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author:("Tian, taping")
1.  Identification of MicroRNAs Involved in Growth Arrest and Apoptosis in Hydrogen Peroxide-Treated Human Hepatocellular Carcinoma Cell Line HepG2 
Although both oxidative stress and microRNAs (miRNAs) play vital roles in physiological and pathological processes, little is known about the interactions between them. In this study, we first described the regulation of H2O2 in cell viability, proliferation, cycle, and apoptosis of human hepatocellular carcinoma cell line HepG2. Then, miRNAs expression was profiled after H2O2 treatment. The results showed that high concentration of H2O2 (600 μM) could decrease cell viability, inhibit cell proliferation, induce cell cycle arrest, and finally promote cell apoptosis. Conversely, no significant effects could be found under treatment with low concentration (30 μM). miRNAs array analysis identified 131 differentially expressed miRNAs (125 were upregulated and 6 were downregulated) and predicted 13504 putative target genes of the deregulated miRNAs. Gene ontology (GO) analysis revealed that the putative target genes were associated with H2O2-induced cell growth arrest and apoptosis. The subsequent bioinformatics analysis indicated that H2O2-response pathways, including MAPK signaling pathway, apoptosis, and pathways in cancer and cell cycle, were significantly affected. Overall, these results provided comprehensive information on the biological function of H2O2 treatment in HepG2 cells. The identification of miRNAs and their putative targets may offer new diagnostic and therapeutic strategies for liver cancer.
PMCID: PMC5002491  PMID: 27597883
2.  Analyzing Dynamic Changes of Laboratory Indexes in Patients with Acute Heart Failure Based on Retrospective Study 
BioMed Research International  2016;2016:7496061.
Background. Changes of N-terminal probrain natriuretic peptide (NT-proBNP) have been studied whether in the long term or the short term in patients of acute heart failure (AHF); however, changes of NT-proBNP in the first five days and their association with other factors have not been investigated. Aims. To describe the dynamic changes of relevant laboratory indexes in the first five days between different outcomes of AHF patients and their associations. Methods and Results. 284 AHF with dynamic values recorded were analyzed. Changes of NT-proBNP, troponin T, and C-reactive protein were different between patients with different outcomes, with higher values in adverse group than in control group at the same time points (p < 0.05). Then, prognostic use and risk stratification of NT-proBNP were assessed by receiver-operating characteristic curve and logistic regression. NT-proBNP levels at day 3 showed the best prognostic power (area under the curve = 0.730, 95% confidence interval (CI): 0.657 to 0.794) and was an independent risk factor for adverse outcome (odds ratio, OR: 2.185, 95% CI: 1.584–3.015). Classified changes of NT-proBNP may be predictive for adverse outcomes in AHF patients. Conclusions. Sequential monitoring of laboratory indexes within the first 5 days may be helpful for management of AHF patients.
PMCID: PMC4837244  PMID: 27144175
3.  HnRNP A1 is Involved in Deep Vein Thrombosis Patients with Behçet's Disease 
EBioMedicine  2016;6:215-221.
The aim of this study was to verify the hypothesis originated from bioinformatics and literature reviews that hnNRP A1 may be a new immune target of Behçet's disease (BD).
First, bioinformatics was used to show the correlation between hnRNP A1 and A2/B1 in amino acid sequences and three dimensional structures. Second, hnRNP A1 was expressed, purified, and immunologically confirmed by systematic immunology methods including: Western blotting, immunoprecipitation and Dot-ELISA. Then, ELISA was used to screen the anti-hnRNP A1 autoantibodies in newly confirmed clinical samples and the clinical significance was compared between anti-hnRNP A1 antibody positive and negative groups. Finally, the endothelial cells antigen profile of one anti-hnRNP A1 antibody positive BD patient was detected using immunoprecipitation with liquid chromatography tandem mass spectrometry (LC–TMS).
In total 720 subjects enrolled and tested in this study. Our results demonstrated hnRNP A1 as a new immune target of BD. The reactivity of BD serum IgG antibodies against hnRNP A1 was significantly higher than healthy controls (P < 0.0001), and deep vein thrombosis (DVT) showed a significant higher in the anti-hnRNP A1 antibodies positive group (P < 0.05).
•Bioinformatics was used to predict that hnRNP A1 may play a role in BD.•HnRNP A1 was immunologically confirmed as an autoantigen of BD.•Deep vein thrombosis has a close relationship with anti-hnRNP A1 antibody in patients' blood circulation.
Behçet's disease (BD) is a chronic systemic autoimmune disease. The pathogenesis of BD is still not clear, and the diagnosis is based on typical clinical syndromes. Autoantigen identification was considered a key to solve this problem. This study was to verify the hypothesis suggested by bioinformatics that hnRNP A1 may be a new autoantigen of BD. Among the 720 subjects enrolled and systemic tested, our results demonstrated hnRNP A1 as a new autoantigen of BD, and associated with deep vein thrombosis.
