Objective: To prepare an inactivated hepatitis A vaccine using a attenuated strain of hepatitis A virus (HAV) H2 and to analyze its immunizing dose, so as to provide the reference for development and production of inactivated hepatitis A vaccines. Methods: Human embryonic lung diploid cells (KMB17) were infected with attenuated HAV H2 strain to proliferate the virus, then the cells containing viruses were harvested, extracted and purified. The obtained virus concentrate was prepared into vaccine bulk and test vaccines with 1 280 EU/mL antigen content. Vaccine testing was carried out according to the inactivated hepatitis A vaccine standards specified in the Part Ⅲ of the Pharmacopoeia of the People's Republic of China (2020 edition). A total of 110 mice were randomly divided into 11 groups, including 5 dose groups (80, 160, 320, 640 and 1 280 EU/dose) of the test vaccine and the reference vaccine, as well as the adjuvant control group. Mice were immunized twice by intraperitoneal injection, their serum HAV antibodies were detected, and the geometric mean titer (GMT) and positive conversion rate of antibodies were analyzed to evaluate the immunising dose of the vaccine. Results: The antigen content and viral titer of the virus harvest solution were 5 120 EU/mL and 8.33 lgCCID50/mL, respectively. The removal rate of foreign protein reached 98.05% and the recovery rate of antigen was 66.25%. The test vaccine met the requirements of Part Ⅲ of the Pharmacopoeia of the People's Republic of China (2020 edition). The GMTs of HAV antibodies in the test vaccine and the reference vaccine dose groups after the second immunization were more than twice higher than those after the first immunization. Regardless of primary immunization or secondary immunization, the GMTs (log2) of HAV antibodies in the test vaccine groups with doses of 160 EU/dose and above were higher than those in the 80 EU/dose group (all P<0.05), while there was no statistically significant differences between the dose groups of 160 EU/dose and above (all P>0.05). The antibody positive conversion rate of 160 EU/dose and above of the test vaccine was 100.00% after the secondary immunization. Conclusions The inactivated hepatitis A vaccine of attenuated H2 strain tested in this study demonstrates strong immunogenicity in mice, suggesting its potential as a candidate vaccine. The preliminary analysis indicates an immunizing dose of 320 EU/dose for children and 640 EU/dose for adults.

Objective To analyze blood Met、 Phe 、Tyr、 Arg、 Cit、 Orn、 Ser、 Thr、 C0、 C2、 C3、 C14、C14 ∶ 1 , C16 , C16 ∶ 1 , C18 , C18 ∶ 1 and urine 4-OH-PHPLA , 4-OH-PHPPA level of NICCD patient and discuss the application value of diagnosis NICCD. Methods From May 2011 to May 2015, 21 NICCD patient were diagnose in Guangxi Newborn Screening Center. Meanwhile, 100 normal children were selected as the control group. Blood Met, Phe, Tyr and other factors and urine 4-OH-PHPLA, 4-OH-PHPPA level were analyzed by SPSS. Results In the experimental group, blood Met, Phe, Tyr and many other indexes and urine 4-OH-PHPLA, 4-OH-PHPPA level were higher and blood Orn/Cit were lower than the control group(P < 0.05), while blood C2and Cit/Arg were increased (P > 0.05). Conclusion NICCD patient has abnormal biochemical index. Blood test by TMS and urine test by GC-MS are very important in NICCD diagnosis.

[Objective] The objective of this study was to evaluate the diagnostic accuracy of the 2013 edition of Breast Imaging Reporting and Data System (BI-RADS) ultrasound lexicon in diagnosing breast categories 3-5 lesions.[Methods] Using our breast ultrasound database from June 2014 to June 2016,we identified 4428 BI-RADS category 3 to 5 lesions with a known pathological diagnosis in 4 428 adult women.The positive predictive value (PPV) of each BI-RADS category was calculated based on the pathological diagnoses and compared with the reference range provided by the American College of Radiology (ACR).[Results] 4 428 lesions from 4428 patients were included in this study.The PPV of each BI-RADS category waswithin the reference range provided by the ACR in 2013.1198 (27.1％) pathological malignant/borderline results were found in the 4 428 lesions,the other 3 230 (72.9％)lesions were diagnosed with benign results.Among the malignant/borderline lesions,the rate of lymph node metastasis gradually increased as the BI-RADS categories were upgraded.Malignant lesions with a diagnosis ofinvasive ductal carcinoma or invasive lobular carcinoma showed an increasing distribution trend from category 3 to 5.[Conclusion] The 2013 editionof BI-RADS ultrasound lexiconhas good diagnostic accuracy and efficiencyin clinical practice.

