Simple, specific, sensitive and rapid loop-mediated method for detecting Yersinia enterocolitica.

Yersinia enterocolitica (YE) is a main pathogenic bacterium causing diarrhea and yersiniosis occurs in both developed and developing countries with high incidence. YE in contaminated food is able to survive for a long duration even under cold storage, thereby enhancing the risk of food infection. In this study, a new loop-mediated isothermal amplification (LAMP) method showing the characteristics of simplicity, rapidity, high specificity and sensitivity was established by targeting outL of pathogenic YE. Two inner-primers and outer-primers were designed and LAMP reaction was optimized for Mg2+, betaine, dNTPs and inner primers concentrations, reaction temperature and time. Sensitivity and specificity of the LAMP assay was evaluated using YE genomic DNA and those of 44 different bacteria strains, respectively. Validation of LAMP detection method was by employing meat samples spiked with varying CFU of YE. The optimized LAMP assay was specific, capable of detecting 97 fg of genomic DNA (equivalent to 37 genome copies) of YE (100-fold more sensitive than PCR) and 80 CFU/ml of YE-spiked meat samples based on ethidium bromide stained amplicon bands on agarose gel-electrophoresis and on GelRed fluorescence of the LAMP reaction solution, respectively. This rapid, sensitive and specific LAMP technique should enable application in field inspection of Y. enterocolitica in food.

Correlation Analysis of Anti-Cardiolipin Antibody/D Dimer/C-Reactive Protein and Coronary Artery Lesions/Multiple-Organ Damage in Children With Kawasaki Disease.

Aim: Kawasaki disease (KD) is a systemic vasculitis with unknown etiology. In addition to cardiovascular system involvement, it can also have other multiple organs involved. This study is aimed at investigating the correlation between anti-cardiolipin antibody (ACA)/D dimer/C reactive protein (CRP) and coronary artery lesions (CAL)/multiple-organ lesions in children with KD. Methods: Retrospective analysis was performed in 284 KD/IKD patients from May 2015 to April 2016. Among them, 175 were males (61.6%), with average age of 2 years and 5 months old. Patients were divided into ACA+ group and ACA- group, elevated D dimer group (DDE) and normal D dimer group (DDN), and coronary artery injury (CAL) group and non-coronary artery injury (NCAL) group. Results: ACA was most likely tested positive in younger KD children (p < 0.05). ACA+ and hypoproteinemia were correlated with CAL, thrombocytosis, and granulocytopenia (p < 0.05-0.01). Levels of cTnI and CK in the CAL group were significantly higher than those in the NCAL group (p < 0.05). CAL was more frequently detected in younger patients and patients with prolonged fever, later IVIG treatment, and elevated CRP over 100 mg/l, but there was no statistically significant difference (all p > 0.05). In the KD with DDE group, the incidence of granulopenia, thrombocytosis, myocardial damage, cholestasis, hypoproteinemia, and aseptic urethritis was significantly higher than that in the KD with DDN group (p < 0.05-0.01). However, elevated D dimer was not associated with CAL. CRP elevation was highly correlated with D dimer, but not with CAL. Conclusion: Higher incidence of CAL and myocardial damage occurred in KD patients with positive ACA and hypoproteinemia. In the current study, ACA was only tested for positive and negative, which is a limitation to this study. To further elucidate the association, ACA titers would establish its significance in drawing a conclusion for the significance of ACA in CAL and myocardial damages. In addition, higher incidence of CAL occurred in younger patients. The higher D dimer was associated with increased multiple-organ damage (MOD). CRP was closely correlated with D dimer, but not correlated with ACA and CAL.

The Complementary Relationship Between Echocardiography and Multi-Slice Spiral CT Coronary Angiography in the Diagnosis of Coronary Artery Thrombosis in Children With Kawasaki Disease.

Aim: To compare the diagnostic values by using transthoracic echocardiography (ECHO) and multi-slice spiral CT coronary angiography (CTCA) for identifying coronary artery thrombosis in children with Kawasaki disease (KD). Methods: Total 97 KD children with coronary artery dilation complications in our hospital from June 2012 to December 2020 were included in the study. CTCA and ECHO were performed after over 1 month of illness. Results: Coronary artery thrombosis was found in 14 out of 97 patients. Among them, 10 were identified as positive by CTCA, 9 were identified as positive by ECHO, and 5 were identified as positive by both CTCA and ECHO. Conclusion: Both CTCA and ECHO can be used to diagnose coronary artery thrombosis. ECHO has advantage in identifying low-density thrombus, and CTCA is better for the clot in distal coronary artery. They can complement each other.

