ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang（LJZT）， and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， and the resulting compounds were identified by using databases， such as MassBank， PubChem， ChemSpider， Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform（TCMSP）， and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT， including liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature， database and MS information， a total of 79 compounds were identified from LJZT， including 31 flavonoids， 15 terpenoids， 14 nitrogen-containing compounds， 6 phenylpropanoids， 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges， and the precision， stability， reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5， 2.602 1， 0.082 6， 0.128 1， 1.778 6， 0.015 7， 0.006 7， 0.030 4， 0.003 2 mg·g^-1， respectively. ConclusionThe established method can quickly， sensitively and accurately analyze the chemical constituents in LJZT， clarify that the material basis of LJZT is mainly flavonoids， terpenoids and nitrogen-containing compounds， and simultaneously determine the contents of the 9 components， which can lay a foundation for the research on quality control， mechanism and clinical application of LJZT.

Objective To evaluate the diagnostic value of myocardial work related parameters in coronary ischemia patients with coronary artery disease(CAD)coronary ischemia using non-invasive left ventricular pressure strain loop(PSL),taking fraction flow reservation(FFR)as the gold standard.Methods From December 2020 to December 2021,53 clinically suspected CAD patients were prospectively enrolled.All patients underwent echocardiography,invasive coronary angiography and FFR measurement.According to the results of coronary angiography,patients were divided into myocardial ischemia group(n=24,FFR≤0.80)and non-myocardial ischemia group(n=29,FFR＞0.80).PSL was used for off-line analysis to obtain the global work index(GWI),global constructive work(GCW),global wasted work(GWW),global work efficiency(GWE),global positive work(GPW),and global systolic constructive work(GSCW)and other myocardial work parameters.The differences of parameter values between the two groups were compared.The diagnostic efficacy of work parameters in myocardial ischemia was analyzed by ROC curve.Results Compared with the non-myocardial ischemia group,GWI,GCW,GPW and GSCW were significantly decreased in the myocardial ischemia group at the 18-,16-,and 12-segment levels(P＜0.001).The ROC curve showed that the AUC results of GWI,GCW,GPW,GSCW at the 18-segment level were 0.803(95％CI 0.679-0.927),0.807(95％CI 0.687-0.928),0.822(95％CI 0.708-0.936),0.819(95％CI 0.703-0.935).The optimal cut-off value of GWI was 1 676.3 mmHg％,and the sensitivity,specificity and accuracy of predicting myocardial ischemia were 70.8％,86.2％and 79.2％,respectively.The optimal cut-off value of GCW was 1 999.4 mmHg％,and the sensitivity,specificity and accuracy of predicting myocardial ischemia were 75.0％,82.8％and 79.2％,respectively.Conclusions Analyzing myocardial work using PSL has good significance for screening suspected myocardial ischemia in CAD patients.

Objective:To explore the pathogenesis of flunarizine-induced parkinsonism from gut-brain axis perspective.Methods:Thirty male C57BL/6 mice were randomly divided into control group and flunarizine group ( n=15). Mice in the control group were given 0.1 mL 50% polyethylene glycol 400+50% saline by gavage once/d for 2 weeks, while mice in the flunarizine group were given 6 mg/mL flunarizine+50% polyethylene glycol 400+50% saline by gavage at a daily dose of 30 mg/kg for 2 weeks. Body mass was recorded 1, 3, 5, 7, 10 and 14 d after drug administration, and motor function was assessed by rotarod test 14 d after drug administration; 16s RNA sequencing was performed in the feces to observe the intestinal flora; intestinal transit function was detected by Evans blue by gavage; and then, the mice were sacrificed and homogenate or frozen sections (brain and intestinal tissues) were prepared; dopamine-ergic neuron expression was detected by Western blotting; RT-qPCR was applied to detect the expressions of inflammatory factors in the substantia nigra, and immunofluorescent staining was used to detect the expressions of ZO-1 and Claudin-5 in the intestinal epithelial tissues. Results:Compared with the control group, the flunarizine group had lower body mass ratio 1, 3, 5, 7, 10 and 14 d after drug administration (ratio to body mass before drug administration). Compared with the control group, the flunarizine group had significantly shortened residence time in rod rotating and lower rotational speed when falling ( P<0.05). Compared with the control group, the flunarizine group had decreased tyrosine hydroxylase protein in the substantia nigra without significant difference ( P>0.05). Compared with the control group, the flunarizine group had significantly increased interleukin-6 and tumor necrosis factor-α in the substantia nigra (1.00±0.00 vs. 2.79±0.83; 1.00±0.00 vs. 3.39±1.37), significantly lower intestinal Evans blue propulsion rate (80.67%±4.51% vs. 50.67%±6.03%), and statistically decreased ZO-1 and Claudin-5 expressions in the colonic epithelial tissues (27.01±1.41 vs. 16.32±2.83; 37.00±2.80 vs. 24.52±2.12, P<0.05). Totally, 576 microorganisms were noted in both control group and flunarizine group, 744 in the control group alone, and 634 in the flunarizine group alone. The intestinal flora β diversity indices in the 2 groups were significantly different based on weighted Unifrac-principle coordinates analysis (PCoA, PCoA1: 39.88%; PCoA2: 30.69%). Compared with the control group, the microbial colony structure of mice in flunarizine group was dominated by phylum thick-walled bacteria and phylum warty microbacteria, and by families Muribaculaceae, Lachnospiraceae and Akkermansiaceae. Compared with the control group, the flunarizine group had significantly decreased relative abundance of Ackermannia spp. and Lactobacillus spp. in the intestinal flora ( P<0.05). Conclusion:Flunarizine may contribute to the pathogenesis of DIP by causing structural disturbances in the intestinal flora and inducing neuroinflammation based on the gut-brain axis.

