Objective To provide the initiative experimental reference for resource utilization of Cynanchum komarovii by biological technology. Methods Using root, stem, and leaf of C. komarovii as explants, the proper media for inducing of callus were optimized by orthogonal test. In culturing process for proliferation of callus, the fresh and dry weights were determined at various times and callus growth curve was drawn. Meanwhile, division test of callus was taken. Results Root callus test showed the best effect in successful inducing percentage of 90%-100%. The best media combination of stem and leaf were MS+2,4-D 1.0 mg/L+6-BA 2.0 mg/L+NAA 0.5 mg/L and MS+2,4-D 0.5 mg/L+6-BA 0.5 mg/L+NAA 0.5 mg/L. The stem segament was more easily induced than leaf. The callus induced by various treatments is in loose character and fresh yellow colour with no brown phenomenon. At day 10 the growth entered the exponent period of callus offspring, the maximal fresh weight to 4.961 g and the dry weight to 0.496 g. The division rate of callus was rather low and not easy to shoot. However, the nail root was commonly seen in the test. Conclusion The root is proved to be the best explants in the test of inducing callus, which shows the best medium of stem and leaf for forming the callus. The growth of callus is bigger, but the division is not easy.

ABSTRACT:Objective To evaluate the features and related factors of decremental response in amyotrophic lateral sclerosis (ALS)patients to low-frequency repetitive nerve stimulation (RNS)in proximal nerves.Methods We performed RNS studies in proximal axillary and accessory nerves with recording in deltoid and trapezius mus-cle respectively in 87 ALS patients.Decremental compound muscle action potential (CMAP)and related factors were analyzed prospectively,and abnormal group of decremental response in ALS patients was compared with 39 pa-tients with myasthenia gravis.Results ① Abnormal decremental responses were found in 43.7% and 49.4% of ALS patients with deltoid and trapezius muscle recording respectively.They were found more frequent in trapezius muscle than those of deltoid muscle.② There was no relationship of decremental response with gender,age,onset or course of disease,ALSFRS-r scores,or rate of disease progression in ALS patients.③ There was significant rela-tionship between decremental response and fluctuating muscle weakness.Decremental responses decreased more ob-viously in ALS patients with fluctuating muscle weakness than in those with nonfluctuating muscle weakness.④ Dec-remental responses were greater in patients with myasthenia gravis than that in ALS patients.Conclusion Decre-mental response with proximal muscle recording is not an uncommon feature in ALS patients;therefore,it should not be treated as a criterium to rule out ALS.Abnormal decremental response of trapezius muscle is found more fre-quent than that of deltoid muscle.Decremental response range in patients with myasthenia gravis is significantly lar-ger than that in ALS patients.One should be more careful when diagnosing ALS patients with fluctuating muscle weakness and abnormal decremental response.

Objective To compare the curative effect and complication of traditional open thyroid operation and minimally invasive laryngeal endoscopic thyroid operation.Methods 80 cases with thyroid disease were randomly divided into group I and groupⅡof 40 cases in each group,the group Ⅰ received the minimally invasive laryngeal endoscopic thyroid operation, while the Ⅱ group was treated with traditional open thyroid operation.The operation time,intraoperative amount of bleeding,postoperative drainage,hospitalization time and complications of two groups were compared.Results The operation time,hospitalization time of groupⅠwere (63.1 ±0.2)min,(5.7 ±0.6)d, which were significantly shorter than those of the control group[(90.4 ±1.1) min,(8.4 ±0.2) d].Amount of bleeding,postoperative drainage volume after operation in groupⅠwere (19.2 ±0.9) mL,(30.4 ±2.6) mL,which were significantly less than those of the control group[(42.1 ±1.8)mL,(46.2 ±1.2)mL] (t=9.819,12.168, 11.182,9.928,all P <0.05);The incidence of postoperative complications of group Ⅰ was 2.5%,lower than 15.0%of groupⅡ,the difference was statistically significant between the two groups(χ2 =13.197,P <0.05). Conclusion Minimally invasive laryngeal endoscopic thyroid operation has advantages in little trauma,short opera-tion time,rapid postoperative recovery.