PMCID: PMC4856785  PMID: 27211563
BD, Behçet's disease; DVT, deep vein thrombosis; SLE, systemic lupus erythematosus; AECA, anti-endothelial cell antibodies; LC–TMS, liquid chromatography tandem mass spectrometry; PVDF, polyvinylidene fluoride membranes; PBS, Phosphate buffered saline; Bioinformatics; Behçet's disease; Autoantigen; HnRNP A1
4.  Evaluation of 29 indicators for the prognosis of advanced non-small cell lung cancer with cytokine-induced killer cell therapy combined with chemotherapy 
The aim of the present study was to evaluate 29 whole blood or serum indicators to identify factors able to predict clinical outcome following cytokine-induced killer (CIK) cell therapy combined with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC), and to evaluate the 5-year prognosis of the patients. From March 2008 to October 2013, 42 patients with advanced NSCLC (stages III and IV) were enrolled in the study. These patients were from a single hospital, and had been treated with CIK therapy combined with chemotherapy. Evaluation of the correlation between prognosis and age, gender, tumor stage, surgery resection status, number of CIK therapy cycles, tumor subtype, and the differential whole blood or serum indicators were analyzed by Kaplan-Meier methods and the log-rank test. The prognostic factors were analyzed by Cox proportional models. The median progression-free survival (mPFS) time of patients with high expression levels of albumin [20.0 months; 95% confidence interval (CI): 17.4–22.6 months] was significantly longer than the mPFS for patients with low expression levels of albumin (36.0 months; 95% CI: 24.7–47.3 months) (P=0.034). Other factors demonstrated no significant difference. Following analysis using the Cox proportional hazards regression model, the number of CIK therapy cycles (P=0.041) and the expression level of albumin (P=0.038) were revealed to be independent prognostic factors following the use of CIK cell therapy combined with chemotherapy for patients with advanced NSCLC. The risk of adverse outcomes in patients receiving ≥4 CIK therapy cycles and in patients with increased expression levels of albumin were 0.38 (95% CI: 0.14–1.13) and 0.32 (95% CI: 0.10–1.24)-fold those of patients receiving <4 CIK therapy cycles and with decreased expression levels of albumin, respectively. The serum albumin concentration may therefore be a predictor of the 5-year survival rate of patients with advanced NSCLC treated with CIK cell therapy combined with chemotherapy; patients with high expression levels of albumin may have a better prognosis in comparison with patients with low expression levels of albumin.
PMCID: PMC4840551  PMID: 27168779
non-small cell lung cancer; cytokine-induced killer; chemotherapy; prognosis; albumin
5.  The clinical performance evaluation of novel protein chips for eleven biomarkers detection and the diagnostic model study 
We aimed to develop and validate two novel protein chips, which are based on microarray chemiluminescence immunoassay and can simultaneously detected 11 biomarkers, and then to evaluate their clinical diagnostic value by comparing with the traditional methods. Protein chips were evaluated for limit of detection, specificity, common interferences, linearity, precision and accuracy. 11 biomarkers were simultaneously detected by traditional methods and protein chips in 3683 samples, which included 1723 cancer patients, 1798 benign diseases patients and 162 healthy controls. After assay validation, protein chips demonstrated high sensitivity, high specificity, good linearity, low imprecision and were free of common interferences. Compared with the traditional methods, protein chips have good correlation in the detection of all the 13 kinds of biomarkers (r≥0.935, P<0.001). For specific cancer detection, there were no statistically significant differences between the traditional method and novel protein chips, except that male protein chip showed significantly better diagnostic value on NSE detection (P=0.004) but significantly worse value on pro-GRP detection (P=0.012), female chip showed significantly better diagnostic value on pro-GRP detection (P=0.005). Furthermore, both male and female multivariate diagnostic models had significantly better diagnostic value than single detection of PGI, PG II, pro-GRP, NSE and CA125 (P<0.05). In addition, male models had significantly better diagnostic value than single CA199 and free-PSA (P<0.05), while female models observed significantly better diagnostic value than single CA724 and β-HCG (P<0.05). For total disease or cancer detection, the AUC of multivariate logistic regression for the male and female disease detection was 0.981 (95% CI: 0.975-0.987) and 0.836 (95% CI: 0.798-0.874), respectively. While, that for total cancer detection was 0.691 (95% CI: 0.666-0.717) and 0.753 (95% CI: 0.731-0.775), respectively. The new designed protein chips are simple, multiplex and reliable clinical assays and the multi-parameter diagnostic models based on them could significantly improve their clinical performance.
PMCID: PMC4723802  PMID: 26884957
Cancer biomarkers; protein array analysis; diagnostic tests; early detection of cancer; logistic models; ROC curve
6.  Determination of reference intervals of serum levels of human epididymis protein 4 (HE4) in Chinese women 
To determine reference intervals for serum levels of human epididymis protein 4 (HE4) in Chinese women.
