Objective To evaluate the immune effect of group C meningococcal polysaccharide(GCMP) conjugates using protein D(PD) of Haemophilus influenzae type b(Hib) as vector, in order to determine the feasibility of PD protein as GCMP protein vector.Methods The recombinant PD and tetanus toxin(TT) were conjugated to GCMP by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP) activation method to prepare conjugates respectively. The female BALB/c mice were subcutaneously immunized with GCMP and GCMP conjugates, which were divided into GCMP, GCMP-PD, PD, GCMP-TT,TT and negative control group(normal saline), with 12 mice in each group for 3 times of immunization. The blood samples were collected from the rim of eye before each booster immunization and 7 d after the last immunization, and the sera were separated for the detection of antibody levels and serum bactericidal assay(SBA). In addition, the spleen samples were taken7 d after the last immunization for the detection of cellular immune levels.Results The conjugates of protein and GCMP enhanced the level of anti-GCMP IgG. After the third immunization, serum anti-GCMP IgG levels in GCMP-PD and GCMP-TT groups were significantly higher than those in GCMP group(F = 5. 294 and 14. 917, respectively, each P < 0. 05), and the immune memory was produced to induce IgG1, while the IgG1 levels in GCMP-PD group were lower than those in GCMP-TT group(F = 5. 828, P < 0. 05). The levels of anti-PD IgG in serum of GCMP-PD and PD groups increased significantly after each immunization, and the levels of anti-PD IgG in GCMP-PD group were significantly higher than those in PD group after the third immunization(F = 15. 426, P < 0. 01), while those in GCMP-TT group were lower than those in TT group(F =211. 207, P < 0. 001). The results of SBA showed that the titers of bactericidal antibody in GCMP-PD group were higher than those in GCMP group(F = 11. 797, P < 0. 05), while lower than those in GCMP-TT group(F = 8. 688, P < 0. 05). The results of flow cytometry showed that the ratio of Th1 and Th2 cell subsets in GCMP-PD group had no significant difference from that in GCMP and GCMP-TT groups(F = 0. 073 and 3. 021, respectively, each P > 0. 05).Conclusion The conjugation of recombinant PD with GCMP can not only enhance the immunogenicity of polysaccharide, improve the humoral immune response, but also trigger the cellular immune response. The bactericidal effect of the immunized serum is remarkable, suggesting that PD is feasible as GCMP protein carrier.

Objective To understand serotype and fimbriae-genotype of B. pertussis vaccine strains and isolates from different periods in China. Methods Serotype of eighty isolates and three vaccine strains were determined using anti-fim2 and fim3 monoclonal antibodies compared with polyclonal antisera. Fim2 and fim 3 genes were amplified by PCR and the amplified products were sequenced and analyzed . Results The serotype of three vaccine strains and all isolates but only one tested by the slide agglutination and micro-plate assay of anti-fim2 and fim3 monoclonal antibodies were the same in comparison with that of the slide agglutination of polyconal antisera. In this study, seventeen isolates and vaccine strains CS and P3S10 were fim2&3 serotype, and forty-eight isolates were tim2 serotype while fifteen isolates and vaccine strain 18530 were fim3 serotype. The predominant serotypes were fim2 and fim2&3 before Expanded Program on Immuni-zation in 1978, while the find became the most popular serotype after nation-wide pertussis vaccination in China. The fim2-1 and fim3-A genotype was the most common type, which was identified in 92.5% and 95.0% of the isolates, respectively. The genotype of vaccine strain 18530 was fim2-2 and fim3-A while oth-er vaccine strains were fim2-1 and fim3-A. The isolates contained fim3-B and fim3-D subtypes were found since 2000. These data indicated that the serotype and fimbriae genotype of B. pertussis isolates have been changed for immune environment of national-wide pertussis vaccination in China. Conclusion The validity and specificity of anti-fim2 and fim3 monoclonal antibodies have been validated for serotyping of B. pertussis strains. The information of serotype and fimbirae genotype of B. pertussis vaccine strains and isolates from dif-ferent time periods have been obtained. These data can facilitate the studies on quality control of vaccine strain, epidemiology and the evolution of B. pertussis in China.

Objective To establish a rapid,accurate,specific quantitative assay for detecting B.pertussis,and apply to clinical diagnosis.Methods According to the specific sequence of B.pertussis IS481 gene,the primers and the fluorescence probe were designed and synthesized.Then a fluorescence quantitative PCR for detecting B.pertussis was developed.The specificity,sensitivity and reproducibility of the method were evaluated.255 specimens including 225 nasopharyngeal swabs from suspected pertussis patients and 30 normal nasopharyngeal swabs were detected by fluorescence quantitative PCR.Results A rapid specific quantitative method for detecting B.pertussis was established.The standard curve of the method indicated that there was a good linear relationship between the CT value and the template concentration with the correlation coefficient being 0.998.The linear range of the system was from 102 to 108 copies/μl and the minimum was 102 copies.It had a high sensitivity and good specificity.The intra.and inter-assay coefficients of variation were 5.78%-16.7% and 8.25%-14.9% respectively.The fluorescence quantitative PCR identified 41 positive results for specimens from suspected pertussis patients and results of 30 normal specimens were all negative.Conclusions The method can quantitatively detect the B.pertussis rapidly with high sensitivity and specificity,it can be applied to clinical diagnosis.

