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1.  Changes of HMGB1 and sRAGE during the recovery of COPD exacerbation 
Journal of Thoracic Disease  2014;6(6):734-741.
Background
Acute exacerbation of chronic obstructive pulmonary disease is associated with increased airway and systemic inflammation. However, the correlation between acute exacerbation/convalescence of chronic obstructive pulmonary disease (COPD) and simultaneous changes of high mobility group protein B1 (HMGB1) and soluble RAGE (sRAGE) levels has not been clearly clarified. The aim of this study was to assess these issues.
Methods
A total of 44 COPD patients were recruited. Following a structured interview, plasma levels of HMGB1, sRAGE, fibrinogen and serum level of high-sensitivity C-reactive protein (hsCRP) were measured in patients with acute exacerbation of COPD (AECOPD) within 24 h of hospitalization and pre-discharge (convalescence). All patients were examined with spirometry in convalescence of COPD.
Results
There was a significant decline in plasma HMGB1 (P<0.01), sRAGE (P<0.05), fibrinogen (P<0.01) and serum hsCRP (P<0.01) levels from acute exacerbation to convalescence phase of COPD. Changes of sRAGE was significantly correlated with changes of HMGB1 (r=0.4, P=0.007). COPD disease status correlated with the ratio of HMGB1/sRAGE, but not gender, age, course of disease, smoking history and FEV1% pred. Levels of HMGB1 and sRAGE were the highest in the current smoker group, and significantly decreased in ex-smoker group in both acute exacerbation and convalescence phase of COPD, however, their levels in never smoker group were higher than ex-smoker group in either phase of COPD.
Conclusions
HMGB1 and sRAGE levels were dynamically changed between exacerbation and convalescence phase of COPD, HMGB1 and sRAGE were likely not only a potential marker in COPD exacerbation but also a therapeutic target for COPD treatment.
doi:10.3978/j.issn.2072-1439.2014.04.31
PMCID: PMC4073385  PMID: 24976997
Chronic obstructive pulmonary disease (COPD); high mobility group protein B1 (HMGB1); soluble RAGE (sRAGE); biomarker; exacerbation; convalescence
2.  Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein 
BioMed Research International  2014;2014:368581.
Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.
doi:10.1155/2014/368581
PMCID: PMC4034427  PMID: 24895571
3.  A preliminary investigation on the relationship between virtues and pathological internet use among Chinese adolescents 
Background
Pathological Internet Use (PIU) has become a global issue associated with the increasing number of Internet users. Previous studies concerned both the interpersonal and intrapersonal vulnerable factors and the corresponding models. However, a limited amount of research has explored the relationship between positive factors and PIU.
Objective
The current investigation attempted to clarify the relationship between virtues and PIU among Chinese adolescents; it also sought to explore the specific contributions of the three virtues. Virtue was the core concept in positive psychology and the Values in Action Classification. A recent study demonstrated that there might be three universal virtues (relationship, vitality, and conscientiousness).
Methods
A cross-sectional sample of adolescents aged 12-17 years were recruited in 2013. A total of 674 adolescents (males = 302, females = 372; junior high school = 296, senior high school = 378) from eight junior and senior high schools in four provinces of Mainland China completed a package of psychological inventories, including the Chinese Virtues Questionnaire (CVQ) and the Adolescent Pathological Internet Use Scale (APIUS). The mean age of the current sample was 15.10 years (SD = 1.81) with an average of 5.31 years’ length (SD = 2.09) of Internet use.
Results
A total of 9.50% participants exhibited significant symptoms of PIU. Male students (Mmale = 2.50) had significantly higher scores on PIU than female students (Mfemale = 2.25). Relationship (β = -.24) and conscientiousness (β = -.21) negatively predicted PIU, whereas vitality (β = .25) positively predicted PIU. Dominance analysis further revealed that relationship and conscientiousness could explain 81% variance of PIU, and vitality only accounted for another 19%.
Conclusions
Relationship and conscientiousness were possible protective factors of pathological Internet users, while vitality was vulnerable. The results could be helpful in screening “at-risk” Internet users (low relationship and conscientiousness as well as high vitality). Future intervention strategies could focus on how to enhance relationship and conscientiousness and on how to reduce vitality.
doi:10.1186/1753-2000-8-8
PMCID: PMC3995982  PMID: 24594317
Virtue; Pathological Internet use; Vitality; Conscientiousness; Relationship
4.  Association of Biomarkers of Inflammation with Dyslipidemia and Its Components among Mongolians in China 
PLoS ONE  2014;9(2):e89023.
