Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

Objective: To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini(OV)-associated CCA in OV/dimethylnitrosamine(DMN)-induced CCA hamster model. Methods: Nine Syrian hamsters were divided into 3 groups as follows(n=3 each): normal(healthy control group); OV group; and OV/DMN group(CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavilin A(Con A)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LCMS/MS. Results: Among the 24 Con A-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1(NDRG1) and fetuin-B(FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN(CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform x3(NSFL1C) was found only in the samples from OV/DMN(CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. Conclusions: NDRG1, FETUB and NSFL1 C glycoproteins isolated by Con A-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1 C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

Identification of novel biomarkers for adult-onset-immunodeficiency(AOID)syndrome using serum proteomics

Objective:To identify the candidate protein biomarkers of adult-onset-immunodeficiency(AOID) syndrome using serum proteomics. Methods:Screening and verification phases were performed in the study. A total of 97 serum samples were classified into three groups:AOID patients with opportunistic infections(active AOID),AOID patients without opportunistic infections(inactive AOID),and healthy control. In the screening phase,pooled sera collected from patients and healthy control in each group were separated by 2D-gel electrophoresis,analyzed for differentially expressed proteins and identified for biomarkers using LC/MS. In the verification phase,the protein candidates were selected for confirmation by western blotting. Results:The analysis revealed 35 differentially expressed proteins. Three proteins including haptoglobin,gelsolin,and transthyretin,were selected for verification. The results showed that the levels of haptoglobin in both active and inactive AOID groups were significantly higher than that in the control group,while the levels of gelsolin in the active AOID group were significantly lower than that in the inactive AOID group. The level of transthyretin in the active AOID group was also significantly lower than that in the control group. Conclusions:The comparison of serum proteins between the three groups revealed three candidates which are related to chronic inflammatory diseases. Haptoglobin and transthyretin biomarkers could be applied in clinical assessment for monitor of disease outcome,including for the study of AOID pathogenesis.

Nevirapine induces apoptosis in liver(HepG2) cells

Objective: To generate insights into the mechanism of NVP induced hepatotoxicity. Methods: Liver(HepG2) cells were cultured with various concentrations of NVP. This cell line was chosen because it has low expression of cytochrome P450, allowing evaluation of the effects of NVP rather than specific metabolites. Cytotoxicity was determined using a proliferation assay and cell numbers were monitored using trypan blue exclusion assay for long term culture experiments and apoptosis induction was determined by morphological and biochemical investigation. Results: HepG2 cells treated with the highest concentration of NVP tested(819 μM) initially showed a rounded morphology and all cells had died by week three of exposure. Nuclear condensation and fragmentation, increased Annexin V/propidium iodide staining and caspase 9 activation all supported the induction of apoptosis in HepG2 cells in response to NVP treatment. Conclusions: There is a clear induction of apoptosis in response to NVP which suggests that NVP has significant cytotoxicity, over and above any cytotoxicity of metabolites and may contribute directly to patient hepatotoxicity.

Purification and Identification of Glutathione S-transferase in Rice Root under Cadmium Stress

Cadmium (Cd) contamination in paddy soils poses a serious threat to the production and quality of rice.Among various biochemical processes related to Cd detoxification in rice,glutathione S-transferase (GST) plays an important role,catalyzing Cd complexation with glutathione (GSH) and scavenging reactive oxygen species (ROS) in cells.In this study,a hydroponic experiment was conducted to investigate the response of GST isozymes in rice roots upon Cd exposure.Results showed that the GST activity in rice roots was clearly enhanced by 50 μmol/L Cd treatment for 7 d.The GST isozymes were purified by ammonium sulphate precipitation,gel filtration chromatography and affinity chromatography.After being separated by SDS-PAGE and visualized by silver staining,GSTU6 was identified by in-gel digestion,MALDI-TOF-MS analysis and peptide mass fingerprint.The results confirm the vital function of tau class rice GST in Cd detoxification.