Objective: To identify the structure of the complementarity determining region (CDRs), the V(D)J rearrangement and somatic hypermutational characteristics of the heavy and light chains of a red blood cell blood group-specific monoclonal antibody. Methods: The hybridoma cell line secreting human IgM κ monoclonal anti-P antibody was used as the research object. Total RNA was extracted from cultured monoclonal cell line, and cDNA was obtained by reverse transcription PCR (RT-PCR) using random hexamers primers. It was then amplified and sequenced using primers specific for variable regions of the immunoglobulin heavy and light chains encoding the anti-P antibody. The sequences were aligned against the NCBI database using online Immunoglobulin BLAST (Ig-BLAST) tool. Results: The study determined the structure of the CDRs and framework regions (FRs) of the variable regions of human monoclonal anti-P immunoglobulin, as well as the characteristics of V(D)J rearrangement. Moreover, the closest VH, VD, and VJ germline alleles for the heavy chain and VL and VJ germline alleles for the light chain were also identified. The IgH gene rearrangment pattern of the monoclonal anti-P was IGHV6-1 * 01—IGHD5-18 02—IGHJ4 02 and IgL gene was IGκV1-12 01—IGκJ3 01. Nine base mutations occurred within the germline gene IGHV6-1 01 in variable region of heavy chain, whereas 5 base mutations were found in the germline gene IGκV1-12 01 in variable region of light chain, respectively. Conclusion: This study characterized the CDR structure in monoclonal antibody cell line targeting the high-frequency red blood cell P antigen, and provided a foundation for the construction of recombinant antibody expressing plasmids and transfomation of the immunoglobulin type.

Objective To observe the electrophysiological characteristics of anterior cingulate cortex(ACC)neurons in mice with orofacial pain induced by complete Freund's adjuvant(CFA).Methods Eighteen mice were randomly divided into Sham group and CFA group.CFA was injected under the right facial beard pad in mice to construct a model of orofacial pain.Normal saline was injected in the Sham group.Von-Frey filament was used to detect the threshold of mechanical orofacial pain.The excitatory postsynaptic current(EPSC)and action potential(AP)of ACC neurons were observed in the Sham group and CFA group by patch clamp method.Results The threshold of mechanical pain in the CFA group was significantly lower than that in the Sham group after injection(P<0.05).The frequency and amplitude of the EPSC of ACC neurons in the CFA group were significantly higher than those in the Sham group(P<0.05).AP results showed that when the stimulus current was 100,125,150,175 and 200 pA,the discharge frequency of ACC neurons in the CFA group was significantly higher than that in the Sham group(P<0.05).Conclusion The activity of ACC neurons increases in the development and prgression of orofacial pain in mice and may be closely related to the formation of orofacial pain.

Objective:To describe the clinical features and prognosis of congenital anomalous origin of coronary artery(AOCA) in children to increase our understanding of the disease.Methods:This retrospective study included children diagnosed with AOCA using computed tomography coronary angiography(CTCA) admitted to the Department of Cardiology, Beijing Children′s Hospital, Capital Medical University, from January 1, 2014 to December 31, 2019.The clinical presentations, laboratory results, imaging analyses, treatments, and prognoses of these patients were analyzed.Results:A total of 208 children, including 105 boys and 103 girls, we evaluated the ages(9.03±4.18)years old with AOCA.Of these, 157 cases(75.5%) presented with cardiac symptoms, such as chest tightness, palpitations, dizziness, syncope, fatigue, and decreased endurance.Three cases(1.4%) had atypical symptoms of paroxysmal crying, dyspnoea and cyanosis, and 48 cases(23.1%) were asymptomatic.Levels of serological markers of myocardial injury were elevated in 59 cases(28.4%), and 140 cases(67.3%) had predominant ST-T abnormalities on electrocardiograms.Transthoracic echocardiography identified 27 cases (13%) with cardiac enlargement and ten cases(4.8%) with left ventricular systolic dysfunction.There were 126 cases(60.6%) with the anomalous origin of the left coronary artery revealed by CTCA, 50 cases(24.0%) with the anomalous origin of the right coronary artery and 32 cases(15.4%) with bilateral coronary arteries of anomalous origin.Five children underwent surgical treatment, and the remaining 203 children were treated conservatively with drugs.The whole group was successfully treated, and no death case was recorded during the follow-up period.Conclusion:AOCA may cause different degrees of myocardial ischemia.Diverse clinical presentations and diagnostic limitations of transthoracic echocardiography often lead to missed diagnosis or misdiagnosis.In contrast, CTCA has high diagnostic accuracy and can be used to identify the location and course of the coronary ostia.Hence, the management of AOCA should be tailored on a case-to-case basis, taking into consideration of the specific type of coronary origin, with surgical intervention being warranted if necessary.