Objective
This study aims to examine the association between inflammatory biomarkers and dyslipidemia and its components among Mongolians in China.
Methods
Data were obtained from 2544 Mongolians via standard questionnaires and blood samples in Inner Mongolia, China. High sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) as well as blood lipids were examined.
Results
Individuals with dyslipidemia had higher levels of hsCRP, sICAM-1 and sE-selectin than those without dyslipidemia (all P values<0.001). Compared to the lowest quartile of inflammatory biomarkers, individuals with the highest quartile were more likely to have dyslipidemia (odds ratio, 95% confidence interval: 3.215, 2.551–4.116 for hsCRP; 1.575, 1.253–1.980 for sICAM-1; 1.495, 1.193–1.873 for sE-selectin). Moreover, hsCRP was associated with all the components of dyslipidemia, whereas, sICAM-1 was not related to high density lipoprotein cholesterol (HDL-c) or triglycerides (TAG). Additionally, sE-selectin was just associated with TAG.
Conclusion
Our study indicated that elevated plasma levels of hsCRP, sICAM-1 and sE-selectin were positively and significantly associated with increased risk of dyslipidemia among Mongolians. However, the associations were not identical for different inflammatory biomarkers with the components of dyslipidemia.
doi:10.1371/journal.pone.0089023
PMCID: PMC3928392  PMID: 24558466
5.  Glycerol Affects Root Development through Regulation of Multiple Pathways in Arabidopsis 
PLoS ONE  2014;9(1):e86269.
Glycerol metabolism has been well studied biochemically. However, the means by which glycerol functions in plant development is not well understood. This study aimed to investigate the mechanism underlying the effects of glycerol on root development in Arabidopsis thaliana. Exogenous glycerol inhibited primary root growth and altered lateral root development in wild-type plants. These phenotypes appeared concurrently with increased endogenous glycerol-3-phosphate (G3P) and H2O2 contents in seedlings, and decreased phosphate levels in roots. Upon glycerol treatment, G3P level and root development did not change in glycerol kinase mutant gli1, but G3P level increased in gpdhc1 and fad-gpdh mutants, which resulted in more severely impaired root development. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H2O2 levels, leading to increased tolerance to exogenous glycerol, which suggested that FAD-GPDH plays an important role in modulating this response. Free indole-3-acetic acid (IAA) content increased by 46%, and DR5pro::GUS staining increased in the stele cells of the root meristem under glycerol treatment, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1 and PIN7 expression, β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) fluorescence in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1 and ARF7 were involved in regulating root growth under glycerol treatment. Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1 decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. Our data demonstrated that glycerol treatment altered endogenous levels of G3P, phosphate and ROS, affected auxin distribution and cell division in the root meristem, and eventually resulted in modifications of root development.
doi:10.1371/journal.pone.0086269
PMCID: PMC3899222  PMID: 24465999
6.  Association between Human Urotensin II and Essential Hypertension—A 1:1 Matched Case-Control Study 
PLoS ONE  2013;8(12):e81764.
Objective
We aimed to evaluate the controversial association between human urotensin II and essential hypertension in untreated hypertensive cases and normotensive controls.
Methods
197 newly diagnosed hypertensive patients and 197 age- and sex-matched normotensive controls were studied. Plasma urotensin II, nitric oxide metabolites, and other traditional biomarkers were examined.
Results
Hypertensive patients had higher urotensin II [median (interquartile rang): 9.32 (7.86–11.52) ng/mL vs 8.52 (7.07–10.41) ng/mL] and lower nitric oxide metabolites [19.19 (2.55–38.48) µmol/L vs 23.83 (11.97–43.40) µmol/L] than normotensive controls. Urotensin II was positively correlated with systolic blood pressure (r = 0.169, P<0.001) and diastolic blood pressure (r = 0.113, P = 0.024) while negatively correlated with nitric oxide metabolites (r = −0.112, P = 0.027). In multivariate regression analysis, subjects in the highest quartile of urotensin II were more likely to have hypertension than those in the lowest quartile (OR, 2.58; 95% CI, 1.21–5.49). Sub-group analyses in 106 pairs of cases and controls with either both normal or both abnormal nitric oxide metabolites levels showed that the association between urotensin II levels and hypertension persisted (P value for trend = 0.039).
