In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.
Animals ; Chlorocebus aethiops ; Clone Cells ; DNA, Complementary ; Distemper Virus, Canine/genetics* ; Plasmids/genetics* ; Vero Cells

Sijunzi decoction (SJZD) is a Chinese classical formula to treat spleen qi deficiency syndrome (SQDS) and has been widely used for thousands of years. However, the quality control (QC) standards of SJZD are insufficient. Chinmedomics has been designed to discover and verify bioactive compounds of a variety of formula rapidly. In this study, we used Chinmedomics to evaluate the SJZD's efficacy against SQDS to discover the potential quality-markers (q-markers) for QC. A total of 56 compounds in SJZD were characterized in vitro, and 23 compounds were discovered in vivo. A total of 58 biomarkers were related to SQDS, and SJZD can adjust a large proportion of marker metabolites to normal level and then regulate the metabolic profile to the health status. A total of 10 constituents were absorbed as effective ingredients that were associated with overall efficacy. We preliminarily determined malonyl-ginsenoside Rb2 and ginsenoside Ro as the q-markers of ginseng; dehydrotumulosic acid and dihydroxy lanostene-triene-21-acid as the q-markers of poria; glycyrrhizic acid, isoglabrolide, and glycyrrhetnic acid as the q-markers of licorice; and 2-atractylenolide as the q-marker of macrocephala. According to the discovery of the SJZD q-markers, we can establish the quality standard that is related to efficacy.

Objective:To investigate the correlation between acoustic radiation force impulse imaging(ARFI) and reserve function of liver and the feasibility of liver reserve function assessment by ARFI.Methods:According to the inclusion criteria, 74 patients were screened and 71 patients were finally enrolled from Henan Province People′s Hospital during June 2017 and June 2019. The portal vein diameter (D PV), spleen length (L SP) were measured by two-dimensional ultrasound. The liver shear wave velocity (LSWV) and spleen shear wave velocity (SSWV) were measured by ARFI. Serological markers were checked, and the indocyanine green (ICG)15-minute retention rate (ICG R15) was measured by excretion test. The patients were divided into ICG R15<10% group and ICG R15≥10% group, the difference of the measurements between two groups were calculated, the correlations of the measurements and ICG R15 were analyzed. LSWV diagnostic performance for liver reserve function was evaluated by the ROC curve. Results:There were significant differences in LSWV, D PV, SSWV, L SP, aspartate transaminase(AST), alkaline phosphatase(ALP), y-glutamyl transpeptidase(γ-GGT) and albumin (ALB) between ICG R15<10% group and ICG≥10% group( P<0.05), but no significant differences in other measurements( P>0.05). The correlations between ICG R15 and LSWV( r=0.673, P<0.001), D PV( r=0.355, P<0.05), SSWV( r=0.384, P<0.05), L SP( r=0.403, P<0.001), ALP( r s=0.245, P<0.05) and ALB( r s=-0.390, P<0.05) were statistically significant. The ROC curve showed high diagnostic performance for liver reserve function assessment by LSWV. The area under the ROC curve was 0.903 (95% CI=0.810-0.961, P<0.01), and the cut-off value was 2.15 m/s (sensitivity 84.6%, specificity 86.7%). Conclusions:The LSWV can evaluate the reserve function and it is a useful supplement to the ICG excretion experiment.

Objective:To assess the feasibility of delayed-enhancement MRI in contouring the lumpectomy cavity (LC) for patients with invisible seroma or a low cavity visualization score (CVS≤2) in the excision cavity after breast-conserving surgery (BCS).Methods:Twenty-six patients with stage T 1-2N 0M 0 who underwent prone radiotherapy after BCS were recruited. The LC delineated on CT simulation images was denoted as LC CT. The LCs delineated on T 2WI, as well as on different delayed phases (2-, 5-and 10-minute) of delayed-enhancement T 1WI were defined as LC T2, LC 2T1, LC 5T1 and LC 10T1, respectively. Subsequently, the volumes and locations of the LCs were compared between CT simulation images and different sequences of MR simulation images using deformable image registration. Results:The volumes of LC T2, LC 2T1, LC 5T1 and LC 10T1 were all larger than that of LC CT. A statistical significance was found between the volume of LC CT and those of LC 2T1 or LC 5T1, respectively (both P<0.05). The conformal index (CI), degree of inclusion (DI), dice similarity coefficient (DSC) and the distance between the center of mass of the targets (COM) of LC CT-LC 10T1 were better than those of LC CT-LC T2, LC CT-LC 2T1 and LC CT-LC 5T1, however, there was no statistical difference among them (all P>0.05). Conclusions:It is feasible to delineate the LC based on prone delayed-enhancement MR simulation images in patients with low CVS after BCS. Meanwhile, the LCs derived from prone delayed-enhancement T 1WI of 10-minute are the most similar with those derived from prone CT simulation scans using titanium clips, regardless of the volumes and locations of LCs.