In this multicenter (n = 9) study, 618 healthy women, 767 patients with non-malignant diseases, and 951 patients with malignant tumors were enrolled. Serum levels of HE4 were measured in all patients using electrochemiluminescence immunoassays. The influence of age, menopause, malignancy status and other characteristics on the levels of HE4 was evaluated using univariate and multivariate analyses. Confidence intervals (2.5–97.5 %) were determined in different populations.
There were significant differences in HE4 levels among groups with different ages, menopause or malignancy status. Higher levels of HE4 were detected in elder compared to younger, post- compare to pre- menopause and malignant compared to benign subjects. Multivariate analysis showed that menopause and malignancy status, as well as smoking and pelvic masses were independent factors involved in serum HE4 levels. In pre-menopause stage, the reference ranges of HE4 level were 29.30–68.79, 28.12–1284.83 and 34.75–981.91 pmol/L in healthy, benign and malignant populations, respectively. In post-menopause stage, the reference ranges are 35.96–114.43, 39.11–2208.70 and 39.40–1678.13 pmol/L for those populations.
The present study has established the reference intervals of HE4 levels in pre- and post-menopause populations with different malignancy status.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-015-0201-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4637994  PMID: 26552478
Human epididymis protein 4; Epithelial ovarian cancer; Pelvic masses
7.  Role of serum polyunsaturated fatty acids in the development of colorectal cancer 
We aimed to investigate the role of serum levels of polyunsaturated fatty acid (PUFA) in the development of colorectal cancer (CRC). Serum levels of n-3 and n-6 PUFA in 69 healthy control (Ctrl), 62 benign colorectal polys (CRP) and 100 CRC patients were detected by gas chromatograph. The adjusted odds ratio (OR) by quartiles of n-3 and n-6 PUFA were analyzed. During the process of Ctrl to CRP, total n-3 PUFA (OR=0.159, P<0.001), total n-6 PUFA (OR=0.190, P<0.001), C20:5 n-3 (OR=0.263, P=0.030), C22:6 n-3 (OR=0.125, P<0.001), and C18:2 n-6 (OR=0.299, P=0.025) were inversely associated with CRP risk. The ratio of total n-6 PUFA and total n-3 PUFA (OR=4.667, P=0.002), and the ratio of C20:4 n-6 and (C20:5 n-3+C22:6 n-3) (OR=6.000, P<0.001) were positively associated with CRP risk. During the process of CRP to CRC, total n-3 PUFA (OR=4.059, P=0.007), total n-6 PUFA (OR=8.146, P<0.001), C22:6 n-3 (OR=3.789, P=0.048), and C18:2 n-6 (OR=3.667, P=0.045) were positively associated with CRC risk. The ratio of C20:4 n-6 and (C20:5 n-3+C22:6 n-3) (OR=0.588, P=0.001) was inversely associated with CRC. In conclusion, our results found that the total n-3 PUFA, C22:6 n-3, the total n-6 PUFA, C18:2 n-6, and the ratio of C20:4 n-6 and (C20:5 n-3 +C22:6 n-3) played controversy role in the process of CRP and the process of CRC, and may provide nutritional intervention suggestions for the clinical practice.
PMCID: PMC4658982  PMID: 26629093
Polyunsaturated fatty acids; colorectal polys; colorectal cancer; odds ratio
8.  Comparisons of Different Metabolic Syndrome Definitions and Associations with Coronary Heart Disease, Stroke, and Peripheral Arterial Disease in a Rural Chinese Population 
PLoS ONE  2015;10(5):e0126832.
We estimated the prevalence of metabolic syndrome (MetS) and compared associations of different MetS definitions with coronary heart disease (CHD), stroke, and peripheral arterial disease (PAD) in a rural Chinese population.
Among 4,748 residents (2,145 men and 2,603 women) aged 30+ years in rural China from 2006 to 2007, the prevalence of MetS was estimated by using five different definitions: modified World Health Organization (WHO), Chinese Diabetes Society (CDS), the updated National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) for Asian-Americans, International Diabetes Federation (IDF), and Joint Interim Statement (JIS). Multivariable logistic regression analyses were implemented to estimate the association between MetS and the prevalence of CHD, stroke and PAD, respectively.
