Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Deoxynivalenol (DON) degradation and peroxidase enzyme activity in submerged fermentation

This work aims to evaluate deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in a submerged fermentation system and to correlate it to the activity of oxydo-reductase enzymes. The submerged medium consisted of sterile distilled water contaminated with 50 μg of DON and 4 × 10(6) spore.mL-1 inoculum of Aspergillus oryzae and Rhizopus oryzae species, respectively in each experiment. Sampling was performed every 24 hours for monitoring the peroxidase specific activity, and every 48 hours for determining mycotoxin levels. Results showed that the fungi species were able to decrease DON levels as the peroxidase activity increased. The 48 hours fermentation interval presented the highest peroxidase specific activity (ΔABS/minute.μg.protein-1), 800 and 198, while the highest DON degradation velocity was 10.8 and 12.4 ppb/hour, respectively in both cases for Rhizopus oryzae and Aspergillus oryzae.

Influence of the degree of hydrolysis and type of enzyme on antioxidant activity of okara protein hydrolysates

Abstract The objective of this work was to evaluate the antioxidant activity of protein hydrolysates obtained by the enzymatic hydrolysis of okara using an endopeptidase (Alcalase) and exopeptidase (Flavourzyme). The reaction was monitored by the pH-stat procedure in which five aliquots were collected during the hydrolysis by each enzyme, corresponding to different degrees of hydrolysis (DH). The antioxidant activities of the aliquots were evaluated by the ABTS, DPPH and FRAP methods. For the hydrolysates obtained using Alcalase, the antioxidant activities increased from: 68.6 to 99.5% (ABTS), 14.5 to 17.7% (DPPH) and 222.6 to 684.9 µM Trolox (FRAP), when the DH varied from 0 to 33.6%. With respect to Flavourzyme, the results were: 67.2 to 88.2% (ABTS), 9.5 to 18.5% (DPPH) and 168.0 to 360.3 µM Trolox (FRAP), when the DH increased up to 5.8%. The results showed that the protein hydrolysates had antioxidant capacities, which were influenced by the degree of hydrolysis and the type of enzyme.