Objective:To investigate the efficacy of Sishen pill compound combined with mesalazine in the treatment of mild to moderate ulcerative colitis and its effect on the circadian rhythm of symptoms. Methods:A total of 136 patients with mild to moderate ulcerative colitis who received treatment in Hospital of Chengdu University of Traditional Chinese Medicine from January 2018 to December 2020 were included in this prospective randomized controlled trial. These patients were divided into a treatment group ( n = 68) and a control group ( n = 68). The treatment group was treated with Sishen pill compound combined with mesalazine. The control group was treated with mesalazine alone. All patients were treated for 12 weeks. Clinical efficacy, as well as morning abdominal pain grade, morning diarrhea score, fecal trait score, Mayo score, hemoglobin, and hypersensitive C-reactive protein pre- and post-treatment, were compared between the two groups. Results:Total response rate in the treatment group was significantly higher than that in the control group [91.18% (62/68) vs. 72.06% (49/68), χ2 = 8.28, P < 0.05]. After treatment, morning diarrhea score, morning abdominal pain score, fecal trait score, Mayo score, hemoglobin, and hypersensitive C-reactive protein in the treatment group were (0.47 ± 0.56) points, (0.53 ± 0.56) points, (3.01 ± 0.72) points, (7.13 ± 1.38) points, (108.04 ± 12.21) g/L, (4.00 ± 2.19) mg/L, respectively, and they were (0.84 ± 0.56) points, (1.12 ± 0.56) points, (4.40 ± 0.76) points, (3.25 ± 1.44) points, (102.15 ± 12.61) g/L, and (6.07 ± 3.66) mg/L respectively in the control group. There were significant differences in these indexes between the treatment and control groups ( t = 3.59, 5.95, 10.06, 9.62, 2.78, 3.99, all P < 0.05). Conclusion:Sishen pill compound combined with mesalazine can effectively reduce clinical symptoms of active ulcerative colitis, increase hemoglobin level, decrease C-reactive protein level, improve the efficiency of treatment, reduce symptoms and the number of diarrhea rhythms, and improve stool symptoms of mild to moderate ulcerative colitis patients.

OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro-vascular endothelial cells(CMEC)after oxygen-glucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemia-reperfusion model,and were divided into normal,model,low(10 μmol·L-1),medium(20 μmol·L-1)and high(40 μmol·L-1)ICA group,and high ICA+ inhibitor group(40 μmol·L-1+20 nmol·L-1).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col-lect ICA-related targets,the GeneCards data-base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar-get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft-ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec-tive effect at 10-160 μmol·L-1;the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P＜0.01,P＜0.01);and the results of tube-for-mation assay showed that each group of ICA could significantly promote the generation of branches(P＜0.01)and the capillary length exten-sion(P＜0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar-gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich-ment analysis found 53 results,involving AGE-RAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu-lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra-tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha-nism of action of ICA against myocardial isch-emia-reperfusion injury may be related to the reg-ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.