Conclusion
Human urotensin II is markedly associated with essential hypertension, and the association is independent of nitric oxide metabolites. Our results indicated that urotensin II might be an independent risk factor for essential hypertension.
doi:10.1371/journal.pone.0081764
PMCID: PMC3858253  PMID: 24339964
7.  Nucleosome-binding protein HMGN2 exhibits antitumor activity in oral squamous cell carcinoma 
Oncology Letters  2013;7(1):115-120.
Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) serve as effectors in the antitumor response. High mobility group nucleosomal binding domain 2 (HMGN2) is a candidate effector molecule involved in CTL and NK cell function. In the current study, recombinant human HMGN2 was isolated and purified from transformed Escherichia coli. Tca8113 cells, an oral squamous cell carcinoma line, were treated with a variety of HMGN2 protein concentrations and cell growth was analyzed. HMGN2 significantly inhibited the growth of Tca8113 cells and was predicted to arrest cells in the S phase. Moreover, HMGN2 treatment increased the apoptosis rate of Tca8113 cells. Western blotting indicated the upregulation of p53 and Bax proteins, whereas Bcl-2 was significantly downregulated. In addition, caspase-3 was found to be activated. Furthermore, the HMGN2 protein may suppress the growth of Tca8113 cells in vivo. The results of the current study indicated that the HMGN2 protein may inhibit the growth of oral squamous cell carcinoma and HMGN2 may represent an antitumor effector molecule of CTL or NK cells.
doi:10.3892/ol.2013.1665
PMCID: PMC3861564  PMID: 24348831
HMGN2; oral squamous cell carcinoma; apoptosis; antitumor activity; cell cycle
8.  JAK1 truncating mutations in gynecologic cancer define new role of cancer-associated protein tyrosine kinase aberrations 
Scientific Reports  2013;3:3042.
Cancer-associated protein tyrosine kinase (PTK) mutations usually are gain-of-function (GOF) mutations that drive tumor growth and metastasis. We have found 50 JAK1 truncating mutations in 36 of 635 gynecologic tumors in the Total Cancer Care® (TCC®) tumor bank. Among cancer cell lines containing JAK1 truncating mutations in the Cancer Cell Line Encyclopedia databank, 68% are gynecologic cancer cells. Within JAK1 the K142, P430, and K860 frame-shift mutations were identified as hot spot mutation sites. Sanger sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed the JAK1 mutations and showed that these mutations are somatic. JAK1 mediates interferon (IFN)-γ-regulated tumor immune surveillance. Functional assays show that JAK1 deficient cancer cells are defective in IFN-γ-induced LMP2 and TAP1 expression, loss of which inhibits presentation of tumor antigens. These findings identify recurrent JAK1 truncating mutations that could contribute to tumor immune evasion in gynecologic cancers, especially in endometrial cancer.
doi:10.1038/srep03042
PMCID: PMC3807107  PMID: 24154688
9.  microRNA-449a functions as a tumor-suppressor in gastric adenocarcinoma by targeting Bcl-2 
Oncology Letters  2013;6(6):1713-1718.
microRNAs (miRNAs or miRs) may function as oncogenes or tumor suppressors. The present study identified that miR-449a was downregulated in human gastric cancer. The overexpression of miR-449a inhibited gastric adenocarcinoma cell growth and promoted cell apoptosis in the MGC-803 and SGC-7901 gastric adenocarcinoma cell lines. Subsequently, Bcl-2 was identified as a potential miR-449a target by bioinformatics analysis. It was also shown that Bcl-2 was negatively regulated by miR-449a at the post-transcriptional level, via a specific target site within the 3′-untranslated region (3′UTR), by luciferase reporter assay. The expression of miR-449a induced cell apoptosis, as observed by TdT-mediated dUTP nick end labeling and caspase 3/7 assays, and was rescued by Bcl-2 expression. Therefore, these observations indicate that miR-449a acts as a tumor suppressor by targeting the Bcl-2 gene and that it promotes gastric adenocarcinoma cell apoptosis via Bcl-2. The findings of this study contribute to or current understanding of the functions of miR-449a in gastric adenocarcinoma.
doi:10.3892/ol.2013.1609
PMCID: PMC3833858  PMID: 24260067
miR-449a; Bcl-2; gastric adenocarcinoma; caspase 3; caspase 7; apoptosis
10.  A novel hypothesis: up-regulation of HO-1 by activation of PPARγ inhibits HMGB1-RAGE signaling pathway and ameliorates the development of ALI/ARDS 
Journal of Thoracic Disease  2013;5(5):706-710.
