The use of slaughterhouse-obtained small intestinal tissue as control material in histological studies should be applied with prudence

This study aimed to evaluate the reliability of slaughterhouse-obtained small intestinal tissue as control material in equine colic research where molecular stress responses in small intestinal tissue are investigated. For this purpose, small intestinal samples from colic horses were collected during surgery or immediately after euthanasia at the oral border of strangulation resection sites and routinely processed for histopathology (i.c. rinsed with 4°C Krebs' solution, fixated overnight with 4% neutral buffered formaldehyde (FH) at room temperature). Control samples consisted of pieces of mid-jejunum, collected at the slaughterhouse and routinely processed for histopathology under 4 different conditions. The 4 conditions differed with regard to incubation and fixation temperature and whether or not oxygenated Krebs' solution was used. Histological scoring revealed that slaughterhouse samples had a higher mean lesion score (P<0.001) than colic samples. In addition, more slaughterhouse samples had a higher mean inflammation score than colic samples (P=0.001). The inflammatory cells in the small intestine consisted mostly of eosinophils and as such were very suggestive for parasitic infestation. Hypoxia-inducible factor-1α (HIF1α) nuclear immunoreactivity was more pronounced in slaughterhouse tissue, probably as a result of the delay between slaughter and sampling (P=0.034). The histopathological score (P=0.291), the inflammation score (P=0.248) and the HIF1α nuclear immunoreactivity (P=0.538) did not differ between the different collection protocols. It is concluded that slaughterhouse-obtained small intestinal tissue shows distinct alterations and that its use as control tissue when evaluating molecular stress responses should be applied with prudence.     

The development of Escherichia coli and Listeria monocytogenes variants resistant to high‐pressure carbon dioxide inactivation

Aims: The objective of this study was to investigate whether bacterial cells could develop resistance (as a part of their adaptation strategy) to high‐pressure CO₂ (HPCD) inactivation. Methods and Results: Alternating cycles of exposure to pressurized CO₂ (10·5 MPa, 35°C, 400 min^?1, 70% working volume ratio during 10 min) and re‐growth of the surviving subpopulation were used to investigate possible increases in the resistance of Escherichia coli and Listeria monocytogenes to HPCD. The results show an increased resistance of both pathogens tested after seven cycles of inactivation. Increase in the resistance after 15 cycles resulted in a difference of 2·4 log CFU ml^?1 in log N₀/N_i when parental (N₀) and treated cultures (N_i) of E. coli and L. monocytogenes were compared. Conclusions: Current findings indicate the ability of micro‐organisms to adapt to HPCD preservation technology. Significance and Impact of the Study: The occurrence of HPCD‐resistant micro‐organisms could pose a new hazard to the safety and stability of HPCD‐processed foods.     

FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant, a sighted variant of the FVB/N mouse strain suitable for behavioral analysis

Mice of the FVB/N strain are severely visual impaired as a result of tyrosinase gene defects, leading to a deficiency of the key enzyme for melanin synthesis in skin and eye and of cyclic guanosine monophosphate phosphodiesterase gene defects, which results in albinism (Tyr(c/c)) and retinal degeneration (Pde6b(rd1/rd1)), respectively. Nevertheless, FVB/N mice are commonly used for the generation of transgenic animals because of their large, strong pronuclei and high breeding performance. However, due to visual impairment of the FVB/N animals, the resulting transgenic animals cannot be used in tests that depend on vision, including tests of cognitive behavior. Therefore, we have bred a sighted version of the FVB/N strain by an outcross between FVB/N and 129P2/OlaHsd, followed by repeated backcrosses to FVB/N mice while selecting against albinism and homozygosity of the retinal degeneration mutation. After 11 generations of backcrossing, sighted animals were intercrossed to generate the congenic FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant strain, which is pigmented (Tyr(c-ch)/(c-ch)) and devoid of the genetic predisposition to retinal degeneration. The accurate visual abilities of the FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant mice, for which we propose the name FVBS/Ant, demonstrated a clear visual evoked potential in the presence of normal eye histology and improved performance in the Morris water maze test.     

Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies

The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p < 0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significantly increased proteins: alpha actin, tropomyosin alpha chain and creatine kinase M chain (CKM). CKM was represented by multiple spots probably due to posttranslational modification, one of which appeared to be unique for tying-up. Since changes in the rates of synthesis and degradation of proteins are likely to lead to pathological conditions, identification of differentially expressed proteins in acute tying-up might result in the finding of more specific diagnostic markers and in new hypotheses for the common mechanisms that result in this condition. Our findings point to a specific isoform of CKM as a novel biomarker for tying-up suggesting that altered energy distribution within muscle cells is part of the disease etiology.     

Mouse models of Japanese encephalitis virus infection: A systematic review and meta-analysis using a meta-regression approach

BackgroundJapanese encephalitis (JE) virus (JEV) remains a leading cause of neurological infection across Asia. The high lethality of disease and absence of effective therapies mean that standardised animal models will be crucial in developing therapeutics. However, published mouse models are heterogeneous. We performed a systematic review, meta-analysis and meta-regression of published JEV mouse experiments to investigate the variation in model parameters, assess homogeneity and test the relationship of key variables against mortality.Methodology/ Principal findingsA PubMed search was performed up to August 2020. 1991 publications were identified, of which 127 met inclusion criteria, with data for 5026 individual mice across 487 experimental groups. Quality assessment was performed using a modified CAMARADES criteria and demonstrated incomplete reporting with a median quality score of 10/17. The pooled estimate of mortality in mice after JEV challenge was 64.7% (95% confidence interval 60.9 to 68.3) with substantial heterogeneity between experimental groups (I^2 70.1%, df 486). Using meta-regression to identify key moderators, a refined dataset was used to model outcome dependent on five variables: mouse age, mouse strain, virus strain, virus dose (in log₁₀PFU) and route of inoculation. The final model reduced the heterogeneity substantially (I^2 38.9, df 265), explaining 54% of the variability.Conclusion/ SignificanceThis is the first systematic review of mouse models of JEV infection. Better adherence to CAMARADES guidelines may reduce bias and variability of reporting. In particular, sample size calculations were notably absent. We report that mouse age, mouse strain, virus strain, virus dose and route of inoculation account for much, though not all, of the variation in mortality. This dataset is available for researchers to access and use as a guideline for JEV mouse experiments.     

HERG channel (dys)function revealed by dynamic action potential clamp technique

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.     

Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells,an In Vitro Model for the Porcine Gastrointestinal Tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H₂DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.