This paper aimed at evaluating the therapeutic effects of acupuncture and moxibustion in treatment of heart-spleen deficiency and observing the improvement effect for insomnia patients′ sleep quality. A total of 70 cases of heart-spleen deficiency were selected as the research data, to the all cases were divided into acupuncture group and controlgroup. Acupuncture group were treated with acupuncture and moxibustion in many acupoints, the control group weretreated with estazolampo. After 3 courses of treatment, the PSQI and TESS of the two groups were observed. The totaleffective rate of the acupuncture group was 94.3％, the total effective rate of the control group was 77.1％ (P ＜ 0.05), andthe acupuncture group was more ideal than the control group (P ＜ 0.05) . After treatment the PSQI score and TESS scorein acupuncture group were decreased compared with control group (P ＜ 0.05) . Acupuncture and moxibustion in thetreatment of heart-spleen deficiency insomnia curative effect is ideal and reliable, can effectively decrease the PSQI andTESS score, and has lower auxiliary work compared with taken estazolam.

Objective To investigate the click and tone burst evoked auditory brainstem responses (ABR)in normal Wistar rat,and to establish the standards of ABR testing method,and to provide a reference for studies rat audition.Methods Fifteen male Wistar rats(30 ears)were used in this sutdy.The latency and amplitude of ABR e-voked by click and TB at 80,50 and 20 dB SPL were measured.Results The occurrence rate of wave Ⅱand Ⅳat low levels(20 dB SPL)was nearly the same according to the amplitude.The cABR (dB peSPL)threshold was 21.83± 4.45 and tbABR (dB SPL)thresholds were 2.02±0.09,2.88±0.16,3.77±0.25,4.69±0.29,and 5.78±0.41, respectively.80 dB stimulus evoked cABR (peSPL)wave I,I b,II,III,IV and V latency (ms)were 1.76±0.12, 2.13±0.11,2.67±0.16,3.49±0.28,4.39±0.29,and 5.45±0.41,respectively.tbABR (SPL)of wave I,Ib, II,III,IV and V latency (ms)at 4 kHz were 2.02±0.09,2.88±0.16,3.77±0.25,4.69±0.29,and 5.78± 0.41,respectively.At 8 kHz they were 1.76±0.07,2.28±0.10,2.63±0.16,3.49±0.21,4.44±0.28,and 5.48±0.43;while at 12 kHz were1.76±0.08,2.24±0.12,2.61±0.25,3.53±0.25,4.46±0.32,and 5.52± 0.45;at 16 kHz were 1.79±0.10,2.25±0.12,2.70±0.18,3.62±0.27,4.52±0.37,and 5.61±0.49;at 24 kHz were 1.75±0.09,2.27±0.11,2.67±0.16,3.60±0.27,4.52±0.38,and 5.60±0.51;at 32 kHz were 1.77±0.10,2.24±0.12,2.64±0.20,3.59±0.34,4.52±0.40,and 5.61±0.52,respectively.Conclusion Wave Ⅳ was the best wave to determine threshold of click and tone burst evoked auditory brainstem response in rat.

To analyze the molecular mechanisms of cross-host transmission of the Aleutian mink disease vi rus (ADV), the hypervariable region fragment of the VP2 gene of the ADV in Jilin Province (China) was amplified. Sequencing analyses showed diversity at residue 174 by comparison with other VP2 genes in GenBank. The phylogenetic tree indicated that the ADV-JL strain had a close relationship with the highly pathogenic strain from Denmark: ADV-K. Results implied that residue 174 may be associated with ADV infectivity.
Aleutian Mink Disease ; virology ; Aleutian Mink Disease Virus ; chemistry ; classification ; genetics ; isolation & purification ; Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; China ; Mink ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment ; Sequence Analysis