Suppression of inflammation in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) by activation of peroxisome proliferator-activated receptor (PPAR)-γ has been well demonstrated in animal model studies. However, the molecular mechanisms underlying this effect remain largely unknown. The induction of heme oxygenase-1 (HO-1) exerts antioxidant, anti-apoptotic, and immunomodulatory functions in various situations. Recent studies have indicated that activation of PPARγ induces expression of HO-1, suggesting that HO-1 is a downstream target of PPARγ. Meanwhile, study has shown that activation of PPARγ ameliorates inflammatory response of cells by inhibiting high mobility group box 1 (HMGB1) release. In pulmonary system, binding of HMGB1 to its receptor for advanced glycation end-products (RAGE) triggers the production of pro-inflammatory cytokines, chemokines, adhesion molecules and reactive oxygen species, promoting the development of ALI/ARDS. Based on the recent findings that induction of HO-1 protects tissues and cells from extracellular stress by reducing HMGB1 production, we propose the hypothesis that HO-1 may mediate the protective effects of PPARγ on inhibition of HMGB1-RAGE signaling pathway to attenuate the development of ALI/ARDS.
doi:10.3978/j.issn.2072-1439.2013.08.69
PMCID: PMC3815721  PMID: 24255785
Peroxisome proliferator-activated receptor (PPAR)-γ; heme oxygenase-1 (HO-1); high mobility group box 1 (HMGB1); receptor for advanced glycation end-products (RAGE); acute lung injury/acute respiratory distress syndrome (ALI/ARDS)
11.  Preventative effect of Astragalus flavescens on hepatic fibrosis in rats and its mechanism of action 
The aim of this study was to investigate the preventative effect of Astragalus flavescens on hepatic fibrosis in rats and its mechanism of action. A total of 60 rats were randomly divided into normal control, model control, high-dose treatment and low-dose treatment groups, and a hepatic fibrosis model was established. The high- and low-dose treatment groups were treated with 2 g/100 g and 0.5 g/100 g Astragalus flavescens, respectively, once a day. Eight weeks following the initiation of treatment, the liver specimens of the rats were stained and observed under a light microscope. Hepatic fibrosis indices, specifically, type III precollagen (PC III), type IV collagen (C IV), hyaluronic acid (HA) and laminin (LN), were detected. Furthermore, the expression and localization of the hepatic fibrosis-related factors transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and platelet-derived growth factor-BB (PDGF-BB) were determined. The serum levels of hepatic fibrosis indices, and the liver tissue levels of hepatic fibrosis-related factors and collagen surface density in the model control group and the high- and low-dose treatment groups were significantly higher compared with those of the normal control group (P<0.05). In addition, the values in the two treatment groups were significantly lower compared with those of the model control group (P<0.05). The present study demonstrated that Astragalus flavescens effectively prevents hepatic fibrosis in rats. A possible mechanism for this is that it may reduce the expression levels of TGF-β1, PDGF-BB and CTGF, thereby inhibiting the activation of hepatic stellate cells and specifically blocking the signal transduction pathway of hepatic fibrosis.
doi:10.3892/etm.2013.1232
PMCID: PMC3797310  PMID: 24137287
Astragalus flavescens; hepatic fibrosis; prevention; mechanism
12.  Association between Vitamin D Insufficiency and Elevated Serum Uric Acid among Middle-Aged and Elderly Chinese Han Women 
PLoS ONE  2013;8(4):e61159.
Background
Association between vitamin D insufficiency and hyperuricemia has not been reported so far. We aimed to study the association of vitamin D insufficiency with elevated serum uric acid among middle-aged and elderly Chinese Han women.
