To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Objective·To investigate the therapeutic effect of telomerase gene therapy(JV001)on heart failure induced by transverse aortic constriction(TAC)in mice.Methods·Male C57BL/6J mice were randomly divided into sham operation group(Sham group),model group(TAC group),and JV001 treatment group(TAC+JV001 group).An adeno-associated virus 9(AAV9)vector containing the catalytic inactivation(D868A)and nuclear export phosphorylation(Y707F)mutations in the human telomerase reverse transcriptase(hTERT)gene(AAV9-modhTERT,named JV001)was intravenously administered to TAC animals at a single dose of 4×1012 gc/kg.The Sham group and the TAC group received equivalent volumes of saline via tail vein injection.Six weeks after administration,the expression of JV001 in the heart was determined by real-time quantitative PCR(RT-qPCR).Murine cardiac functions were assessed using echocardiography.Physiological indexes of mice were recorded for calculating heart weight/body weight ratio(HW/BW)and heart weight/tibial length ratio(HW/TL).Cardiac hypertrophy was assessed using hematoxylin-eosin(HE)staining and wheat germ agglutinin(WGA)staining.Cardiac collagen deposition was observed by Masson staining.The expressions of myocardial hypertrophy-related genes(Nppa,Nppb,Myh7,Myh6)and myocardial fibrosis-related genes(Col1a1,Col3a1,Ctgf)were detected by RT-qPCR.Results·High levels of modhTERT mRNA were expressed in the hearts of mice at 6 weeks post-injection.Compared with sham-operated mice,TAC mice exhibited significantly reduced left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Compared to TAC mice,TAC+JV001 mice exhibited a significant improvement in LVEF and LVFS.Concurrently,there was a downregulation in the HW/BW and HW/TL in TAC+JV001 mice compared to TAC mice.Furthermore,JV001 treatment reduced the mean cardiomyocyte cross-sectional area and improved the expression levels of myocardial hypertrophy-related genes,including Nppa,Nppb,Myh7 and Myh6.Additionally,JV001 treatment ameliorated the TAC-induced increase in myocardial interstitial fibrosis and reduced the elevated expression levels of myocardial fibrosis-related genes,including Col1a1,Col3a1,and Ctgf.Conclusion·AAV9-modhTERT treatment can alleviate TAC-induced cardiac dysfunction,cardiac hypertrophy,and fibrosis in mice.

