A globotetraosylceramide (Gb4) receptor-based ELISA for quantitative detection of Shiga toxin 2e

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb₄) receptor by ELISA (Gb₄-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb₄-ELISA increased linearly with Stx2eB concentration in the range of 20–2,500 ng/ml. The Gb₄-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.     

The high prevalence of hepatitis E virus infection in wild boars in Ibaraki Prefecture,Japan

Hepatitis E virus (HEV) is known as a causative agent of zoonosis and food poisoning. Pigs and some species of wild animals, including wild boar, are known to be a reservoir of HEV. In this study, we investigated the situation regarding HEV infection in wild boars in Ibaraki Prefecture, Japan. Serum, liver and feces samples from 68 animals were collected, and the presence or absence of HEV genomic RNA and HEV antibodies were analyzed. The viral genome was detected in samples from 7 (10.3%) animals, with all HEVs classified as genotype 3, subtype 3b. HEV antibodies were detected in samples from 28 (41%) animals. This report demonstrates for the first time the high prevalence of HEV infection in wild boars in Ibaraki Prefecture.     

Analysis of the effect of feedback feeding on the farm-level occurrence of porcine epidemic diarrhea in Kagoshima and Miyazaki Prefectures,Japan

When a large-scale epidemic of porcine epidemic diarrhea (PED) occurred in 2013 in Japan, feedback feeding (feeding feces and gut tissues of infected piglets) was attempted to impart immunity to sows and immunize nursing piglets via breastfeeding. This study evaluated the effect of feedback feeding on PED control at 172 farms in Kagoshima and Miyazaki Prefectures. Univariable and multivariable generalized linear models were used to analyze the associations between conduct of feedback feeding and damage from the outbreak (outbreak period and the number of piglet deaths) at the farm level. The within-farm outbreak period shortened over time after the regional outbreak began on Kyushu Island (P=0.009) and was longer on large-scale farms (mean 66.0 days, P=0.003) than small-scale farms (29.4 days) and on farms that used feedback feeding (145.2 days, P=0.059) than those that did not (66.0 days). The number of dead piglets decreased over time since the first regional case (P<0.001) and was higher at farrow-to-finish farms (3.8 piglets/sow, P<0.001) than reproduction farms (0.7 piglets/sow). The effect of feedback feeding on the number of dead piglets was not significant, but its interaction term with farm style had a significant effect (5.0 more piglet deaths at reproduction farms than fallow-to-finish farms, P=0.001). These results suggest that feedback feeding made the damage from PED worse, though it was well established at a later stage of the regional PED epidemic.     

Morphometric evaluation of canine hepatocellular carcinoma using computed tomography: a promising tool for predicting malignancy

The size of canine focal liver lesions (FLLs) is known to be one of the predicting criteria for malignancy. However, there are discrepancies for the measurement of maximum lesion size, resulting in contradicting results among studies and incidences of false positive outcomes. Thus far, the morphometric changes of FLLs for distinguishing malignancy from benignancy remains undocumented. This study aimed to investigate morphometric characteristics of FLLs using computed tomography (CT). CT images of 40 dogs with histopathological confirmation of 49 liver lesions, including 39 hepatocellular carcinomas and 10 nodular hyperplasias were retrospectively reviewed. The morphometric parameters including size (long and short axis diameters measured on transverse image), shape (measured by long to short axis (L/S) ratio), volume, and surface appearance of a liver lesion were evaluated using univariate and stepwise multivariate analyses, respectively. The results of univariate analysis showed that long and short axis diameters, L/S ratio, volume, and surface appearance of a lesion were significantly different between hepatocellular carcinomas and nodular hyperplasias. Multivariate analysis revealed that short axis diameter (>3.30 cm; odds ratio (OR): 36.1, 95% confidence interval (CI): 3.36–387.05, P=0.0031) and L/S ratio (>1.23; OR: 18.1, 95% CI: 1.61–205.12, P=0.0191) were independent predictors of malignancy, with the area under the curve of 0.9154. These results suggest that the combination of short axis diameter and L/S ratio is a promising tool for predicting liver malignancy with outstanding discriminating ability.     