Methods
We collected data from participants residing in Jinchang district of Suzhou from January to May, 2010. Serum uric acid, 25-hydroxy vitamin D and other traditional biomarkers including fasting plasma glucose and blood lipids were determined in 1726 women aged above 30 years. Association between vitamin D insufficiency and elevated uric acid was analyzed in premenopausal and postmenopausal women, respectively.
Results
Among postmenopausal women, 25-hydroxy vitamin D level of participants with elevated uric acid was lower than that of those with normal uric acid (median [interquartile range]: 35[28–57] vs 40[32–58], µg/L; P = 0.006). Elevated uric acid was more prevalent in participants with vitamin D insufficiency compared to those without vitamin D insufficiency (16.50% vs 8.08%; P<0.001). Association between vitamin D insufficiency and elevated uric acid was not significant among premenopausal women. However, participants with vitamin D insufficiency were more likely to have elevated uric acid compared with those without vitamin D insufficiency among postmenopausal women (OR, 95% CI: 2.38, 1.47–3.87). Moreover, after excluding individuals with diabetes and/or hypertension, the association of vitamin D insufficiency with elevated uric acid was still significant (OR, 95% CI: 2.48, 1.17–5.44).
Conclusions
Vitamin D insufficiency was significantly associated with elevated uric acid among postmenopausal Chinese Han women. This study suggested that a clinical trial should be conducted to confirm the association of vitamin D insufficiency with hyperuricemia.
doi:10.1371/journal.pone.0061159
PMCID: PMC3621974  PMID: 23585876
13.  High frequency of HIV mutations associated with HLA-C suggests enhanced HLA-C-restricted CTL selective pressure associated with an AIDS-protective polymorphism 
Delayed HIV-1 disease progression is associated with a single nucleotide polymorphism upstream of the HLA-C gene that correlates with differential expression of the HLA-C antigen. This polymorphism was recently shown to be a marker for a protective variant in the 3′UTR of HLA-C that disrupts a microRNA binding site, resulting in enhanced HLA-C expression at the cell surface. Whether individuals with ‘high’ HLA-C expression show a stronger HLA-C-restricted immune response exerting better viral control than that of their counterparts has not been established. We hypothesised that the magnitude of the HLA-C-restricted immune pressure on HIV would be greater in subjects with highly expressed HLA-C alleles. Using a cohort derived from a unique narrow source epidemic in China, we identified mutations in HIV proviral DNA exclusively associated with HLA-C which were used as markers for the intensity of the immune pressure exerted on the virus. We found an increased frequency of mutations in individuals with highly expressed HLA-C alleles which also correlated with IFN-γ production by HLA-C-restricted CD8+ T-cells. These findings show that immune pressure on HIV is stronger in subjects with the protective genotype and highlights the potential role of HLA-C-restricted responses in HIV control. This is the first in vivo evidence supporting the protective role of HLA-C-restricted responses in non-Caucasians during HIV infection.
doi:10.4049/jimmunol.1103472
PMCID: PMC3378658  PMID: 22474021
14.  Biomarkers on melanoma patient T Cells associated with ipilimumab treatment 
Background
Ipilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4+ and CD8+ T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.
Methods
Microarray analysis of flow-cytometry purified CD4+ and CD8+ T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.
Results
Ipilimumab up-regulated Ki67 and ICOS on CD4+ and CD8+ cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8+ at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4+ and CD8+ cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES+CD8+, GranzymeB+EOMES+CD8+ and decreased Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67+CD8+ T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67+EOMES+CD8+ T cells were associated with relapse (p ≤ 0.001), and low Ki67+EOMES+CD4+ T cells were associated with irAE (p ≤ 0.008).
Conclusions
Up-regulation of proliferation and activation signals in CD4+ and CD8+ T cells were pharmacodynamic markers for ipilimumab. Ki67+EOMES+CD8+ and Ki67+EOMES+CD4+T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.
doi:10.1186/1479-5876-10-146
PMCID: PMC3527361  PMID: 22788688
CTLA-4; Antibody; Biomarker; Melanoma
15.  Admission clinical characteristics and early clinical outcomes among acute ischemic stroke patients 
Journal of Biomedical Research  2012;26(3):152-158.