Objective·To investigate the therapeutic effect of telomerase gene therapy(JV001)on heart failure induced by transverse aortic constriction(TAC)in mice.Methods·Male C57BL/6J mice were randomly divided into sham operation group(Sham group),model group(TAC group),and JV001 treatment group(TAC+JV001 group).An adeno-associated virus 9(AAV9)vector containing the catalytic inactivation(D868A)and nuclear export phosphorylation(Y707F)mutations in the human telomerase reverse transcriptase(hTERT)gene(AAV9-modhTERT,named JV001)was intravenously administered to TAC animals at a single dose of 4×1012 gc/kg.The Sham group and the TAC group received equivalent volumes of saline via tail vein injection.Six weeks after administration,the expression of JV001 in the heart was determined by real-time quantitative PCR(RT-qPCR).Murine cardiac functions were assessed using echocardiography.Physiological indexes of mice were recorded for calculating heart weight/body weight ratio(HW/BW)and heart weight/tibial length ratio(HW/TL).Cardiac hypertrophy was assessed using hematoxylin-eosin(HE)staining and wheat germ agglutinin(WGA)staining.Cardiac collagen deposition was observed by Masson staining.The expressions of myocardial hypertrophy-related genes(Nppa,Nppb,Myh7,Myh6)and myocardial fibrosis-related genes(Col1a1,Col3a1,Ctgf)were detected by RT-qPCR.Results·High levels of modhTERT mRNA were expressed in the hearts of mice at 6 weeks post-injection.Compared with sham-operated mice,TAC mice exhibited significantly reduced left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Compared to TAC mice,TAC+JV001 mice exhibited a significant improvement in LVEF and LVFS.Concurrently,there was a downregulation in the HW/BW and HW/TL in TAC+JV001 mice compared to TAC mice.Furthermore,JV001 treatment reduced the mean cardiomyocyte cross-sectional area and improved the expression levels of myocardial hypertrophy-related genes,including Nppa,Nppb,Myh7 and Myh6.Additionally,JV001 treatment ameliorated the TAC-induced increase in myocardial interstitial fibrosis and reduced the elevated expression levels of myocardial fibrosis-related genes,including Col1a1,Col3a1,and Ctgf.Conclusion·AAV9-modhTERT treatment can alleviate TAC-induced cardiac dysfunction,cardiac hypertrophy,and fibrosis in mice.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Objective To explore the beneficial effects and mechanisms of neutrophil elastase (NE) and myeloperoxidase (MPO) on lead-induced hepatic inflammation in mice. Methods The specific pathogen free male C57BL/6 mice were randomly divided into four groups： control group, lead-exposed group, NE inhibitor group, and MPO inhibitor group, with three mice in each group. The mice in lead-exposed group, NE inhibitor group, and MPO inhibitor group were intraperitoneally injected with a dose of 10 mg/kg body mass of lead acetate solution, while the mice of control group received an equal volume of 0.9% saline three times per week for four weeks. In the last seven days, mice in both inhibitor groups were intraperitoneally injected with a dose of 40 mg/kg NE inhibitor sivelestat sodium or MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) once per day. Mouse body weight and liver histopathological changes were observed. The mRNA expression of genes associated with inflammation, such as tumor necrosis factor-α (Tnfa), interleukin-1β (Il1b), interleukin-6 (Il6), and nucleotide-binding oligomerization domain-like receptor protein 3(Nlrp3), apoptosis-associated speck-like protein (Asc) and cysteinyl aspartate specific proteinase (Caspase1) in the mouse liver tissues was detected by real-time quantitative polymerase chain reaction. The protein expression of NLRP3, ASC, and CASPASE-1 was detected using Western blotting. Results The activities of mice in all four groups were generally normal, and there was no significant difference in body weight (P>0.05). The results of hematoxylin-eosin staining showed that the cell size of hepatocytes varied in the lead-exposed mice, with indistinct cell boundaries, indicating early inflammatory responses in liver tissues. After intervention with NE or MPO inhibitors, the early inflammatory responses improved in the liver tissues of the mice in both inhibitor groups, with a better improvement observed in MPO inhibitor group compared with the NE inhibitor group. The mRNA expression of Tnfa, Il1b, Il6, Nlrp3, Asc, and Caspase1, as well as the protein expression of ASC, and CASPASE-1 in the livers of mice in the lead-exposed group was higher compared with those in the control group (all P<0.05). Compared with the lead-exposed group, the relative mRNA expression of Tnfa, Il1b, Il6, Nlrp3 and Asc was decreased in the liver tissues of mice in the NE inhibitor group (all P<0.05), while the relative expression of mRNA of Tnfa, Il1b, Il6, Caspase1 and the protein expression of ASC and CASPASE-1 were decreased in the liver tissues of mice in the MPO inhibitor group (all P<0.05). Conclusion Lead induce hepatic inflammation in mice by activating NLRP3 inflammasome. The inhibition of NE or MPO improve the lead-induced hepatic inflammatory responses in mice by alleviating NLRP3 inflammasome activation.

Objective To study the effects of mindfulness behavior training on coping style and illness uncertainty in patients of shoulder hand syndrome (SHS) after ischemic stroke.Methods 65 cases with SHS were randomly divided into two groups by digit method: the control group (n=32) and experimental group (n=33).Patients in control group only received routine rehabilitation, while patients in experimental group also received mindfulness behavior training.The daily life, medical coping style, illness uncertainty and mindfulness were evaluated respectively by Barthel Index (BI) , Medical Coping Style Questionnaire (MCMQ) , Illness Uncertainty Scale (IUS) and Mindful Attention Awareness Scale (MAAS).Results Before training, there were no significant difference in BI,MCMQ,IUS and MAAS (P＞0.05).After treatment, scores in BI improved significantly in the experimental group than that in the control group(72.4± 11.6 vs 62.9±10.1) ,scores in IUS improved significantly in the experimental group than that in the control group(69.3±9.3 vs 86.9±7.2) and scores in MCMQ and MAAS improved significantly in the experimental group than that in the control group(P＜0.05).26 cases whose Barthel index was more than 60 points in experimental group after treatment were found while 17 cases whose Barthel index was more than 60 points in control group were done after treatment (x2 =6.415, P＜0.05).Conclusion Mindfulness behavior training can regulate the patient coping style and weaken illness uncertainty,and improve functions rehabilitation.

OBJECTIVETo construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.

METHODSThe gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining.

RESULTS AND CONCLUSIONWe established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157:H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD60 value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157:H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD60 value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective To investigate the effect of Lokomat Robotic Gait Training System on motor, gait and range of motion (ROM) for stroke patients with hemiplegia. Methods 80 stroke patients with hemiplegia were divided into treatment group (n=40) and control group (n=40). The control group accepted routine rehabilitation and the treatment group accepted robotic gain training. They were assessed with Fugl-Meyer Assessment, gait parameter and ROM of hip and knee before training and after 10 weeks of treatment. Results There was no significant difference on all the index before treatment (P>0.05), and improved more in the treatment group than in the control group after training (P<0.05). Conclusion Lokomat Robotic Gait Training System intervention can promote the recovery of walking in stroke patients with hemiplegia.