Initiator Integrated Poly(dimethysiloxane)-Based Microarray as a Tool for Revealing the Relationship between Nonspecific Interactions and Irreproducibility

Nonspecific interactions (NSIs) and irreproducibility greatly reduce the accuracy of antigen–antibody screening, which is key to the discovery of monoclonal antibody drugs and biomarkers identification. We previously developed a solid supporting material, polymer-coated initiator integrated poly­(dimethysiloxane) (iPDMS), which is able to provide near-zero background for microarray screening. Here, we applied two monoclonal antibodies (mAbs), namely, anti-FLAG and HM1, to screen an iPDMS-based peptide microarray with 2083 peptides from 62 proteins to evaluate NSIs and irreproducibility. In addition to recognizing their cognate epitopes, the two mAbs also cross-reacted with random sequences, especially when they were used at high concentrations. At 50 μg mL^–1, 295 peptides (14.2% of the peptide library) had positive reactions to anti-FLAG and only 39 peptides (1.9%) reacted positively to HM1. Virtually all cross-reactions disappeared when the [mAbs] reached 0.01 μg mL^–1. Reproducible experiments of 404 peptides at various [mAbs] showed that only specific interactions, molecular mimicry, and mimotope were reproducible between different experiments. These findings suggest that irreproducibility was at least partially caused by NSIs. We also demonstrated that repeating tests and mAb dilution could effectively avoid NSI-related irreproducibility in serological screening. This will not only largely simplify the data analysis, but will also make immunoassays more reliable for clinical research

Serum GP73 concentration was related with levels of different biochemical marker.

<p>A and B: serum GP73 concentration was correlated with ALT in patients with ALT ≥ 80 U/L, but nearly normal ALT was not. Although different HBV DNA levels had their different GP73 concentration (C), the correlation was not significant (D). Sample number may be one of most important causes. GP73 were also correlated with total bilirubin (F), especially, significantly correlated with serum ALB negatively (E).</p

Effects of gp73 recombinant protein on LX2 cells.

<p>Effects of gp73 recombinant protein on LX2 cells.</p

The Multivariate ordinal logistic regression analysis for the factors assocaited with Fibrogenesis.

<p>Note: Adjusted the factors including Sex, Age, ALT, total bilirubin, albumin, and platelet.</p

GP73 were stained in different liver tissue.

<p>GP73 was stained in brown. Arrow indicated positive cells. A: mild fibrosis (S1); B: significant fibrosis (S2); C: severe fibrosis (S3–4); D: cirrhosis (S4).</p

Serum GP73 concentration was correlated with liver stiffness (761 patients).

<p>A: Different GP73 levels were observed in patients with different groups of liver stiffness. B: serum GP73 concentration was correlated with liver stiffness. C and D: the ROC analysis of GP73 was performed on diagnosis of significant fibrosis and liver cirrhosis. The numbers after symbols “<”or “ = ” are p value.</p

Serum GP73 concentration related with liver stiffness ( FibroScan), ALT, Fibrosis grading, and serum HBV DNA.

a<p><b>.</b> 761 patients received liver stiffness measurements; <b>^b.</b> 472 patients with nearly normal ALT; ^c. 633 patients with chronic hepatitis B infections; * <i>P</i><0.05 Compared with patients with HBV DNA less than 4 Log.</p

Serum GP73 was correlated with grading of patients.

<p>A: serum GP73 was correlated with grading of 633 patients. B and C: serum GP73 was correlated with grading of 472 patients with nearly normal ALT. D, E, F: ROC analysis of GP73 was performed on diagnosing S2(D), G2(E), and cirrhosis (F) respectively.</p

Gp73 recombinant protein prompted LX2 cells proliferation.

<p>A: when the concentration of GP73 recombinant protein was above 20 ng/ml, the LX2 proliferation was prompted. B: GP73 recombinant protein up-regulated collagen III expression, but collagen I was not. C: GP73 expression evaluated in different cells <i>in vitro</i>.</p

A systematic investigation based on microRNA-mediated gene regulatory network reveals that dysregulation of microRNA-19a/Cyclin D1 axis confers an oncogenic potential and a worse prognosis in human hepatocellular carcinoma

<div><p>MicroRNAs (miRNAs) contribute to a wide variety of human diseases by regulating gene expression, leading to imbalances in gene regulatory networks. To discover novel hepatocellular carcinoma (HCC)-related miRNA-target axes and to elucidate their functions, we here performed a systematic investigation combining biological data acquisition and integration, miRNA-target prediction, network construction, functional assay and clinical validation. As a result, a total of 117 HCC differentially expressed miRNAs were identified, and 728 high confident target genes of these miRNAs were collected. Then, the interaction network of target genes was constructed and 221 key nodes with topological importance in the network were identified according to their topological features including degree, node-betweenness, closeness and K-coreness. Among these key nodes, Cyclin D1 had the highest node-betweenness, implying its bottleneck role in the network. Luciferase reporter assay confirmed that miRNA-19a, which was one of HCC downregulated miRNAs, directly targeted Cyclin D1 in HCC cells. Moreover, miR-19a might play inhibitory roles in HCC malignancy via regulating Cyclin D1 expression. Further clinical evidence also highlighted the prognostic potential of miR-19a/Cyclin D1 axis in HCC. In conclusion, this systematic investigation provides a framework to identify featured miRNAs and their target genes which are potent effectors in the occurrence and development of HCC. More importantly, miR-19a/Cyclin D1 axis might have promising applications as a therapeutic target and a prognostic marker for patients with HCC.</p></div