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.
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.
Subclinical atherosclerosis; Lipid ratio; Intima-media thickness; Plaque
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.
Bacillus subtilis; aminopeptidase; substrate specificity; saturation mutagenesis; protein hydrolysis
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.
Biomarker discovery; Nanoporous silica film; Peptide; MALDI-TOF MS; Hepcidin
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.
leptin; lung adenocarcinoma; cisplatin/pemetrexed; prognosis
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.
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.
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.
ATF4; cytokine; MyD88; TLR4 signaling pathway; TRIF
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.
Coronary artery disease; Gene expression profiling; Multiplex polymerase chain reaction; GeXP
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.
Primary kidney disease; IgA nephropathy; Multiparameter analysis
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.
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.
MALDI-TOF MS; mini-profile; PCA; proteomics
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.
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
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.
innate immunity; tumour; leukocytes; polymorphonuclear leucocytes; distribution; healthy people
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.