The purpose of the present study was to investigate the association between admission clinical characteristics and outcomes at discharge among acute ischemic stroke patients in the Chinese population. A total of 2,673 patients with acute ischemic stroke were included in the present study. The clinical characteristics at admission and other study variables were collected for all patients. The study outcome was defined as neurological deficiency (National Institute of Health Stroke Scale score ≥10) at discharge or in-hospital death. Compared with the subjects without neurological deficiency at discharge or in-hospital death, the subjects with neurological deficiency at discharge or in-hospital death had a significantly higher prevalence of hyperglycemia or history of atrial fibrillation at admission. Age ≥ 80 years, hyperglycemia, hypertension, and history of atrial fibrillation were significantly associated with neurological deficiency at discharge or in-hospital death after adjustment for other variables. It is concluded that old age (≥80 years), hyperglycemia, hypertension and history of atrial fibrillation are significantly associated with neurological deficiency at discharge or in-hospital death among patients with acute ischemic stroke.
doi:10.7555/JBR.26.20110129
PMCID: PMC3596064  PMID: 23554744
acute ischemic stroke; clinical characteristics; death; neurological deficiency; discharge outcome
16.  The Rice HGW Gene Encodes a Ubiquitin-Associated (UBA) Domain Protein That Regulates Heading Date and Grain Weight 
PLoS ONE  2012;7(3):e34231.
Heading date and grain weight are two determining agronomic traits of crop yield. To date, molecular factors controlling both heading date and grain weight have not been identified. Here we report the isolation of a hemizygous mutation, heading and grain weight (hgw), which delays heading and reduces grain weight in rice. Analysis of hgw mutant phenotypes indicate that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea, both composing the spikelet hull that is known to determine the size and shape of brown grain. Molecular cloning and characterization of the HGW gene showed that it encodes a novel plant-specific ubiquitin-associated (UBA) domain protein localized in the cytoplasm and nucleus, and functions as a key upstream regulator to promote expressions of heading date- and grain weight-related genes. Moreover, co-expression analysis in rice and Arabidopsis indicated that HGW and its Arabidopsis homolog are co-expressed with genes encoding various components of ubiquitination machinery, implying a fundamental role for the ubiquitination pathway in heading date and grain weight control.
doi:10.1371/journal.pone.0034231
PMCID: PMC3311617  PMID: 22457828
17.  Multilayered defence in HLA-B51 associated HIV viral control 
Polymorphism in the Human Leukocyte Antigen (HLA) region of chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, B27 and B51. Taking advantage of a unique cohort infected with clade B’ HIV-1 through contaminated blood, in which many variables, such as the length of infection, the infecting viral strain and host genetic background are controlled, we performed a comprehensive study in order to understand HLA-B51 associated HIV-1 control. We focused on the T-cell responses against three dominant HLA-B51 restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in Position 1 of epitope LI9 was seen in most B51+ individuals, followed by V289X in Position 8 of the TI8, then A328S in Position 2 of the NI9 epitope was also seen in some B51+individuals. Good control of viral load and higher CD4+ counts were significantly associated with at least one detectable T cell response to un-mutated epitopes, whereas lower CD4+ counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T-cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defence against the development of immune escape mutations.
doi:10.4049/jimmunol.1100316
PMCID: PMC3166850  PMID: 21670313
18.  DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer 
Molecular Ecology Resources  2011;11(6):1002-1011.
Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.
doi:10.1111/j.1755-0998.2011.03041.x
PMCID: PMC3195333  PMID: 21689384
cytochrome c oxidase subunit I; DNA barcoding; internal transcribed spacer; oomycete; species identification
19.  DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer 
Molecular ecology resources  2011;11(6):1002-1011.
Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.
doi:10.1111/j.1755-0998.2011.03041.x
PMCID: PMC3195333  PMID: 21689384
cytochrome c oxidase subunit I; DNA barcoding; internal transcribed spacer; oomycete; species identification
20.  Association of inflammation and endothelial dysfunction with metabolic syndrome, prediabetes and diabetes in adults from Inner Mongolia, China 
Background
We examined the association of biomarkers of inflammation and endothelial dysfunction with diabetes and metabolic syndrome (MetS) in persons from Inner Mongolia.