Objective To clone the mature peptide gene of seo, construct two kinds of prokaryotic fusion expression vector, obtain recombinant staphylococcal enterotoxin O(SEO) and evaluate its biological activity. Methods The seo gene was amplified from ZNZ2-3 strain of Staphylococcus xylosus by designed primers and it was cloned into fusion vector pGEX-6P-1 and pET28a. The soluble recombinant protein(GST-SEO) was expressed in E. coli BL21 host cells, the purified GST-SEO was obtained by a single step of affini-ty chromatography using Glutathinione Sepharose 4B colume. Another fusion protein(P28-SEO) with his-tag was expressed in E. coli BL21 ( DE3), the purified P28-SEO protein was harvested by nickel chelate affinity chromatography method. The antigenicity of recombinant SEO was tested by Western blot. The stimulating effects of recombinant SEO on mouse lymphocytes was tested by MTT. The activity of Caspase 3 and the ap-optosis of DNA ladder were tested. Results The nucleotide sequence of the cloned seo gene was the same as that of reported in GenBank. The soluble recombinant protein GST-SEO was expressed at 25% expression level, and P28-SEO was 22%. The Western blot by GST-SEO-positive sera demonstrated that the recombi-nant SEO had good immunogenicity. The MTY assay of the enterotoxin activity showed that low dose of re-combinant SEO stimulated the proliferation of mice spleen lymphocytes, higher doses will lead to apoptosis. Conclusion Two of the recombinant SEO still have superantigen activity, and can lead to apoptosis of mice splenocytes.

Objective To understand the usage of national medical insurance negotiated drugs(hereinafter referred to as"negotiated drugs")at Tianjin Second People's Hospital and to provide references for optimizing and adjusting the hospital's drug catalog.Methods A retrospective study was conducted on the usage of negotiated drugs from January 1,2018 to December 31,2022 to compare changes in the unit price of drugs,the quantity and amount of sales,the usage frequency(DDDs)and the daily average cost(DDC),etc before and after the negotiation.Results Between 2018 and 2022,the varieties of negotiated drugs used in the hospital increased from the original 5 to 24.Among the 13 drugs analyzed for comparison,the unit prices of 11 drugs had been reduced after negotiation,and 7 drugs were included in the medical insurance and outpatient-specific disease payment directory.The average decrease in DDC was 36.43%,and the average increase in DDDs was 1 770.31%.The implementation of this policy had increased the accessibility of medication for patients and significantly increased sales quantity.Conclusion The quantity of sales of negotiated drugs significantly increased through reducing the unit price of drugs and including them in the scope of medical insurance payment,etc.These increase the pharmacoeconomic viability of negotiated drugs,effectively reduce the burden on patients,promote rational drug use in hospitals,and improve the access and efficiency of drugs.

OBJECTIVETo explore the molecular etiology for a Chinese family affected with isolated methylmalonic acidemia (MMA).

METHODSPotential mutations of MUT, MMAA and MMAB genes in the proband were screened by PCR and Sanger sequencing. The pathogenicity of identified mutations was analyzed using Polyphen2, SIFT, HSF, DNAMAN 6.0 and Swiss-PdbViewer4.1.0 software.

RESULTSTwo novel mutations of the MUT gene, including c.581C>T (p.P194L) and c.1219A>T (p.N407Y), were discovered in the proband, which were inherited respectively from his mother and father. Bioinformatics analysis suggested that both mutations were damaging. The affected codons P194 and N407, both located in the (beta, alpha) 8 barrel domain and to which the substrate methylmalonyl-CoA is bound, are highly conserved across various species. Both mutations can disrupt the space conformation of its protein product, affecting the function of the MCM protein.

