicken lines having a varying degree of resistance to MD.
In order to study the mechanisms of resistance and susceptibility to MD, we performed genome-wide temporal expression analysis in spleen tissues from MD-resistant line 63, susceptible line 72 and recombinant congenic strain M (RCS-M) that has a phenotype intermediate between lines 63 and 72 after MDV infection. Three time points of the MDV life cycle in chicken were selected for study: 5 days post infection (dpi), 10dpi and 21dpi, representing the early cytolytic, latent and late cytolytic stages, respectively. We observed similar gene expression profiles at the three time points in line 63 and RCS-M chickens that are both different from line 72. Pathway analysis using Ingenuity Pathway Analysis (IPA) showed that MDV can broadly influence the chickens irrespective of whether they are resistant or susceptible to MD. However, some pathways like cardiac arrhythmia and cardiovascular disease were found to be affected only in line 72; while some networks related to cell-mediated immune response and antigen presentation were enriched only in line 63 and RCS-M. We identified 78 and 30 candidate genes associated with MD resistance, at 10 and 21dpi respectively, by considering genes having the same trend of expression change after MDV infection in lines 63 and RCS-M. On the other hand, by considering genes with the same trend of expression change after MDV infection in lines 72 and RCS-M, we identified 78 and 43 genes at 10 and 21dpi, respectively, which may be associated with MD-susceptibility.
By testing temporal transcriptome changes using three representative chicken lines with different resistance to MD, we identified 108 candidate genes for MD-resistance and 121 candidate genes for MD-susceptibility over the three time points. Genes included in our resistance or susceptibility genes lists that are also involved in more than 5 biofunctions, such as CD8α, IL8, USP18, and CTLA4, are considered to be important genes involved in MD-resistance or -susceptibility. We were also able to identify several biofunctions related with immune response that we believe play an important role in MD-resistance.
PMCID: PMC3269463  PMID: 21992110
21.  Differential Gene Expression by RamA in Ciprofloxacin-Resistant Salmonella Typhimurium 
PLoS ONE  2011;6(7):e22161.
Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways.
PMCID: PMC3139621  PMID: 21811569
22.  Bacterial diversity of subgingival plaque in 6 healthy Chinese individuals 
The subgingival microbial ecology is complex, and little is known regarding its bacteria species composition in healthy Chinese individuals. This study aimed to identify the subgingival microbiota from 6 healthy Chinese subjects. Subgingival samples from 6 volunteers were collected, the 16S rRNA gene was amplified using broad-range bacterial primers, and clone libraries were constructed. For the initial 2,439 sequences analyzed, 383 species-level operational taxonomic units (SLOTUs) belonging to seven phyla were identified, estimated as 51% [95% confidence interval (CI) 44–55] of the SLOTUs in this ecosystem. Most (85%) of the bacterial sequences, falling into 228 types of species, corresponded to known and cultivated species. However, 146 (6%) sequences, comprising 104 phylotypes, had <97% similarity to prior database sequences. Ten bacterial genera were conserved among all 6 individuals, comprising 2,000 (82%) of the 2,439 clones analyzed. Ten species were noted in all of the 6 subjects, comprising 1,435 (58.8%) of the 2,439 clones. Streptococcus infantis was the species most frequently cloned. Furthermore, certain species which may participate in the pathogenesis of periodontal disease were present in the 6 subjects. Although the initial subgingival plaque community of each subject was unique in terms of diversity and composition, 10 common key species were found in the 6 Chinese individuals. These ten species of bacteria in the human subgingival plaque in the 6 healthy individuals may be key species which, to some extent, affect periodontal health. Destruction of these key species in subgingival bacteria may break the microbiota balance and may easily lead to over-breeding conditions resulting in pathogenic oral disease.
PMCID: PMC3440835  PMID: 22977615
subgingival plaque; microbiota; microbial diversity; small subunit rRNA genes; healthy individuals
23.  Down-regulation of promoter methylation level of CD4 gene after MDV infection in MD-susceptible chicken line 
BMC Proceedings  2011;5(Suppl 4):S7.