Methods
A cross-sectional study was conducted among 2,536 people aged 20 years and older from Inner Mongolia, China. Overnight fasting blood samples were obtained to measure plasma concentrations of high sensitivity C-reactive protein (hsCRP), soluble inter-cellular adhesion molecule-1 (sICAM-1), sE-selectin, angiotensin II, high density lipoprotein cholesterol, triglycerides, and blood glucose. Waist circumference and blood pressure were measured by trained staff. MetS was defined according to the modified ATP III definition for Asians. Elevated level of the biomarker was defined as values in the upper tertile of the distribution. Participants were categorized into one of four groups based on the presence or absence of metabolic and glycemic abnormalities: 1) free of prediabetes, diabetes and MetS (reference group), 2) prediabetes or diabetes only, 3) MetS without prediabetes or diabetes, and 4) MetS plus prediabetes or diabetes. The multivariable models are adjusted for age, gender, smoking, drinking, family history of hypertension, and body mass index.
Results
Among study participants, 18.5% had prediabetes, 3.6% had diabetes, and 27.4% of the entire study population had 3 or more components of the MetS. Elevated hsCRP was associated with an increased odds of prediabetes or diabetes only, MetS without prediabetes or diabetes, and MetS plus prediabetes or diabetes with multivariable adjusted odds ratios (95% confidence intervals) of 2.3 (1.7-3.1), 3.0 (2.4-3.8), and 5.8 (4.5-7.5), respectively. Elevated sICAM-1 was associated with increased odds (95% CI) of prediabetes or diabetes only (2.1, 1.6-2.9) and MetS plus prediabetes or diabetes (4.2, 3.2-5.3) but was not associated with MetS alone. Elevated sE-selectin was associated with a modestly increased risk of MetS (OR 1.7, 95% CI 1.4-2.2). Elevated levels of Angiotensin II were not associated with the MetS plus prediabetes or diabetes in this study.
Conclusions
Diabetes and the MetS are common in the Inner Mongolia population. The biomarkers of inflammation and endothelial dysfunction are associated with increased risk for diabetes and MetS in this population. These results are consistent with results from other populations.
doi:10.1186/1472-6823-11-16
PMCID: PMC3204247  PMID: 21989115
metabolic syndrome; diabetes; inflammation; endothelial dysfunction; C-reactive protein; intercellular adhesion molecule-1; E-selectin
21.  Structural Basis for Sequence Specific DNA Binding and Protein Dimerization of HOXA13 
PLoS ONE  2011;6(8):e23069.
The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of genes during embryonic morphogenesis. Here we present the NMR structure of HOXA13 homeodomain (A13DBD) bound to an 11-mer DNA duplex. A13DBD forms a dimer that binds to DNA with a dissociation constant of 7.5 nM. The A13DBD/DNA complex has a molar mass of 35 kDa consistent with two molecules of DNA bound at both ends of the A13DBD dimer. A13DBD contains an N-terminal arm (residues 324 – 329) that binds in the DNA minor groove, and a C-terminal helix (residues 362 – 382) that contacts the ATAA nucleotide sequence in the major groove. The N370 side-chain forms hydrogen bonds with the purine base of A5* (base paired with T5). Side-chain methyl groups of V373 form hydrophobic contacts with the pyrimidine methyl groups of T5, T6* and T7*, responsible for recognition of TAA in the DNA core. I366 makes similar methyl contacts with T3* and T4*. Mutants (I366A, N370A and V373G) all have decreased DNA binding and transcriptional activity. Exposed protein residues (R337, K343, and F344) make intermolecular contacts at the protein dimer interface. The mutation F344A weakens protein dimerization and lowers transcriptional activity by 76%. We conclude that the non-conserved residue, V373 is critical for structurally recognizing TAA in the major groove, and that HOXA13 dimerization is required to activate transcription of target genes.
doi:10.1371/journal.pone.0023069
PMCID: PMC3148250  PMID: 21829694
22.  The Antiviral Efficacy of HIV-Specific CD8+ T-Cells to a Conserved Epitope Is Heavily Dependent on the Infecting HIV-1 Isolate 
PLoS Pathogens  2011;7(5):e1001341.
A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef90–97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A–H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.