Prevalence of MetS in men was 11.5% (WHO), 14.8% (CDS), 32.4% (NCEP-ATP III), 27.5% (IDF) and 39.7% (JIS) and in women was 15.7% (WHO), 20.7% (CDS), 54.2% (NCEP-ATP III), 51.5% (IDF) and 54.2% (JIS), respectively. Respective ORs (95% CI) for associating MetS with CHD in men were 1.79 (1.02-3.17), 1.25 (0.69-2.26), 1.61 (1.01-2.58), 1.84 (1.14-2.96), and 1.53 (0.96-2.43). Corresponding ORs (95% CI) for stroke in men were 2.18 (95% CI 1.20 to 3.97), 2.20 (95% CI 1.25 to 3.89), 1.71 (95% CI 1.02 to 2.84), 1.30 (95% CI 0.77 to 2.23), and 1.61 (95% CI 0.97 to 2.68), respectively. In women, CHD and stroke were significantly associated with MetS using all five definitions of MetS. In addition, PAD was associated with all five MetS definitions in men, but not in women. Only hyperglycemia and BMI were significantly associated with PAD in women.
In this rural Chinese population, the JIS, IDF and CDS criteria may not be more suitable than WHO and updated NCEP-ATPIII definitions for screening high-risk individuals and estimating the risk of CHD and stroke from MetS, especially in men.
PMCID: PMC4427409  PMID: 25961739
9.  Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet’s Disease 
PLoS ONE  2015;10(4):e0124760.
Behçet’s disease (BD) is a chronic inflammatory disease with multisystem involvement, and it is listed as a rare disease in the United States but is common in the Middle East, China, and Japan. The aim of this study was to identify novel autoantigens in Chinese patients with BD. First, the candidate autoantigens were screened by Western blotting, and the sequences of putative antigens were identified by LC-MALDI-TOF/TOF mass spectrometry. Next, the screened protein was cloned, expressed and purified. Then, an optimized ELISA was developed, and the serological criteria were evaluated using a large number of confirmed patients. One antigen with a molecular weight of approximately 28 kDa was identified as electron transfer flavoprotein subunit beta (ETFB). Positive reactivity was detected in recombinant human ETFB sera from 38 of 92 BD patients (41 %) and 1 of 90 healthy controls (1 %).
PMCID: PMC4410958  PMID: 25915519
10.  Annexin A2 as a target endothelial cell membrane autoantigen in Behçet's disease 
Scientific Reports  2015;5:8162.
Cell membrane proteins are believed to play a critical role in the pathogenesis of autoimmune diseases. However, few membrane autoantigens have been linked with Behçet's disease. Here, a cell-chip was performed to identify autoantibody target cells, and the suspected autoantigens were detected using immunoblotting. The amino acid sequences of the detected proteins were determined using LC-MALDI-TOF/TOF. Putative proteins were recombinantly expressed and purified, and a corresponding ELISA was developed and clinically validated using real clinical samples. It was found that a 36-kDa membrane protein - annexin A2 - was detected in approximately one-third of the patients' blood circulation. The immunohistochemistry results showed that annexin A2 was highly expressed in vascular endothelial cells. Moreover, vascular involvement was significantly higher in the anti-annexin A2 antibody-positive group versus the anti-annexin A2 antibody-negative group among all the clinical samples analyzed, indicating that annexin A2 is a novel endothelial cell membrane antigen involved in Behçet's disease.
PMCID: PMC4313095  PMID: 25641213
11.  The correlation between serum lipid profile with carotid intima-media thickness and plaque 
It is indicated that non-HDL cholesterol and lipid ratios, including total/HDL cholesterol and LDL/HDL cholesterol ratios, are risk indicators with greater predictive value for coronary atherosclerotic progression or regression compared with conventional lipid profile. However, there have been few reports about the correlation between serum lipid profile with carotid intima-media thickness (IMT) and plaque in Chinese general people.
We examined 402 subjects without apparent diseases in a cross-sectional study (mean age 50.16 years; 36.07% female). Demographics, anthropometrics, and laboratory data were collected. The presence of carotid plaque and intima-media thickness were evaluated by ultrasonography.
Univariate correlations showed carotid IMT was correlated with LDL-C (r = 0.137, p = 0.009), non-LDL-C levels (r = 0.140, p = 0.008) and LDL-C/HDL-C ratio (r = 0.169, p = 0.001). After adjustment for potential covariates, LDL-C/HDL-C ratio (β = 0.132, p < 0.001) were independent variables that interacted on carotid IMT. Other risk factors including age and systolic blood pressure were independently associated with carotid IMT. LDL-C levels, non-HDL-C levels, TC/HDL-C and LDL-C/HDL-C ratios were significantly higher, but HDL-C levels were significantly lower in subjects with carotid plaque than those without it. The subsequent multiple logistic regression analysis showed that LDL-C (OR; 1.325, 95% CI; 1.046-1.821, p = 0.033) and HDL-C levels (OR; 0.093, 95% CI; 0.038-0.227, p < 0.001) were significantly associated with the presence of carotid plaque after adjustment of age. Furthermore, LDL-C combined with HDL-C levels showed the highest area under the curve (0.788, 95% CI; 0.740–0.837, p < 0.001).
Serum LDL-C/HDL-C ratio represents as an independent index associated with increased carotid IMT and LDL-C combined with HDL-C levels may be useful markers for predicting the presence of carotid plaque in the Chinese general population.