Abstract: Objective To investigate the preventive and therapeutic effects of anti-idiotypic monoclonal antibodies (Ab2β) of lactadherin on neonatal mice infected with human rotavirus (HRV), and to analyze the underlying mechanism. Methods Hybridoma technology was used to prepare Ab2β of lactadherin. One hundred and twenty 7-day-old Kunming mice were randomly divided into groups A, B, C and control, each consisting of 30 mice. Groups A, B, and C were all infected with HRV via oral gavage. Group A received no treatment, group B was orally administered lactadherin for 7 days prior to infection, and group C was orally administered lactadherin for 7 days after infection, the control group was orally administered cell culture medium that did not contain the virus. The clinical manifestations (diarrhea, body weight) at different time points after infection of the neonatal mice in each group were observed, and the content of rotavirus antigen in the feces of neonatal mice was detected by enzyme-linked immunosorbent assay (ELISA). After HRV infection for 7 days, immunohistochemical staining was used to examine the expression level of intercellular adhesion molecule-1 (ICAM-1) in mouse small intestinal tissues in each group. Results No diarrhea occurred in the control group at any time point. Groups A, B, and C showed diarrhea symptoms after HRV challenge for 1 day. The degree of diarrhea in groups B and C was lower than that in group A at 2-4 days after HRV challenge, and the difference was statistically significant (P<0.05). The HRV antigen content in the feces of the neonatal mice in groups B and C was lower than that in group A at 1-5 days after HRV challenge, and the difference was statistically significant (P<0.05). There was no significant difference in the degree of diarrhea and HRV antigen content between groups B and C at each time point (P>0.05). There was no significant difference in the body weight of the neonatal mice in each group before infection and 1 day after infection (P>0.05); the weight of neonatal mice in groups B and C was higher than that in group A at 3, 5 and 7 days after HRV challenge, and the difference was statistically significant (P<0.05), and there was no significant difference in body weight between groups B and C at each time point after HRV challenge (P>0.05). The number of ICAM-1 expressing cells in the small intestine of the three groups A, B, and C was higher than that of the control group after HRV challenge for 7 days, and the difference was statistically significant (P<0.05). The cell number and gray value of ICAM-1 expressing cells in groups B and C were lower than those in group A, and the difference was statistically significant (P<0.05). Conclusions Anti-idiotypic monoclonal antibodies (Ab2β) of lactadherin has a good preventive and therapeutic effects on human rotavirus infection in neonatal mice, and can significantly improve diarrhea symptoms and reduce HRV viral load. Its specific mechanism may be related to the inhibition of ICAM-1.

Abstract: Objective To investigate the preventive and therapeutic effects of anti-idiotypic monoclonal antibodies (Ab2β) of lactadherin on neonatal mice infected with human rotavirus (HRV), and to analyze the underlying mechanism. Methods Hybridoma technology was used to prepare Ab2β of lactadherin. One hundred and twenty 7-day-old Kunming mice were randomly divided into groups A, B, C and control, each consisting of 30 mice. Groups A, B, and C were all infected with HRV via oral gavage. Group A received no treatment, group B was orally administered lactadherin for 7 days prior to infection, and group C was orally administered lactadherin for 7 days after infection, the control group was orally administered cell culture medium that did not contain the virus. The clinical manifestations (diarrhea, body weight) at different time points after infection of the neonatal mice in each group were observed, and the content of rotavirus antigen in the feces of neonatal mice was detected by enzyme-linked immunosorbent assay (ELISA). After HRV infection for 7 days, immunohistochemical staining was used to examine the expression level of intercellular adhesion molecule-1 (ICAM-1) in mouse small intestinal tissues in each group. Results No diarrhea occurred in the control group at any time point. Groups A, B, and C showed diarrhea symptoms after HRV challenge for 1 day. The degree of diarrhea in groups B and C was lower than that in group A at 2-4 days after HRV challenge, and the difference was statistically significant (P<0.05). The HRV antigen content in the feces of the neonatal mice in groups B and C was lower than that in group A at 1-5 days after HRV challenge, and the difference was statistically significant (P<0.05). There was no significant difference in the degree of diarrhea and HRV antigen content between groups B and C at each time point (P>0.05). There was no significant difference in the body weight of the neonatal mice in each group before infection and 1 day after infection (P>0.05); the weight of neonatal mice in groups B and C was higher than that in group A at 3, 5 and 7 days after HRV challenge, and the difference was statistically significant (P<0.05), and there was no significant difference in body weight between groups B and C at each time point after HRV challenge (P>0.05). The number of ICAM-1 expressing cells in the small intestine of the three groups A, B, and C was higher than that of the control group after HRV challenge for 7 days, and the difference was statistically significant (P<0.05). The cell number and gray value of ICAM-1 expressing cells in groups B and C were lower than those in group A, and the difference was statistically significant (P<0.05). Conclusions Anti-idiotypic monoclonal antibodies (Ab2β) of lactadherin has a good preventive and therapeutic effects on human rotavirus infection in neonatal mice, and can significantly improve diarrhea symptoms and reduce HRV viral load. Its specific mechanism may be related to the inhibition of ICAM-1.

Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.