Author Summary
One of the greatest challenges to developing an effective HIV vaccine is the ability of HIV to rapidly alter its viral sequence. Such variation in viral sequence enables the virus to frequently evade recognition by the host immune system. To counteract this problem, there has been increasing interest in developing HIV vaccines that target T-cell responses to the regions of the virus that are highly conserved between strains of HIV. However, previous studies have focused on identifying amino acid variation predominantly within a single viral isolate, or have focused on classical within-epitope escape mutation. Our study assessed T-cell recognition of a conserved epitope shared by a total of 13 HIV strains. Strikingly, we show that only a small proportion of the viral strains were effectively recognised and targeted by the T-cells. In contrast, differences in amino acid sequence in the region flanking the epitope impaired the intracellular processing and presentation of epitope in the majority of HIV strains tested. Thus, our findings highlight that a large proportion of HIV strains may evade epitope-specific T-cell recognition despite absolute epitope conservation. This has important implications for both vaccine design and evaluation of vaccine efficacy.
doi:10.1371/journal.ppat.1001341
PMCID: PMC3093356  PMID: 21589893
23.  Characteristics of CD8+ T cell subsets in Chinese patients with chronic HIV infection during initial ART 
Background
CD8+ T cells may play an important role in protecting against HIV. However, the changes of CD8+ T cell subsets during early period of ART have not been fully studied.
Methods
Twenty-one asymptomatic treatment-naive HIV-infected patients with CD4 T+ cells less than 350 cells/μl were enrolled in the study. Naïve, central memory(CM), effective memory(EM) and terminally differentiated effector (EMRA) CD8+ cell subsets and their activation and proliferation subsets were evaluated in blood samples collected at base line, and week 2, 4, 8 and 12 of ART.
Results
The total CD8+ T cells declined and the Naïve and CM subsets had a tendency of increase. Activation levels of all CD8+ T cell subsets except EMRA subset decreased after ART. However, proliferation levels of total CD8+ T cells, EMRA, EM and CM subsets increased at the first 4 weeks of ART, then decreased. Proliferation level of the naïve cells decreased after ART.
Conclusion
The changes of CD8+ T cell subsets during initial ART are complex. Our results display a complete phenotypical picture of CD8+ cell subsets during initial ART and provide insights for understanding of immune status during ART.
doi:10.1186/1742-6405-8-15
PMCID: PMC3073867  PMID: 21435275
24.  Analysis of DNA Methylation in Various Swine Tissues 
PLoS ONE  2011;6(1):e16229.
DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively.
In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.
doi:10.1371/journal.pone.0016229
PMCID: PMC3025005  PMID: 21283691
25.  Exposure to Cocaine Dynamically Regulates the Intrinsic Membrane Excitability of Nucleus Accumbens Neurons 
Drug-induced malfunction of nucleus accumbens (NAc) neurons underlies a key pathophysiology of drug addiction. Drug-induced changes in intrinsic membrane excitability of NAc neurons are thought to be critical for producing behavioral alterations. Previous studies demonstrate that following short-term (2d) or long-term (21d) withdrawal from non-contingent cocaine injection, the intrinsic membrane excitability of NAc shell (NAcSh) neurons is decreased, and decreased membrane excitability of NAcSh neurons increases the acute locomotor response to cocaine. However, animals exhibit distinct cellular and behavioral alterations at different stages of cocaine exposure, suggesting that the decreased membrane excitability of NAc neurons may not be a persistent change. Here, we demonstrate that the membrane excitability of NAcSh neurons is differentially regulated depending on whether cocaine is administered contingently or non-contingently. Specifically, the membrane excitability of NAcSh MSNs was decreased at 2d after withdrawal from either 5-day intraperitoneal (i.p.) injections (15 mg/kg) or cocaine self-administration (SA). At 21d of withdrawal, the membrane excitability of NAcSh MSNs, which remained low in i.p.-pretreated rats, returned to a normal level in SA-pretreated rats. Furthermore, upon a re-exposure to cocaine after long-term withdrawal, the membrane excitability of NAcSh MSNs instantly returned to a normal level in i.p.-pretreated rats. On the other hand, in SA-pretreated rats, the re-exposure elevated the membrane excitability of NAcSh MSMs beyond the normal level. These results suggest that the dynamic alterations in membrane excitability of NAcSh MSNs, together with the dynamic changes in synaptic input, contribute differentially to the behavioral consequences of contingent and non-contingent cocaine administration.
doi:10.1523/JNEUROSCI.4063-09.2010
PMCID: PMC2853189  PMID: 20220002
membrane excitability; cocaine; nucleus accumbens; action potential; withdrawal; synapse

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