Objective: To establish a functional antibody detection method for acellular pertussis vaccines in order to conveniently and effectively evaluate the production consistency and potency of acellular pertussis vaccine bulks and final products. Methods: Chinese hamster ovary (CHO) cell clustering assay was optimized and used to measure titers of neutralizing antibodies against pertussis toxin in mouse immune serum samples. Results: Vaccine samples were determined to be immunized intraperitoneally with 1/5 the human dose to ten female NIH mice (20-24 g, 5-week-old). Four weeks after immunization, blood samples were collected to isolate serum. Serially diluted serum samples were used to neutralize 0.1 IU/ml of pertussis toxin national reference product for 2 hours. Results of clustering were determined after 48 hours of incubation in pre-cultured CHO cell wells. The geometric mean of the serum dilution of the final unclustered wells was the neutralizing antibody titer of vaccine sample. There were significant differences in the titers of neutralizing antibodies elicited by acellular pertussis vaccines prepared with different manufacturing processes. Vaccine samples succeed or failed the modified intracerebral challenge assay (MICA) were easily distinguishable by neutralizing antibodies. Conclusion The method of detecting neutralizing antibodies to pertussis toxin greatly reduces the amount of animals used in research. CHO cell clustering assay that has better repeatability and precision can be used for monitoring and initial evaluation of the consistency and potency of the bulks and final products of pertussis vaccines prepared with different manufacturing processes.

Objective:To construct the risk prediction model of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and verify its effectiveness based on deep learning and back propagation algorithm neural network (BP neural network).Methods:Based on the relevant data of 1 326 patients with chronic obstructive pulmonary disease (COPD) in the team's previous clinical study, the acute exacerbation, and its risk factors during the stable period and 6 months of follow-up were recorded and analyzed. Combined with previous clinical research data and expert questionnaire results, the independent risk factors of AECOPD after screening and optimization by multivariate Logistic regression including gender, body mass index (BMI) classification, number of acute exacerbation, duration of acute exacerbation and forced expiratory volume in one second (FEV1) were used to build the BP neural network by Python 3.6 programming language and Tensorflow 1.12 deep learning framework. The patients were randomly selected according to the ratio of 4∶1 to generate the training group and the test group, of which, the training group had 1 061 sample data while the test group had 265 pieces of sample data. The training group was used to establish the prediction model of neural network, and the test group was used for back-substitution test. When using the training group data to construct the neural network model, the training group was randomly divided into training set and verification set according to the ratio of 4∶1. There were 849 training samples in the training set and 212 verification samples in the verification set. The optimal model was screened by adjusting the parameters of the neural network and combining the area under the receiver operator characteristic curve (AUC), and the sample data of the test group was substituted into the model for verification.Results:The independent risk factors including gender, BMI classification, number of acute exacerbation, duration of acute exacerbation and FEV1 were collected from the team's previous clinical research, and the AECOPD risk prediction model was constructed based on deep learning and BP neural network. After 10 000 training sessions, the accuracy of the AECOPD risk prediction model in the validation set of the training group was 83.09%. When the number of training times reached 8 000, the accuracy basically tended to be stable and the prediction ability reached the upper limit. The AECOPD risk prediction model trained for 10 000 times was used to predict the risk of the validation set data, and the receiver operator characteristic curve (ROC curve) analysis showed that the AUC was 0.803. When using this model to predict the risk of the data of the test group, the accuracy rate was 81.69%.Conclusion:The risk prediction model based on deep learning and BP neural network has a medium level of prediction efficiency for acute exacerbation within 6 months in COPD patients, which can evaluate the risk of AECOPD and assist the clinic in making accurate treatment decisions.

Objective: To investigate the current basic situation of the staff of ultrasound departments in Shanghai′s medical institutions, for providing references in making management policy of these professionals. Methods: Questionnaire surveys on human resource and service ability were made to all the ultrasound departments of medical institutions in Shanghai in December 2013 and November 2018 respectively. Data of the two surveys were compared and analyzed, and were descriptively analyzed by mean and percentage. Results: The number of ultrasound professionals per 10 000 people in Shanghai was 1.04 in 2018. Tertiary hospitals had advantages in the number of the professionals, and the proportion of professional qualification, age, education background and professional title of the professionals. Compared to those data in 2013, the number of ultrasound professionals had increased 31.8% in 2018. The proportion of medical practitioners with medical imaging specialty was 95.6%(2 063/2 158), and had increased by 4.7 percent. The medical services workload of ultrasound was 19.82 million person-time, and had increased 45.8%. Conclusions Development of ultrasound departments was rapid, but the development of professionals was unbalanced with the development of medical services. It is suggested to strengthen training of ultrasound professionals and improve the system of hierarchical medical system.

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.