Objective To evaluate the disease onset regions and spreading patterns in sporadic amyotrophic lateral sclerosis (ALS)patients and related influencing factors.Methods We performed a prospective analysis of 1 58 ALS patients.The disease-onset was confirmed according to the patients’self-reports,neurological examination results and electromyogram study.We followed up 1 5 1 patients with the second or other affected body regions during the disease progression.Data were analyzed according to the different groups of onset regions.Results 1.In 94.3% (149/1 58)of the patients,the early motor manifestations were focally in the bulbar,upper or lower limbs.2.The region of onset was associated with signs of lower motor neuron (LMN)and upper motor neuron (UMN)involvement (P = 0.000 ).The LMN involvement was more distinctive in patients with bulbar onset (65.4%,1 7/26 )group.Patients with cervical onset more frequently showed pure LMN (47.9%,45/94 )or concomitant UMN (52.1%,49/94)signs in the affected limbs.The highest proportion of UMN and LMN signs in the affected lower limb was found in the lumbar onset (83.8%,31/37 )group.3.Spreading patterns:Rostral to caudal spreading pattern was more frequent in bulbar onset patients.For patients with limb onset,there were significant differences between spreading patterns and disease-onset regions (P =0.04).Circular (31.5%,28/89),horizontal (30.3%,31/89)and vertical (21.3%,1 9/89)spreading patterns were more frequent in cervical onset patients whereas circular (47.2%,1 7/36)spreading patterns were more frequent in lumbar onset patients.4.There was a strong association between the rate of progression and age of disease onset (P =0.01 1).Patients aged over 60 had a faster progression.Conclusion ALS is a focal process at motor axis along the spinal cord and cerebral cortex.Different disease-onset can cause different distribution of UMN and LMN signs.Therefore,special attention should be paid to the signs of disease-onset clinically.ALS does start focally and spreads to adjacent regions.Elder patients have a faster disease progression.

Objective: To explore the effect of bulbar involvement time on survival time of patients with spinal-onset amyotrophic lateral sclerosis (ALS). Methods: We followed up 168 patients with spinal-onset ALS admitted to our hospital from January 2, 2011 to December 31, 2017 until December 31, 2018. Univariate and multivariate analyses were performed to evaluate the affecting factors of the ALS patients' survival time. Kaplan-Meier analysis was made to evaluate the effects of bulbar involvement time on survival time. Results: COX multivariate analysis showed that the risk of death in age-onset <55 y patients was 0.72 times that in age-onset ≥55 y (P=0.09), the risk of death in diagnosis delay time <10.98 m patients was 2.64 times that ≥10.98 m (P<0.001); the risk of death in bulbar involvement time ≥11.5 m and bulbar uninvolvement was 0.30 and 0.32 times respectively that bulbar involvement time <11.5 m (P<0.001). Kaplan-Meier analysis showed differences among bulbar involvement time <11.5 m, ≥11.5 m and bulbar uninvolvement groups (median survival time 20.37 m vs. 40.6 m vs. 39.60 m, Test statistic =39.96, P<0.001). The 2-year, 3-year and 5-year survival rates were 32.17%, 10.80% and 0%, respectively, in bulbar involvement time <11.5 m patients; 89.20%, 57.24% and 10.53% in bulbar involvement time ≥11.5 months patients; and 62.16%, 38.39% and 10.53% in bulbar uninvolvement patients. Conclusion: Similar to the diagnosis delay time and whether to have taken riluzole, the occurrence of bulbar involvement at 11.5 month after onset was an independent risk factor affecting survival time in spinal-onset ALS. The median survival time in patients with bulbar involvement time <11.5 months was significantly shorter than that in patients with bulbar involvement time ≥11.5 months and bulbar uninvolvement.

Objective To investigate the status and species of mycobacterial infections in Shenzhen for clinical diagnosis and treatment to provide a reliable scientific basis .Methods 1 096 of samples from patients with suspicious were detection by gene chip .Results Positive rate of microarray detection mycobacterium was 9 .40% (103/1 096) .103 cases of positive were 87 mycobac-teria by gene chip ,and 16 cases of non-tuberculosis(5 cases of M .abscessus ,3 cases of M .intracellulare ,3 cases of M .avium ,2 cases of M .fortuitum ,1 cases of M .kansas ,1 case of M .marinum ,1 case of M .gordonae .Conclusion The mycobacterial infections in Shenzhen ,tuberculosis infection as the main disease types (84 .47% ) .Non-tuberculous mycobacteria′s isolation ratio has reached 15 .53% ,including 7 kinds of species infection and one case of mixed infections .Identification of M ycobacterium by genechip have great significances for individualized treatment .

Objective To determine the influence of depression on C reactive protein in patients with chronic ischemic heart failure (CIHF).Methods 73 patients with CIHF were divided into CIHF without depression group(n=49) and CIHF with depression group (n=24) according to the standard sore of the self-rating depression scale.C reactive protein(CRP)and other clinical characteristics were detected.The geometric means of CRP between two groups were compared,and stepwise multiple linear regression analysis was used to confirm the independent correlation factors of CRP.Results (1) The geometric means of CRP in CIHF without depression group and CIHF with depression group were (1.38± 1.29)mg/L and (1.78±1.53) mg/L respectively.No statistically significant difference was found between two groups(P=0.276).(2) Stepwise multiple linear regression analysis showed that aspirin inversely related with CRP(β=-1.070,P=0.004),but brain natriuretic peptide (BNP)positively related with CRP(β=0.426,P=0.001).Conclusion Depression has no additional effect on CRP in patients with CIHF.