Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2

<p>Abstract</p> <p>Background</p> <p>The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in <it>E. coli</it>, for diagnosis of PCV infection.</p> <p>Results</p> <p>The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%.</p> <p>Conclusions</p> <p>This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera.</p

Calcineurin mediates acetylcholinesterase expression during calcium ionophore A23187-induced HeLa cell apoptosis

AbstractWe previously reported that acetylcholinesterase plays a critical role in apoptosis and its expression is regulated by Ca2+ mobilization. In the present study, we show that activated calpain, a cytosolic calcium-activated cysteine protease, and calcineurin, a calcium-dependent protein phosphatase, regulate acetylcholinesterase expression during A23187-induced apoptosis. The calpain inhibitor, calpeptin, and the calcineurin inhibitors, FK506 and cyclosporine A, inhibited acetylcholinesterase expression at both mRNA and protein levels and suppressed the activity of the human acetylcholinesterase promoter. In contrast, overexpression of constitutively active calcineurin significantly activated the acetylcholinesterase promoter. Furthermore, we identify a role for the transcription factor NFAT (nuclear factor of activated T cells), a calcineurin target, in regulating the acetylcholinesterase promoter during ionophore-induced apoptosis. Overexpression of human NFATc3 and NFATc4 greatly increased the acetylcholinesterase promoter activity in HeLa cells treated with A23187. Overexpression of constitutive nuclear NFATc4 activated the acetylcholinesterase promoter independent of A23187, whereas overexpression of dominant-negative NFAT blocked A23187-induced acetylcholinesterase promoter activation. These results indicate that calcineurin mediates acetylcholinesterase expression during apoptosis

Effect of cooling pad installation on indoor airflow distribution in a tunnel-ventilated laying-hen house

Extra cooling pads on the sidewalls are needed for larger poultry houses using tunnel ventilation system. Preliminary study showed that the airflow velocity going through different aisles varies greatly when the extra pads are installed at the end of sidewalls, making a “[”-shape air inlet. Combined with field tests, the CFD (computational fluid dynamics) technology was used to study the uniformity of airflow distribution in a tunnel-ventilated laying-hen house. The air distribution was first monitored in a layer house to find the main reason resulting in the variations of airflows in different aisles. Then CFD simulations were carried out with different distances (D=2 m, 3 m or 4 m) between the pads on end-wall and the extra pads on side walls. The field test showed that airflow streams from the different groups of cooling pads collided vertically at the house corners, mixed with each other, then flew towards the center of the house. This was the main reason that the wind speed in the middle aisle was much higher than in other aisles, leaving large zones of lower ventilation in the aisles adjacent to the sidewalls. The results of CFD simulations indicated that air distributions could be significantly improved when the extra pieces of pads were moved away for an appropriate distance from the end cooling pads. As far as conventional poultry house with a span of 12 m, the air speeds in different aisles were more uniform when this distance was about 3 m

Proteome analysis of human colorectal cancer tissue using 2-D DIGE and tandem mass spectrometry for identification of disease-related proteins

Laser capture microdissection and two-dimensional difference gel electrophoresis were used to establish the proteomic profiles for tumor and matched adjacent tissues from 12 patients. Differential protein spots were identified by mass spectrometric analysis. The cDNA of the differential protein was transfected into colorectal cancer cells, and the biological behavior of these cells was observed. The proteomic profile in colorectal cancer tissues was significantly different from that in normal adjacent tissues. There was a 1.5-fold difference and 60 differential protein spots between cancer and adjacent tissues. Ten differential protein spots were analyzed. Among them, two protein spots were down-regulated and eight protein spots were up-regulated in the primary tumor tissues. After identification by mass spectrometry, the two down-regulated proteins were carbonic anhydrase II and protein disulfide isomerase, and these eight up-regulated proteins included APC-stimulated guanine nucleotide exchange factor, phosphoglycerate kinase 1, fumarate hydratase, aldolase A, activator protein 2B, glutathione S-transferase A3, Arginase and zinc finger protein 64 homolog. After been transfected with carbonic anhydrase II, the invasive ability, mobility and drug resistance of colon cancer lovo cells were significantly reduced. The proteomic profile was significantly different between colorectal cancer tissues and normal adjacent tissues. The down-regulation of carbonic anhydrase II and protein disulfide isomerase and up-regulation of APC-stimulated guanine nucleotide exchange facto, aldolase A, glutathione S-transferase A3 and arginase were correlated with the onset of colorectal cancer.Key words: Colorectal cancer, proteomics