AIM:To evaluate the effects of atorvastatin on nitric oxide(NO),endothelin-1(ET-1)and myocardial no-reflow in a rabbit model of acute myocardial infarction and reperfusion(AMI/R). METHODS:Twenty-four rabbits were randomized into 3 groups:8 in AMI/R group,8 in atorvastatin-treated group(5 mg·kg~(-1)·d~(-1))and 8 in sham-operated group. Animals in the former two groups were subjected to 60 min of coronary occlusion followed by 120 min of reperfusion. Data on haemodynamics were collected. NO in blood sample,and in normal,and in infarcted reflow and no-reflow myocardium were evaluated respectively by nitrate reductase method. The levels of ET-1 in blood sample,and in normal,infarcted reflow and no-reflow myocardium were determined by radioimmunoassay. RESULTS:(1)Compared to the baselines,the heart rate(HR),systolic blood pressure(SBP),diastolic blood pressure(DBP),left ventricular systolic pressure(LVSP),maximal rate of increase and decline in left ventricular pressure(±dp/dt_(max))and cardiac output(CO)in AMI/R and atorvastatin-treated groups were significantly declined,whereas left ventricular end-diastolic pressure(LVEDP)was increased after 60 min of coronary occlusion and 120 min of reperfusion(P<0.05 or P<0.01). However,in atorvastatin-treated group,LVSP,LVEDP,±dp/dt_(max) and CO at the time point of 120 min of reperfusion recovered more significantly than those at the time point of 60 min of coronary occlusion(P<0.01),which was more significant than those in AMI/R group(P<0.05 or P<0.01). Compared to AMI/R group,the SBP and DBP were significantly heigher in atorvastatin-treated group(P<0.01).(2)In atorvastatin-treated group,the levels of ET-1 in blood sample were significantly lower than those in AMI/R group(P<0.01),and the levels of NO were significantly higher(P<0.01). Moreover,the levels of NO or ET-1 in infarcted reflow myocardium were significantly lower than that in AMI/R group(P<0.05 or P<0.01).(3)Atorvastatin could ameliorate myocardial function. CONCLUSION:Atorvastatin is effective in increasing NO and reducing ET-1 in blood plasma and local myocardium,and in protection of endothelial cells. Atorvastatin also has a beneficial effect on improving left ventricular function during acute myocardial infarction and reperfusion in rabbits.

Objective To explore the distrbution characteristics of glutathione S-transferase T1 and M1 (GSTT1 and GSTM1) gene polymorphism of Daur population in Inner Mongolia region, and provide the relevant data for studying the genotype of minority ethnic group of the Inner Mongolia.Methods Genomic DNA was extracted from peripheral blood and the fragments of GSTM1 and GSTT1 was amplified by internal control allele specific PCR in 220 volunteers who had been residents in Inner Mongolia region. At the same time, the genotype of GSTT1 and GSTM1 was resolved by the density of 2.5% agarose gel electrophoresis.Results In the studied population, the frequencies of GSTM1 (-) and GSTT1 (-) were 50.8% and 71.4% respectively. The frequency of the individuals with combination of GSTM1 (-) and GSTT1 (-) was 31.4%.Conclusion The distribution of GSTM1 and GSTT1 displays gene polymorphism in Chinese Daur population, approximate to Mongolian population in the north of China, but there is significant difference between Chinese Daur population and Han people.

OBJECTIVE: To explore the nature of pathology of sluggishness of lung-defensive qi and to offer objective experimental indexes for weifen syndrome (defensive phase syndrome). METHODS: According to the completely random design, the plasma levels of vasoactive intestinal peptide (VIP) and thromboxane B2 (TX2) of 19 patients with weifen syndrome and 13 patients with qifen syndrome (qi phase syndrome) were detected by radioimmunoassay. The plasma levels of VIP and TX2 at different stages of weifen syndrome and qifen syndrome were observed. RESULTS: The plasma levels of VIP in weifen syndrome and in the late stage of weifen syndrome increased greatly at different stages as compared to qifen syndrome and the blank group (P < 0.01), while the plasma level of TX2 of weifen syndrome was higher only at the late stage than the blank group and qifen syndrome (P < 0.01). As for the levels of VIP and TX2 in weifen syndrome with different internal organs infected, there was no significant difference (P > 0.05). CONCLUSION: VIP may be an index reflecting the pathology of weifen syndrome, and it is one of the material foundations of sluggishness of lung-defensive qi, but it has nothing to do with the infected internal organs. The level of TX2 increases only after the fever of patients with weifen syndrome subsided, so it can not be the basis for diagnosis of the early stage of weifen syndrome. It doesn't increase in qifen syndrome either, the mechanism remains to be further studied.