PMCID: PMC4272763  PMID: 25491329
Subclinical atherosclerosis; Lipid ratio; Intima-media thickness; Plaque
12.  Factors influencing the quality of preconception healthcare in China: applying a preconceptional instrument to assess healthcare needs 
Preconception care is defined as the promotion of the health and well-being of a woman and her partner before pregnancy. Improving preconception health can result in improved reproductive health outcomes. China has issued latest version official guideline for preconception care in 2011. The objective of this cross-sectional study is to determine whether there is a variation in the quality of preconception healthcare services in distinct eastern and northern populations of China, and what factors are associated with such variation.
A cross-sectional survey using our previously developed preconception instrument was conducted. Women at reproductive age planning for pregnancy were surveyed along with their partners at hospitals during their pre-pregnancy health examination. Data collected include general health/life profiles, pregnancy history, alcohol/tobacco/drug exposures, immunizations, micronutrient supplements and the demands in preconception care. After quality assessment, statistical analysis were applied to evaluate the variations in preconception factors between people from Hebei and Jiangsu Provinces.
3202 women of reproductive age in from eastern province, Jiangsu, and in a northern province, Hebei, participated this study. 2806 of them and their partners have completed the questionnaire, at a rate of 87.6%, 1011 were from Jiangsu and 1795 were from Hebei. Statistical significance was obtained for maternal age (P < 0.001), body mass index (u =13.590, P <0.001), education (χ2 = 916.33, P < 0.001), occupation (χ2 = 901.78, P < 0.001), health status/common disease, immunization status, and need for preconception care.
For a country as large as China, the centralized guideline for standardized preconception healthcare does have a very crucial positive role in reproductive healthcare, but it may not be suited for all populations. Regional authorities should consider the demographics and healthcare needs of the local population and modify the centralized guideline accordingly, as well as provide a better education and professional services for the public, to improve the quality of preconception services at both the regional and the national level.
PMCID: PMC4289191  PMID: 25366578
Preconception healthcare; Maternal health; Survey; China
13.  Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase 
Prion  2013;7(4):328-334.
Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.
PMCID: PMC3904319  PMID: 23787698
Bacillus subtilis; aminopeptidase; substrate specificity; saturation mutagenesis; protein hydrolysis
14.  Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids 
Endogenous peptides that represent biological and pathological information of disease have attracted interest for diagnosis. However, the extraction of those low abundance peptides is still a challenge because of the complexity of human bodily fluids (HBF). Hepcidin, a peptide hormone, has been recognized as a biomarker for iron-related diseases. There is no rapid and reliable way to enrich them from HBF. Here we describe a peptides extraction approach based on nanoporous silica thin films to successfully detect hepcidin from HBF. Cooperative functions of nanopore to biomolecule, including capillary adsorption, size-exclusion and electrostatic interaction, were systematically investigated to immobilize the target peptide. To promote this new approach to clinical practices, we further applied it to successfully assay the hepcidin levels in HBF provided by healthy volunteers and patients suffering from inflammation. Our finding provides a high-throughput, rapid, label-free and cost-effective detection method for capturing and quantifying low abundance peptides from HBF.
PMCID: PMC4077980  PMID: 24566273
Biomarker discovery; Nanoporous silica film; Peptide; MALDI-TOF MS; Hepcidin
15.  Prognostic value of serum leptin in advanced lung adenocarcinoma patients with cisplatin/pemetrexed chemotherapy 
Oncology Letters  2014;7(6):2073-2078.
Cisplatin/pemetrexed chemotherapy has been established as a standard treatment in lung adenocarcinoma. However, the response to the cisplatin/pemetrexed combination varies considerably among patients due to individual variations. Thus, novel biomarkers are required to aid the prediction of the response to the cisplatin/pemetrexed combination. We hypothesized that leptin expression may be a determinant for prognosis in lung adenocarcinoma patients with cisplatin/pemetrexed chemotherapy. Serum from consenting patients with lung adenocarcinoma were obtained for the measurement of leptin and associated tumor biomarkers. Leptin expression was measured by radioimmunoassay. Carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), CA15-3, CA125, CA72-4, cytokeratin 19 fragment (CYFRA21-1) and neuron-specific enolase (NSE) expression were determined by electrochemiluminescence immunoassays. Serum squamous cell carcinoma antigen levels were measured using a microparticle enzyme immunoassay. The associations between serum leptin and tumor biomarker expression were evaluated by Spearman’s correlation analysis. Serum CEA, CA19-9, CA15-3, CA125, CA72-4, CYFRA21-1 and NSE levels showed no obvious difference among patients. However, a trend towards an improved prognosis was observed in patients with lower serum leptin at diagnosis and an increase during cisplatin/pemetrexed chemotherapy. The results indicated that the serum leptin level has prognostic indications in patients with advanced lung adenocarcinoma during cisplatin/pemetrexed chemotherapy, which indicates that it may be a useful marker for the prognosis of cancer patients undergoing chemotherapy treatment.