Objective:To analyze the various clinical presentations of anomalous aortic origin of a coronary artery (AAOCA) and determine factors related to myocardial ischemia.Methods:Children diagnosed with AAOCA on CT coronary angiography at Beijing Children′s Hospital, Capital Medical University from 1 January 2014 to 31 December 2022 were classified based on AAOCA type, age and high-risk anatomy.The clinical characteristics of the different AAOCA types and age groups were compared and the correlation between the severity of manifestations and high-risk anatomy was analyzed.Results:A total of 69 children with AAOCA[34 males and 35 females, aged (8.89±4.40) years] were included.Ten (14.5%) patients had anomalous origin of the left coronary artery (ALCA) from the right coronary artery sinus and 57 (82.6%) patients had anomalous origin of the right coronary artery (ARCA) from the left coronary artery sinus.In two (2.9%) patients, AAOCA did not arise from a coronary sinus.Nineteen (27.5%) patients were asymptomatic, including 35 (50.7%) cases presented with minor symptoms (chest tightness, chest pain, fatigue), 13 (18.8%) cases had severe symptoms (syncope), and two (2.9%) cases had atypical symptoms (paroxysmal crying). All children were successfully treated; no deaths were reported during follow-up.There were no significant differences in gender, clinical manifestations, positive myocardial injury markers, electrocardiogram and transthoracic echocardiography findings, and proportion of children with high-risk anatomy among the different AAOCA groups (ALCA, ARCA, and non-coronary sinus AAOCA). Divided by age, there were nine (13.1%) children in infant group, 11 (15.9%) in preschool group, and 49 (71.0%) in school age group.More infants were asymptomatic than preschoolers ( P<0.001). Forty-three (62.3%) patients had a high-risk anatomy: two patients with acute take-off angles, four patients with stenosis or slit-like orifices, and 37 patients with interarterial courses.The remaining patients (37.7%) had non-high-risk anatomies.Children with a high-risk anatomy had severe symptoms and were prone to cardiac syncope ( P<0.05). Logistic multivariate analysis suggested that high-risk anatomy was an independent risk factor for cardiogenic syncope( OR=9.026, 95% CI 1.071~76.084, P=0.043). Conclusion:There are no significant differences in the proportion of high-risk anatomy and clinical characteristics among children with different AAOCA types.Younger children are often misdiagnosed due to atypical or insignificant clinical symptoms.The severity of clinical symptoms is related to the high-risk anatomy.High-risk anatomy is an independent risk factor for cardiogenic syncope in children with AAOCA.

【Objective】 To explore the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in the genotyping of difficult blood typing samples, and to provide evidence for clinical blood transfusion. 【Methods】 Three ambiguous blood group samples, submitted to Shanghai Blood Center by Shanghai regional hospitals, were studied, of which Sample1 included the proband and his parents. Serological methods were used to perform blood group typing, direct antibody test, unexpected antibody screening and identification test. Blood group genotyping was performed by using the MALDI-TOF MS detection systeme stablished in our laboratory. Sanger sequencing was used to confirm gene mutation sites, and serological or flow methods were used to verify specific samples′ phenotype. 【Results】 Serological results indicated the existence of antibodies against high frequency antigens in sample 1 (including proband and her mother), 2 and 3. The genotyping results of MALDI-TOF MS showed that the proband of sample 1 was Di(a+ b+ ), her father was Di(a-b+ ), her mother was Di(a+ b-), sample 2 was p, and sample 3 was Jr(a-). Sequencing results of three samples were consistent with mass spectrometry typing results. Serological results showed that sample 2 had a p phenotype. The flow cytometry results suggested that sample 3 had a Jr(a-) phenotype. 【Conclusion】 For the first time, we applied MALDI-TOF MS technology to blood type genotyping of ambiguous clinical samples in China. Compared with other genotyping methods such as PCR-SSP, MALDI-TOF MS has the advantages of rapid detection, high throughput and high specificity, which would contribute to identification of difficult blood typing samples in the future, as well as rare blood group screening.

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Infant tachycardia is a critical disease, mainly with supraventricular tachycardia and ventricular tachycardia.The treatment of tachycardia in infant is quite different from that of older children, and there is no relevant guidelines at present.Drug therapy in the acute stage of supraventricular tachycardia and atrial fluttery is mainly intravenous adenosine injection.Digoxin is widely used in neonates.Propranolol is the first choice for prophylactic treatment, and landilolol is in the development stage.Ventricular tachycardia can be spontaneously subsided, the treatment is dominated by intravenous lidocaine.For non-drug therapy, heart cardioerter is the emergency treatment for serious rapid arrhythmia.Radiofrequency ablation is used in infants with more severe conditions and where the onset of tachycardia can not be controlled.Bury cardioverter-defibrillator is effective in preventing infant ion channel disease complicated with malignant ventricular tachycardia induced sudden cardiac death.Subcutaneous implantion of a defibrillator may be superior to intravenous implantation in infants and young children.