Inheritance of resistance to brown leaf spot disease in gentians

Brown leaf spot caused by Mycochaetophora gentianae is a serious disease on gentian in Japan. Previous studies revealed that Gentiana triflora cultivars were susceptible to M. gentianae and that G. scabra cultivars and their interspecific hybrid cultivars were resistant. We subsequently analyzed the mode of inheritance of resistance to M. gentianae in gentians using several populations derived from crosses between G. scabra and G. triflora. Parental G. scabra and G. triflora lines, F₁ and BC₁ plants were inoculated with a conidial suspension and assessed for susceptibility to M. gentianae. All tested F₁ plants were resistant to M. gentianae, indicating that the resistance of G. scabra was inherited dominantly. Segregation ratios in backcross progenies using G. scabra line OK fit the Mendelian ratio of 1:1, showing the involvement of a single dominant locus. These results indicate that the resistance to M. gentianae in G. scabra line OK is controlled by a single dominant allele designated as gentian brown leaf spot resistance (GBLS-1). Finding this allele should facilitate future breeding efforts to develop resistant gentian cultivars.     

Differentiation of skeletal muscle Mesenchymal progenitor cells to myofibroblasts is reversible

Accumulation of intramuscular adipose tissue (IMAT) and development of fibrous tissues due to accumulation of collagen both affect meat quality such as tenderness, texture, and flavor. Thus, it is important for the production of high‐quality meat to regulate the amount of adipose and fibrous tissues in skeletal muscle. IMAT is comprised of adipocytes, while collagens included in fibrous tissues are mainly produced by activated fibroblasts. Both adipocytes and fibroblasts are differentiated from their common ancestors, called mesenchymal progenitor cells (MPC). We previously established rat MPC clone, 2G11 cells. As several reports implicated the plasticity of fibroblast differentiation, in the present study, using 2G11 cells, we asked whether myofibroblasts differentiated from MPC are capable of re‐gaining adipogenic potential in vitro. By treating with bFGF, their αSMA expression was reduced and adipogenic potential was restored partially. Furthermore, by lowering cell density together with bFGF treatment, 2G11 cell‐derived myofibroblasts lost αSMA expression and showed the highest adipogenic potential, and this was along with their morphological change from flattened‐ to spindle‐like shape, which is typically observed with MPC. These results indicated that MPC‐derived myofibroblasts could re‐acquire adipogenic potential, possibly mediated through returning to an undifferentiated MPC‐like state.     

Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.     

Simple method for isolation of glyceraldehyde 3‐phosphate dehydrogenase and the improvement of myofibril gel properties

Porcine glycoliytic enzyme, glyceraldehyde 3‐phosphate dehydrogenase (G3PD) was prepared effectively by a combination of ethylene diamine tetra‐acetate (EDTA) pretreatment and affinity purification. After salting out of porcine sarcoplasmic proteins (SP) with ammonium sulfate at 75% saturation, the obtained supernatant (SP‐f3) was treated with EDTA, leaving G3PD in the supernatant (G3PD‐E) and most other SPs in the precipitate. At that time, the separation of G3PD‐E required more than 20 mmol/L EDTA. G3PD‐E was then subjected to affinity purification by batchwise method using blue‐sepharose CL‐6B, and purified G3PD (G3PD‐AP) was obtained using 2 mol/L potassium chloride (KCl) as an eluent. Texture analysis showed that the hardness, adhesiveness and gumminess of the myofibril gel at 0.2‐mol/L NaCl increased with the addition of G3PD‐AP. Scanning electron microscopy revealed that the G3PD‐AP reinforced the gel network of the myofibril. However, scanning electron micrograph analysis showed that the network‐structure of the gel by the addition of G3PD‐AP developed in a different manner from that by adding 0.6 mol/L NaCl. These results showed that glycolytic enzyme, G3PD, contributes to the improvement of the rheological properties of meat products.     

Protein structures forming the shell of primitive bacterial organelles

Bacterial microcompartments are primitive organelles composed entirely of protein subunits. Genomic sequence databases reveal the widespread occurrence of microcompartments across diverse microbes. The prototypical bacterial microcompartment is the carboxysome, a protein shell for sequestering carbon fixation reactions. We report three-dimensional crystal structures of multiple carboxysome shell proteins, revealing a hexameric unit as the basic microcompartment building block and showing how these hexamers assemble to form flat facets of the polyhedral shell. The structures suggest how molecular transport across the shell may be controlled and how structural variations might govern the assembly and architecture of these subcellular compartments.