PMCID: PMC4049753  PMID: 24932291
leptin; lung adenocarcinoma; cisplatin/pemetrexed; prognosis
16.  Enhanced Thermal Stability and Hydrolytic Ability of Bacillus subtilis Aminopeptidase by Removing the Thermal Sensitive Domain in the Non-Catalytic Region 
PLoS ONE  2014;9(3):e92357.
Besides the catalytic ability, many enzymes contain conserved domains to perform some other physiological functions. However, sometimes these conserved domains were unnecessary or even detrimental to the catalytic process for industrial application of the enzymes. In this study, based on homology modeling and molecular dynamics simulations, we found that Bacillus subtilis aminopeptidase contained a thermal sensitive domain (protease-associated domain) in the non-catalytic region, and predicted that deletion of this flexible domain can enhance the structure stability. This prediction was then verified by the deletion of protease-associated domain from the wild-type enzyme. The thermal stability analysis showed that deletion of this domain improved the T50 (the temperature required to reduce initial activity by 50% in 30 min) of the enzyme from 71°C to 77°C. The melting temperature (Tm) of the enzyme also increased, which was measured by thermal denaturation experiments using circular dichroism spectroscopy. Further studies indicated that this deletion did not affect the activity and specificity of the enzyme toward aminoacyl-p-nitroanilines, but improved its hydrolytic ability toward a 12-aa-long peptide (LKRLKRFLKRLK) and soybean protein. These findings suggested the possibility of a simple technique for enzyme modification and the artificial enzyme obtained here was more suitable for the protein hydrolysis in food industry than the wild-type enzyme.
PMCID: PMC3954873  PMID: 24633010
17.  Profiling of Hepatocellular Carcinoma Cell Cycle Regulating Genes Targeted by Calycosin 
BioMed Research International  2013;2013:317926.
We cocultured calycosin with human hepatocellular carcinoma cell line (BEL-7402) to investigate the effect on cell proliferation. Calycosin can markedly block the cell growth in G1 phase (P < 0.01) on the IC50 concentration. There were seventeen genes involved in cell-cycle regulation showing differentially expressed in treated cells detected by gene chip. Eight genes were upregulated and nine genes were downregulated. Downregulated TFDP-1, CDKN2D, and SPK2 and upregulated CDC2 and CCNB1 might affect cell cycle of tumor cells. Furthermore, we checked the transcription pattern using 2D gel method to find different expression of proteins in human hepatocellular carcinoma cells after exposure to calycosin. Fourteen proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Twelve proteins expression were increased such as transgelin 2, pyridoxine 5′-phosphate, stress-induced-phosphoprotein 1, peroxiredoxin 1, endoplasmic reticulum protein 29, and phosphoglycerate mutase 1. Only thioredoxin peroxidase and high-mobility group box1 proteins' expression decreased. Both genes and proteins changes might be relate to the mechanism of antitumor effect under treatment of calycosin. In conclusion, calycosin has a potential effect to inhibit the BEL-7402 cell growth by inhibiting some oncogene expression and increasing anticancer genes expression, what is more, by blocking cell cycle.
PMCID: PMC3884961  PMID: 24455688
18.  ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes 
Toll-like receptors (TLRs) are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following lipopolysaccharide (LPS) stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as IL-6, IL-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.
PMCID: PMC4003182  PMID: 23241898
ATF4; cytokine; MyD88; TLR4 signaling pathway; TRIF
19.  A novel multiplex polymerase chain reaction assay for profile analyses of gene expression in peripheral blood 
Studies have demonstrated that inflammation has a key role in the pathogenesis of atherosclerosis due to the abnormal gene expressions of multiple cytokines. We established an accurate and precise method to observe gene expression in whole blood that might provide specific diagnostic information for coronary artery disease (CAD) and other related diseases.
The fifteen selected CAD-related genes (IL1B, IL6, IL8, IFNG, MCP-1, VWF, MTHFR, SELL, TNFalpha, ubiquitin, MCSF, ICAM1, ID2, HMOX1 and LDLR) and two housekeeping genes (ACTB and GK) as internal references have been measured simultaneously with a newly developed multiplex polymerase chain reaction (multi-PCR) method. Moreover, the precision was evaluated, and a procedure for distinguishing patients from the normal population has been developed based upon analyses of peripheral blood. A total of 148 subjects were divided into group A (control group without plaques), group B (calcified plaques) and group C (non-calcified plaques, and combination group) according dual-source CT criteria. Gene expression in blood was analyzed by multi-PCR, and levels of glucose and lipids measured in 50 subjects to explore the relationship among them.