CONCLUSIONThe novel mutations of MUT gene probably underlie the isolated MMA in this family.

Adult ; Amino Acid Metabolism, Inborn Errors ; enzymology ; genetics ; Amino Acid Sequence ; Animals ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Female ; Humans ; Infant ; Male ; Methylmalonyl-CoA Mutase ; genetics ; Molecular Sequence Data ; Mutation ; Mutation, Missense ; Pedigree ; Point Mutation ; Sequence Alignment

OBJECTIVETo explore the molecular mechanism for a boy suspected with 3-methylcrotonyl-CoA carboxylase deficiency by neonatal screening.

METHODSPCR and Sanger sequencing were used to identify potential mutations of MCCC1 and MCCC2 genes. SIFT and Polyphen-2 software was used to predict the effect of variant on the protein function and conservation of the variant across various species. Human Splicing Finder and Swiss-PdbViewer4.1.0 were applied to analyze the possible mechanism of the variant.

RESULTSFor the proband, a compound heterozygous mutation was discovered in the MCCC1 gene, namely c.539G>T (p.G180V) and c.704_711del (p.A235Vfs*4), which were inherited from his father and mother, respectively. The two mutations have disrupted the protein conformation, which in turn may impact the function of MCC protein.

CONCLUSIONThe compound heterozygous mutations of the MCCC1 gene may contribute to the 3-methylcrotonyl-CoA carboxylase deficiency manifested by the patient.

Amino Acid Sequence ; Base Sequence ; Carbon-Carbon Ligases ; chemistry ; deficiency ; genetics ; DNA Mutational Analysis ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Models, Molecular ; Mutation ; Neonatal Screening ; methods ; Protein Conformation ; Sequence Homology, Amino Acid ; Urea Cycle Disorders, Inborn ; diagnosis ; genetics

Objective:To analyze the correlation between peripheral blood lymphocyte subsets and unex-plained repeated implantation failure(URIF),and to investigate its predictive value for the diagnosis of URIF.Methods:A total of 156 patients with URIF who underwent treatment in the Department of Reproductive Medi-cine,The Second Affiliated Hospital of Kunming Medical University from October 1,2019 to December 1,2021 was selected as the URIF group.Meanwhile,age-matched 41 women with a history of healthy delivery in the past one year were selected as the control group.The percentages of peripheral blood lymphocyte subsets in the two groups were measured by flow cytometry,and the results were statistically analyzed.Receiver operating charac-teristic(ROC)curve was used to evaluate the predictive effect of lymphocyte subsets on URIF.Results:Com-pared with the control group,the percentage of CD3 + CD4 +[(34.03±7.26)%vs.(36.79±6.35)%,P = 0.017]、CD3 +HLA-DR +[(2.60±2.28)%vs.(3.60±2.39)%,P =0.017]、CD3 + CD16 + CD56 +[(1.24±1.04)%vs.(2.62±2.57)%,P<0.000]and CD4 +/CD8 +(1.37±0.48 vs.1.57±0.51,P =0.023)were sig-nificantly increased in URIF group,and were related to the increase of previous failure times to a certain extent.ROC analysis showed that CD3 + CD4 +>35.35%(AUC 0.624),CD3 + HLA-DR +>2.35%(AUC 0.669),CD3 +CD16 +CD56 +>1.86%(AUC 0.660)and CD4 +/CD8 +>1.26(AUC 0.628)could be used as an inde-pendent predictor for the diagnosis of URIF.Among the pair-wise combined indexes,CD3 + HLA-DR + combined with CD3 + CD16 + CD56 + had the highest prediction accuracy(AUC 0.739,Sensitivity 73.3%,Specificity 68.3%).The combination of the four indicators had the highest accuracy(AUC0.767,Sensitivity 68.6%,Specifici-ty 73.2%).Conclusions:There is immune dysfunction in patients with URIF,and the imbalance of peripheral blood lymphocyte subsets may be an important factor leading to embryo implantation failure.CD3 +CD4 +、CD4 +/CD8 +、CD3 +HLA-DR +and CD3 +CD16 +CD56 +could be used as independent indicators for the diagnosis of abnormal peripheral blood lymphocyte subsets in patients with URIF.The combination of them improves the accuracy of prediction,and it has a positive reference significance for dynamic monitoring and early intervention of URIF pa-tients in the process of assisted reproduction technology.