Marek’s disease virus (MDV) is an oncovirus that induces lymphoid tumors in susceptible chickens, and may affect the epigenetic stability of the CD4 gene. The purpose of this study was to find the effect of MDV infection on DNA methylation status of the CD4 gene differed between MD-resistant (L63) and –susceptible (L72) chicken lines.
Chickens from each line were divided into two groups with one group infected by MDV and the other group as uninfected controls. Then, promoter DNA methylation levels of the CD4 gene were measured by Pyrosequencing; and gene expression analysis was performed by quantitative PCR.
Promoter methylation of the CD4 gene was found to be down-regulated in L72 chickens only after MDV infection. The methylation down-regulation of the CD4 promoter is negatively correlated with up-regulation of CD4 gene expression in the L72 spleen at 21 dpi.
The methylation fluctuation and mRNA expression change of CD4 gene induced by MDV infection suggested a unique epigenetic mechanism existed in MD-susceptible chickens.
PMCID: PMC3108237  PMID: 21645322
24.  Estimation of HIV-1 incidence among five focal populations in Dehong, Yunnan: a hard hit area along a major drug trafficking route 
BMC Public Health  2010;10:180.
Since 1989 when the first 146 HIV positives in China were identified, Dehong Prefecture had been one of the areas hardest-hit by HIV in China. The local and national governments have put substantial financial resources into tackling the HIV epidemic in Dehong from 2004. The objective of this study was to track dynamic changes in HIV-1 prevalence and incidence among five focal populations in Dehong and to assess the impact of HIV prevention and control efforts.
Consecutive cross-sectional surveys conducted in five focal populations between 2004 and 2008. Specimens seropositive for HIV were tested with the BED IgG capture enzyme immunoassay to identify recent seroconversions (median, 155 days) using normalized optical density of 0.8 and adjustments.
From 2004 to 2008, estimated annual HIV incidence among injecting drug users (IDUs) decreased significantly [from 15.0% (95% CI = 11.4%-18.5%) in 2004 to 4.3% (95% CI = 2.4%-6.2%) in 2008; trend test P < 0.0001]. The incidence among other focal populations, such as HIV discordant couples (varying from 5.5% to 4.7%), female sex workers (varying from 1.4% to 1.3%), pregnant women (0.1%), and pre-marital couples (0.2 to 0.1%) remained stable. Overall, the proportion of recent HIV-1 infections was higher among females than males (P < 0.0001).
The HIV epidemic in Dehong continued to expand during a five-year period but at a slowing rate among IDUs, and HIV incidence remains high among IDUs and discordant couples. Intensive prevention measures should target sub-groups at highest risk to further slow the epidemic and control the migration of HIV to other areas of China, and multivariate analysis is needed to explore which measures are more effective for different populations.
PMCID: PMC2858119  PMID: 20374618
25.  Calibration of Mutation Rates Reveals Diverse Subfamily Structure of Galliform CR1 Repeats 
Chicken Repeat 1 (CR1) repeats are the most abundant family of repeats in the chicken genome, with more than 200,000 copies accounting for ∼80% of the chicken interspersed repeats. CR1 repeats are believed to have arisen from the retrotransposition of a small number of master elements, which gave rise to the 22 CR1 subfamilies as previously reported in Repbase. We performed a global assessment of the divergence distributions, phylogenies, and consensus sequences of CR1 repeats in the chicken genome. We identified and validated 57 chicken CR1 subfamilies and further analyzed the correlation between these subfamilies and their regional GC contents. We also discovered one novel lineage-specific CR1 subfamilies in turkeys when compared with chickens. We built an evolutionary tree of these subfamilies and concluded that CR1 repeats may play an important role in reshaping the structure of bird genomes.
PMCID: PMC2817409  PMID: 20333183
CR1 repeats; comparative genomics; chicken genome

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