The precision results of the multi-PCR system revealed within-run and between-run CV values of 3.695–12.537% and 4.405–13.405%, respectively. The profiles of cytokine gene expression in peripheral blood were set: a positive correlation between glucose and MCSF, HMOX1 or TNFalpha were found. We also found that triglyceride levels were negatively correlated with SELL gene expression in 50 subjects. Compared with controls, gene expression levels of IL1B, IL6, IL8 and MCP-1 increased significantly in group C.
A new multiple gene expression analysis system has been developed. The primary data suggested that gene expression was related to CAD. This system might be used for risk assessment of CVDs and other related diseases.
PMCID: PMC3445828  PMID: 22780915
Coronary artery disease; Gene expression profiling; Multiplex polymerase chain reaction; GeXP
20.  A novel differential diagnostic model based on multiple biological parameters for immunoglobulin A nephropathy 
Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis in China. An accurate diagnosis of IgAN is dependent on renal biopsies, and there is lack of non-invasive and practical classification methods for discriminating IgAN from other primary kidney diseases. The objective of this study was to develop a classification model for the auxiliary diagnosis of IgAN using multiparameter analysis with various biological parameters.
To establish an optimal classification model, 121 cases (58 IgAN vs. 63 non-IgAN) were recruited and statistically analyzed. The model was then validated in another 180 cases.
Of the 57 biological parameters, there were 16 parameters that were significantly different (P < 0.05) between IgAN and non-IgAN. The combination of fibrinogen, serum immunoglobulin A level, and manifestation was found to be significant in predicting IgAN. The validation accuracies of the logistic regression and discriminant analysis models were 77.5 and 77.0%, respectively at a predictive probability cut-off of 0.5, and 81.1 and 79.9%, respectively, at a predictive probability cut-off of 0.40. When the predicted probability of the equation containing the combination of fibrinogen, serum IgA level, and manifestation was more than 0.59, a patient had at least an 85.0% probability of having IgAN. When the predicted probability was lower than 0.26, a patient had at least an 88.5% probability of having non-IgAN. The results of the net reclassification improvement certificated serum Immunoglobulin A and fibrinogen had classification power for discriminating IgAN from non-IgAN.
These models possess potential clinical applications in distinguishing IgAN from other primary kidney diseases.
PMCID: PMC3488968  PMID: 22738421
Primary kidney disease; IgA nephropathy; Multiparameter analysis
21.  Study of the Effects of Total Flavonoids of Astragalus on Atherosclerosis Formation and Potential Mechanisms 
Astragalus mongholicus Bunge has long been used to treat cardiovascular disease in Chinese traditional medicine. However, its mechanisms are not fully understood. In this study, we explored potential mechanisms and protective effects of total flavonoids of Astragalus (TFA) on cardiovascular disease using in vitro experiments and diet-induced atherosclerotic rabbits. We identified six components and their proportion in TFA. The animal experiments showed that TFA significantly reduced plasma levels of total cholesterol and LDL cholesterol (P < 0.05 to 0.01), increased HDL cholesterol levels (P < 0.01), and reduced the aortic fatty streak area by 43.6 to 63.6% (P < 0.01). We also found that TFA scavenged superoxide and hydroxyl radicals and this effect increased with higher TFA concentration. In in vivo experiments, TFA effectively inhibited the free radical spectrum in the ischemia-reperfusion module. In conclusion, TFA was the active component of Astragalus mongholicus Bunge, which benefits cardiovascular disease attributing to the potent antioxidant activity to improve the atherosclerosis profile.
PMCID: PMC3306992  PMID: 22496932
22.  The Use of Principal Component Analysis in MALDI-TOF MS: a Powerful Tool for Establishing a Mini-optimized Proteomic Profile 
Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology has been applied to the exploration of biomarkers for early cancer diagnosis, but more effort is required to identify a single sensitive and specific biomarker. For early diagnosis, a proteomic profile is the gold standard, but inconvenient for clinical use since the profile peaks are quantitative. It would therefore be helpful to find a minimized profile, comprising fewer peaks than the original using an existing algorithm and compare it with other traditional statistical methods.
In the present study, principal component analysis (PCA) in the ClinProt-Tools of MALDI-TOF MS was used to establish a mini-optimized proteomic profile from gastric cancer patients and healthy controls, and the result was compared with t-test and Flexanalysis software.
Eight peaks were selected as the mini-optimized proteomic profile to help differentiate between gastric cancer patients and healthy controls. The peaks at m/z 4212 were regarded as the most important peak by the PCA algorithm. The peaks at m/z 1866 and 2863 were identified as deriving from complement component C3 and apolipoprotein A1, respectively.
PCA enabled us to identify a mini-optimized profile consisting of significantly differentiating peaks and offered the clue for further research.
