Introduction: The aim of the experiment was to establish the enterotoxigenic Escherichia coli K88 (ETEC K88)-induced BALB/c mouse duodenum inflammation model. Material and Methods: Mice were administered different concentrations of E. coli K88 (1.0 × 107-109 CFU/mL) for 3 d by means of an esophageal catheter. Results: The results showed that the treated group expressed several significant clinical symptoms, such as reduced dietary demands and weight loss, an increased presence of IL-1α, TNF-α, and MPO in the peripheral blood, and some pathological changes in the duodenum. On the 6th-8th days, the body weight of the mice was the lowest. On the 8th day, there were significant differences in IL-1α, TNF-α, and MPO levels compared to the control group (P < 0.05), the gap between the duodenum mucous layer and the muscular layer had widened, the number of goblet cells was increased, and the inflammatory infiltrate and inflammation changes in the lamina propria and the mucous layer were the most obvious. Conclusion: The duodenum inflammation was the most severe on day 8; thus, the model was successfully established. In addition, varying concentrations of ETEC K88 did not significantly influence the duodenum inflammation (P > 0.05).
Objective To investigate the 23 bp and 12 bp insertion/deletion (indel) mutations within the bovine prion protein (PRNP) gene in Chinese dairy cows, and to detect the associations of two indel mutations with BSE susceptibility and milk performance.
Methods Based on bovine PRNP gene sequence, two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed. The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations. Moreover, based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study, the corrections between the two indel mutations and BSE susceptibility were tested, as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.
Results In the analyzed Chinese Holstein population, the frequencies of two “del” alleles in 23 bp and 12 bp indel muations were more frequent. The frequency of haplotype of 23del-12del was higher than those of 23del- 12ins and 23ins-12del. From the estimated r2 and D’ values, two indel polymorphisms were linked strongly in the Holstein population (D’ = 57.5%, r2 = 0.257). Compared with the BSE-affected cattle populations from the reported data, the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations (P < 0.05 or P < 0.01). Similarly, there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle (P < 0.05 or P < 0.01). Moreover, association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population, but no significant differences were found (P > 0.05).
Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits, which layed the foundation for future selection of resistant animals, and for improving health conditions for dairy breeding against BSE in China.
Differential metabolites (DMs) between cows with inactive ovaries (IO) and oestrous (E) cows were screened and metabolic pathways of DMs associated with IO were determined.
Material and Methods
Cows at 50 to 60 days (d) postpartum from an intensive dairy farm were randomly selected and allocated into an E group (n = 16) or an IO group (n = 16) according to a pedometer and rectal examinations. Their plasma samples were analysed by liquid chromatography–mass spectrometry (LC–MS) to compare plasma metabolic changes between the E and IO groups. Multivariate pattern recognition was used to screen the DMs in the plasma of IO cows.
Compared with normal E cows, there were abnormalities in 20 metabolites in IO cows, including a significantly decreased content (VIP > 1, P < 0.05) of cholic acid, p-chlorophenylalanine, and arachidonic acid, and a significantly increased content (VIP > 1, P < 0.05) of tyramine, betaine, L-phenylalanine, L-glutamate, D-proline, L-alanine, and L-pyrophosphate. Five DMs (cholic acid, D-proline, L-glutamate, L-alanine, and L-pyroglutamic acid) with higher variable importance in projection (VIP) values between groups were validated by ELISA with blind samples of re-selected cows (IO, 50 to 60 d postpartum) and the validated results were consistent with the LC–MS results.
The 20 DMs in IO cows during the peak of lactation indicated that the pathogenesis of IO was involved in complex metabolic networks and signal transduction pathways. This study provides a basis for further exploration of the pathogenesis and prevention of IO in cows in the future.
α-Damascone is widely used in perfumes. However, the manufacture of α-damascone remains challenging owing to the limitations of current production processes. Herein, α-damascone was successfully synthesized from α-ionone using a new route involving only four steps, namely oximization, epoxidation, dehydration, and reduction. The total yield was 54.9% with a final chemical purity of 97% (by GC). Only water, cyclohexane, and ethanol were used in the reactions except in the purification step, and all solvents could be recycled. The structures of the intermediates and target compound were identified by 1H NMR and 13C NMR analyses and MS experiments. This route is a simple and successful method for the industrial preparation of α-damascone.
Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein and a unique 1-Cys Prdx of the peroxiredoxin family. The expression and regulation of Prdx6 are implicated in numerous physiological and pathological processes.
Material and Methods: Eight stepwise truncated DNA fragments obtained from the 5′-flank region of the Prdx6 gene were prepared and subcloned into the pSEAP2-Enhancer vectors. To investigate the transcriptional activity of the truncated DNA fragments, the recombinant plasmids were transfected into the COS-1 cells and the transcriptional activity was measured via assaying the expression of the reporter gene of the secreted alkaline phosphatase.
Results: A 3.4 kb 5′-upstream flank region of the Prdx6 gene was cloned and sequenced. The region from −108 nt to −36 nt of the 5′-flanking region of the Prdx6 gene contained basal transcriptional activity.
Conclusion: This result provides the basis for further studies on the gene regulation of the Prdx6-mediated biological processes and on screening for the transacting factors that interact with cis-acting elements of the Prdx6 gene promoter.
Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection.
Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined.
Results: The core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit.
Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.
Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis.
Material and Methods
Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR.
The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity.
OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.
The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms.
Material and Methods
Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed.
The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R2) of the standard curve was 0.999. The sensitivity of the method was 103 CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude.
In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.