To establish a fluorescence-labeled Corynebacterium pseudotuberculosis(Cp),the super-folded green fluorescent protein gene(sfGFP)fused to the red fluorescent protein gene(mCher-ry)was cloned into pXMJ19 and expressed in Cp.The fluorescent signals of recombinant Cp cul-tured under different pH values,and the visualized detection of bacteria in the macrophages J774A.1 or mice infected with recombinant Cp were evaluated.The results showed that a double fluores-cence labeled XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)was successfully construc-ted by electrotransformation of Cp with pXMJ19-sfGFPmCherry,which containing sfGFP fused to mCherry.The colony morphology of XH02-sfGFPmCherry on fresh blood agar plates was pink.The XH02-sfGFP mCherry showed green and red fluorescence when observed under the fluo-rescence microscope.Compared with cultivation at pH7.0,XH02-sfGFPmCherry showed a signifi-cant decrease in green fluorescence intensity(fluorescence intensity/D600)at pH5.0,but signifi-cantly increased in red fluorescence intensity at pH5.0.Green and red fluorescences of Cp were ob-served in the XH02-sfGFPmCherry-infected J774A.1 in vitro or in the liver and kidney of XH02-sfGFPmCherry-infected mice in vivo,and with good overlap of two kinds of fluorescences.This study demonstrates that the constructed dual fluorescent labeled Cp can efficiently express both red and green fluorescent proteins,and express the dominant fluorescent signals under different pH value conditions,which can be used for Cp monitoring in vitro infection of macrophages and in vivo infection of mice by this pathogen,and providing potential tool for the localization and tracing research of Cp infection.

Objective To observe whether the IL-23/Th17 inflammatory pathway is involved in the development of calcific aortic valve disease,and whether secukinumab can delay the progression of calcific aortic valve disease by inhibiting this pathway.Methods Forty-seven mice were divided into a blank control group,model group,and secukinumab group according to the random number table method.The blank control group was fed normal chow,while the model group and secukinumab group were fed pro-calcification chow for 16 weeks to establish a calcific aortic valve disease model.After intervention with secukinumab for 4 weeks,peak flow velocity changes in the aortic valves were detected under Doppler ultrasonography in all mice.Relevant indexes were determined by hematoxylin and eosin staining,Von Kossa staining,immunohistochemical staining,ELISA,and qPCR.Results Compared with the model group,the secukinumab group showed significantly reduced peak flow velocity(P＜0.05)and serum IL-6,IL-17,and IL-23 levels(P＜0.05)in the aortic valve.Compared with the secukinumab group,the model group's leaflet thickness was significantly increased,and there were more calcium deposits.Immunohistochemical result showed that macrophage infiltration(P＜0.05),IL-17A(P＜0.05)and IL-23(P＞0.05)levels in the valve leaflets were reduced in the secukinumab group compared with the model group.PCR result suggested that the expression of STAT3,BMP-2,and α-SMA mRNA was significantly lower in the secukinumab group than the model group(P＜0.05).Conclusions The IL-23/Th17 inflammatory pathway is involved in the pathogenesis of calcific aortic valve disease.The inflammation,fibrosis,osteogenic differentiation,and calcification of mouse valves were alleviated after intervention with secukinumab,which may delay disease progression by inhibiting the IL-23/Th17 inflammatory pathway.

To establish a fluorescence-labeled Corynebacterium pseudotuberculosis(Cp),the super-folded green fluorescent protein gene(sfGFP)fused to the red fluorescent protein gene(mCher-ry)was cloned into pXMJ19 and expressed in Cp.The fluorescent signals of recombinant Cp cul-tured under different pH values,and the visualized detection of bacteria in the macrophages J774A.1 or mice infected with recombinant Cp were evaluated.The results showed that a double fluores-cence labeled XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)was successfully construc-ted by electrotransformation of Cp with pXMJ19-sfGFPmCherry,which containing sfGFP fused to mCherry.The colony morphology of XH02-sfGFPmCherry on fresh blood agar plates was pink.The XH02-sfGFP mCherry showed green and red fluorescence when observed under the fluo-rescence microscope.Compared with cultivation at pH7.0,XH02-sfGFPmCherry showed a signifi-cant decrease in green fluorescence intensity(fluorescence intensity/D600)at pH5.0,but signifi-cantly increased in red fluorescence intensity at pH5.0.Green and red fluorescences of Cp were ob-served in the XH02-sfGFPmCherry-infected J774A.1 in vitro or in the liver and kidney of XH02-sfGFPmCherry-infected mice in vivo,and with good overlap of two kinds of fluorescences.This study demonstrates that the constructed dual fluorescent labeled Cp can efficiently express both red and green fluorescent proteins,and express the dominant fluorescent signals under different pH value conditions,which can be used for Cp monitoring in vitro infection of macrophages and in vivo infection of mice by this pathogen,and providing potential tool for the localization and tracing research of Cp infection.

Objective To investigate the clinical efficacy of Shenmai injection (SMI) combined with heart transplantation of autologous bone marrow stem cells in the treatment of refractory heart failure(RHF). Methods Two hundred patients with RHF were selected from the Emergency Department of the First Affiliated Hospital of Zhengzhou University and the Cardiology Department of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine. They were randomly divided into control group and treatment 1,2 and 3 groups(each 50 cases). In the control group,heart failure standard treatment was given;group 1 received a standard treatment for heart failure combined with autologous bone marrow stem cell heart transplantation;group 2 received a standard treatment for heart failure with SMI;group 3 received a standard treatment for heart failure combined with autologous bone marrow stem cell heart transplantation and SMI. The mean follow-up was 24 months. The prognosis,readmission rate,clinical efficacy,cardiac function and the change in levels of B-type natriuretic peptide(BNP)of patients in each group were observed during the therapeutic course and observation period. Results During the course of treatment,there were 10 cases dead in the control group,4 in each group 1 and 2 respectively,and 3 in group 3. Readmission rates in group 1,2 and 3 were significantly lower than that in control group(38%,36%,24%vs. 48%, P<0.05 or P<0.01). The rates of total efficiency in group 1,2 and 3 were obviously higher than that in the control group(88%,86%,94%vs. 76%,all P<0.05). After treatment,the left ventricular ejection fraction(LVEF),left ventricular fractional shortening(FS)and left ventricular end-systolic diameter(LVESD)in three therapeutic groups were significantly higher than those before treatment,while the left ventricular end-diastolic diameter(LVEDD)and BNP level were significantly lower than those before treatment. All the above indexes in three therapeutic groups after treatment were much more remarkably improved than those in the control group during the same period,and the group 3 being the most significant〔LVEF:0.477±0.099 vs. 0.396±0.098,FS:(30.0±5.1)%vs.(26.8±7.5)%,LVESD (mm):40.6±9.1 vs. 45.8±9.4,LVEDD(mm):44.9±9.8 vs. 52.8±10.1,BNP(ng/L):515±400 vs. 1 875±400, all P<0.05〕. Conclusion SMI combined with heart transplantation of autologous bone marrow stem cells has obvious therapeutic effect for treatment of RHF.