PMCID: PMC3251008  PMID: 22229059
MALDI-TOF MS; mini-profile; PCA; proteomics
23.  Serum microRNA characterization identifies miR-885-5p as a potential marker for detecting liver pathologies 
Circulating miRNAs (microRNAs) are emerging as promising biomarkers for several pathological conditions, and the aim of this study was to investigate the feasibility of using serum miRNAs as biomarkers for liver pathologies. Real-time qPCR (quantitative PCR)-based TaqMan MicroRNA arrays were first employed to profile miRNAs in serum pools from patients with HCC (hepatocellular carcinoma) or LC (liver cirrhosis) and from healthy controls. Five miRNAs (i.e. miR-885-5p, miR-574-3p, miR-224, miR-215 and miR-146a) that were up-regulated in the HCC and LC serum pools were selected and further quantified using real-time qPCR in patients with HCC, LC, CHB (chronic hepatitis B) or GC (gastric cancer) and in normal controls. The present study revealed that more than 110 miRNA species in the serum samples and wide distribution ranges of serum miRNAs were observed. The levels of miR-885-5p were significantly higher in sera from patients with HCC, LC and CHB than in healthy controls or GC patients. miR-885-5p yielded an AUC [the area under the ROC (receiver operating characteristic) curve] of 0.904 [95% CI (confidence interval), 0.837–0.951, P<0.0001) with 90.53% sensitivity and 79.17% specificity in discriminating liver pathologies from healthy controls, using a cut off value of 1.06 (normalized). No correlations between increased miR-885-5p and liver function parameters [AFP (α-fetoprotein), ALT (alanine aminotransferase), AST (aspartate aminotransferase) and GGT (γ-glutamyl transpeptidase)] were observed in patients with liver pathologies. In summary, miR-885-5p is significantly elevated in the sera of patients with liver pathologies, and our data suggest that serum miRNAs could serve as novel complementary biomarkers for the detection and assessment of liver pathologies.
PMCID: PMC2990200  PMID: 20815808
biomarker; cirrhosis; liver pathology; microRNA; miR-885-5p; serum; AFP, α-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHB, chronic hepatitis B; FNH, focal nodular hyperplasia; GC, gastric cancer; GGT, γ-glutamyl transpeptidase; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocellular carcinoma; LC, liver cirrhosis; Mamm U6, mammalian U6; miRNAs, microRNAs; NC, normal control; qPCR, quantitative PCR; ROC, receiver operating characteristic; AUC, the area under the ROC curve; RT, reverse transcription; RT-preamp-qPCR, RT-preamplification-qPCR; snRNA, small nuclear RNA
24.  A survey on the distribution of healthy people with different anti-tumour ability 
The aim of the study was to explore the distribution of healthy people with different anti-tumour ability.
Material and methods
Leukocytes were separated by the Ficoll-Hypaque density gradient centrifugal method. Then they were mixed with A549, MCF-7 and Hela cells at different ratios. The survival rate for target cells was observed and counted by Fluoroskan. Immune function for 200 healthy people was analysed by flow cytometry.
The results obtained by confocal microscopy revealed that human blood leukocytes possessed direct anti-tumour activity. The survival rate for tumour cells was the lowest in the condition of 20:1 ratio of effector cells to target cells. We speculated that in 200 healthy people the leukocyte capacity for killing MCF-7 cells is stronger than the leukocyte capacity for killing A549 cells and Hela cells. We also found that the distribution for 200 healthy people with different anti-tumour ability was different for different tumour cells. The number of healthy people with the strongest anti-tumour ability was highest when the target cells were MCF-7 cells. Moreover, the survival of A549, MCF-7 and Hela cells was correlated with T, B and NK lymphocytes.
From the above, we can select healthy individuals with strong anti-tumour ability as anti-tumour donors according to their distribution with different anti-tumour ability, which opened up a new direction for fighting human cancer.
PMCID: PMC3298353  PMID: 22419943
innate immunity; tumour; leukocytes; polymorphonuclear leucocytes; distribution; healthy people
25.  A study of health effects of long-distance ocean voyages on seamen using a data classification approach 
Long-distance ocean voyages may have substantial impacts on seamen's health, possibly causing malnutrition and other illness. Measures can possibly be taken to prevent such problems from happening through preparing special diet and making special precautions prior or during the sailing if a detailed understanding can be gained about what specific health effects such voyages may have on the seamen.
We present a computational study on 200 seamen using 41 chemistry indicators measured on their blood samples collected before and after the sailing. Our computational study is done using a data classification approach with a support vector machine-based classifier in conjunction with feature selections using a recursive feature elimination procedure.
Our analysis results suggest that among the 41 blood chemistry measures, nine are most likely to be affected during the sailing, which provide important clues about the specific effects of ocean voyage on seamen's health.
The identification of the nine blood chemistry measures provides important clues about the effects of long-distance voyage on seamen's health. These findings will prove to be useful to guide in improving the living and working environment, as well as food preparation on ships.
PMCID: PMC2846872  PMID: 20219089

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