Objective To investigate the role of neutrophils and their IgA Fc receptor CD89 in the occurrence of Henoch-Sch（o）nlein purpura (HSP),to evaluate their effects on vascular endothelial cell apoptosis,and to explore their mechanisms.Methods Peripheral blood neutrophils were isolated from 30 children with acute HSP and 9 age-matched healthy controls separately.After isolation of serum IgA by Jacalin affinity chromatography,IgA was purified by polypropylene dextran gel chromatography.Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of CD89 on neutrophils respectively,and flow cytometry was conducted to measure the expression of neutrophil activation marker CD11b.Human umbilical vein endothelial cells (HUVEC) were co-cultured with neutrophils isolated from patients with HSP (HSP group) and healthy controls (healthy control group) separately.Moreover,the HSP group were divided into 3 subgroups to be treated with serum IgA isolated from the HSP patients (HSP IgA group),monomeric IgA (mIgA group) and phosphate-buffered saline (blank control group) respectively.Then,flow cytometry was conducted to detect apoptosis of co-cultured HUVEC,and enzyme-linked immunosorbent assay (ELISA)to measure levels of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) in the supernatant of co-cultured cells.Results There was no significant difference in the mRNA expression of CD89 on neutrophils between the patients with HSP and healthy controls (P =0.98),but the protein expression of CD89 was significantly lower in the patients with HSP than in the healthy controls (0.60 ± 0.16 vs.0.83 ± 0.24,P =0.03).The expression of CD1 1b on neutrophils was significantly higher in the patients with HSP than in the healthy controls (1 880.25 ± 388.29 vs.1 109.25 ± 364.25,P ＜ 0.01).The apoptosis rate of co-cultured HUVEC was also significantly higher in the HSP group than in the healthy control group (37.44％ ± 5.49％ vs.17.14％ ± 4.45％,P ＜ 0.01).In addition,the H SP IgA group showed significantly higher apoptosis rate of cocultured HUVEC and levels of IL-8 and TNF-cα in the supematant compared with the mIgA group (all P ＜0.01) and blank control group (P ＜ 0.01,=0.01,=0.02,respectively).Conclusions Peripheral blood neutrophils in patients with HSP are activated,which can induce the apoptosis of vascular endothelial cells.HSP IgA can promote the neutrophil-mediated apoptosis of vascular endothelial cells and secretion of IL-8 and TNF-α,while mIgA may show a certain inhibitory effects.

Objective: To investigate the clinical, biochemical and genetic features of four carnitine-acylcarnitine translocase deficiency cases. Methods: Four cases diagnosed with carnitine-acylcarnitine translocase deficiency from Guangxi Maternal and Child Health Hospital were studied. DNA was extracted from dry blood filter for gene analysis. SLC25A20 gene analysis was performed in 1 case and the whole exon sequence analysis was performed in 3 cases. Results: Retrospective study on unrelated carnitine-acylcarnitine translocase deficiency patients, the age of onset was 1-28 d, the age of death were 1.5-30 d, main clinical features were hypoglycemia (4 cases), arrhythmia (2 cases), sudden death (2 cases). Biochemical test showed hypoglycemia (1.2-2.0 mmol/L) , elevated creatine kinase (955-8 361 U/L) and creatine kinase isozyme(199-360 U/L), normal or decreased free carnitine level (3.70-27.07 μmol/L) , elevated long-chain acylcarnitine (palmityl carnitine 1.85-14.84 μmol/L). The gene tests showed that all 4 cases carried SLC25A20 gene c.199-10T> G homozygous mutation, inherited from their parents. By analyzing the haplotype, we found that the mutation loci of C. 199-10T> G were all in the same haplotype. Conclusion The c.199-10T> G mutation is an important molecular cause of carnitine-acylcarnitine translocase deficiency, which has relatively high frequency in Guangxi population, and is related to the founder effect.