Correlation Between Arrhythmia and the Prognosis in Children With EFE/LVNC/DCM.

Aim: To explore the correlation between different phenotypes of arrhythmia and the prognosis in children with EFE/LVNC/DCM. Methods: A total of 167 children with cardiomyopathy diagnosed and treated in Shengjing Hospital between January 2010 and May 2019 were evaluated. After patient screening, 31 patients with endomyocardial fibroelastosis (EFE), left ventricular non-compaction, or dilated cardiomyopathy with significant arrhythmias were selected. In addition, 42 children with primary EFE were selected to evaluate the prognosis with or without arrhythmia. Follow-up was undertaken 0, 1, 3, 6, 9, and 12 months after treatment. Results: We revealed the outcomes for five types of cardiomyopathy: EFE patients with Wolff-Parkinson-White syndrome B and supraventricular tachycardia, intraventricular block and complete left bundle branch block recovered slower than EFE patients with atrial flutter and atrial fibrillation, even slower than EFE with ventricular tachycardia. The average recovering time for LVEF and LVED in EFE patients without arrythmia was 10 months after diagnosis, while 76.9% (3/13 cases) of those with significant arrythmia hadn't recovered until 24 months after diagnosis. Three of patients died at 6, 7, and 6 and half years after diagnosis. Conclusion: The long-term prognosis in children with cardiomyopathy is associated with the type of arrhythmia and time of intervention. The prognosis of EFE patients with arrhythmia is worse than EFE patients without arrhythmia. Patients with Wolff-Parkinson-White syndrome B, especially a significantly widen QRS complex, carry a poor prognosis if radiofrequency ablation is not undertaken. CLBBB patients have similar poor prognosis if proper pacemaker is not implanted timely.

Cloning and Differential Expression Analyses of Cdc42 from Sheep.

INTRODUCTION: 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. RESULTS: 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. CONCLUSION: OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.

[Experimental study on anti-tumor effect of extractive from Celastrus orbiculatus in vivo].

OBJECTIVE: To study the anti-tumor activity of extractive from Celastrus orbiculatus in vivo. METHOD: Mice bearing transplanted tumor S180 and Heps were used to study the effects of acetoacetate and n-butanol extractive from C. orbiculatus. The changes in serum contents of SOD and malondialdehyde (MDA) content were assayed. RESULT: Acetoacetate and n-butanol extractive from C. orbiculatus significantly inhibited the growth of S180 and Heps tumor in mice. SOD content was obviously increased, MDA content obviously decreased in the serum after extractive treatment. CONCLUSION: Acetoacetate and n-butanol extractive from C. orbiculatus have anti-tumor effects and anti-oxidative capacity.

Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis.

Molecular cloning, expression and characterization of programmed cell death 10 from sheep (Ovis aries).

Programmed cell death 10 (PDCD10) is a highly conserved adaptor protein. Its mutations result in cerebral cavernous malformations (CCMs). In this study, PDCD10 cDNA from the buffy coat of Small Tail Han sheep (Ovis aries) was cloned from a suppression subtractive hybridization cDNA library, named OaPDCD10. The full-length cDNA of OaPDCD10 was 1343bp with a 639bp open reading frame (ORF) encoding 212 amino acid residues. Tissue distribution of OaPDCD10 mRNA determined that it was ubiquitously expressed in all tested tissue samples, and the highest expression was observed in the heart. The differential expression of OaPDCD10 between infected sheep (challenged with Brucella melitensis) and vaccinated sheep (vaccinated with Brucella suis S2) was also investigated. The results revealed that, compared to the control group, the expression of OaPDCD10 from infected and vaccinated sheep was both significantly up-regulated (p<0.05). Moreover, the expression levels of OaPDCD10 from the vaccinated sheep were significantly higher than the infected sheep (p<0.05) after 30days post-inoculation. The recombinant OaPDCD10 (rOaPDCD10) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rOaPDCD10 protein was demonstrated to induce apoptosis and promote cell proliferation. Our studies are intended to discover potential diagnostic biomarkers of brucellosis to discern infected sheep from vaccinated sheep, and OaPDCD10 could be considered as a potential diagnostic biomarker of brucellosis.