【Objective】 To investigate the correlation of serum exosomal microRNA-301a (miR-301a) with cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy. 【Methods】 A total of 211 patients with diabetic nephropathy treated with peritoneal dialysis from June 2019 to June 2020 in the First Hospital Affiliated of Hebei North University were selected as study subjects. Serum exosomal miR-301a was detected by real-time fluorescence quantitative polymerase chain reaction. The patients were divided into high miR-301a group and low miR-301a group based on the median of miR-301a; the clinical data of the two groups were compared. The correlation of miR-301a with high-sensitivity C-reactive protein (hs-CRP) was analyzed by Spearman. Linear regression was applied to analyze the factors associated with the effect of miR-301a. The patients were followed up for two years. Kaplan-Meier and Log-Rank were conducted to compare the cumulative incidence of cardiovascular and cerebrovascular events between the two groups, and COX regression and restricted cubic spline were used to analyze the level-effect relationship between miR-301a and cardiovascular and cerebrovascular events after peritoneal dialysis. 【Results】 Thirty-seven cases (17.54%) of cardiovascular and cerebrovascular events occurred during follow-up. The hs-CRP level and dialysis duration were lower in low miR-301a group than in high miR-301a group (P<0.05). There was a positive correlation between miR-301a and hs-CRP (rs=0.237, P=0.001). Linear regression analysis showed that hs-CRP was independently associated with miR-301a (P<0.05). The cumulative cardiovascular and cerebrovascular event rate in low miR-301a group was 3.70% (4/108), which was lower than that in high miR-301a group [32.04% (33/103), P<0.001]. COX regression analysis showed that high serum albumin level was an independent protective factor for cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy,while high hs-CRP level and miR-301a >1.46 were independent risk factors for cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy. Restricted cubic spline fitting COX regression analysis showed a non-linear relationship between miR-301a and cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy (P<0.05). 【Conclusion】 hs-CRP is independently associated with miR-301a in diabetic nephropathy peritoneal dialysis patients. High miR-301a level suggests a high risk of cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy.

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Animals ; Mice ; Humans ; Adenoviruses, Human/genetics* ; Escherichia coli/genetics* ; HEK293 Cells ; Isopropyl Thiogalactoside ; Blotting, Western ; Immunoglobulin G ; Antibodies, Monoclonal ; Antibody Specificity ; Mice, Inbred BALB C

【Objective】 To study the current situation of apheresis platelets collection in various regions of Gansu province by comparing and analyzing relevant data from blood stations in 14 prefecture-level cities of Gansu province. 【Methods】 The units of collected platelets and rate of double-dose collection in 13 regional blood stations and 1 provincial blood center from 2016 to 2020, as well as the clinical supply and demand was statistically analyzed. 【Results】 From 2016 to 2020, the total units of platelets collected by 13 blood stations and 1 blood center in Gansu increased from 11 255 U to 15 270 U, with the increase rate at 35.7% in 5 years, and mainly were collected by the provincial blood center (74.57%, 50 253/ 67 392). Although the rate of double-dose collection in the province showed a steady upward trend, only 3 blood stations realized annual double-dose collection more than 20%. There was still a gap of about 10% between supply and clinical needs. 【Conclusion】 Although the number of platelet collections and units in each blood station in Gansu is on the rise in general, the units collected varies in each blood station. Therefore, further measures need to be taken from the aspects of publicity, recruitment, optimizing the collection process, improving the rate of double-dose collection, retention of regular blood donors and regional coordination to increase the collection units, narrow down the regional gap and ensure the balance between supply and demand.

Acute leukemia marks the main type of non-solid cancer in children.Studies have proved that long non-coding RNA plays important roles in the pathogenesis of hematological malignancies and can be applied as a biomarker for diagnosis, prognosis and efficacy monitoring of leukemia patients.This article briefly reviews the roles of long non-coding RNA in the tumorigenesis and progression of childhood acute leukemia.

Hyperlipidemia pancreatitis in children is mostly genetic metabolic disease.The incidence of acute pancreatitis in children is only (3-13)/100 000, and pancreatitis caused by hyperlipidemia accounts for 9% of acute pancreatitis.A child suffering from V-type hyperlipidemia pancreatitis was admitted to the Children′s Hospital Affiliated to Nanjing Medical University in July 2019.The missense mutation at position c. 2770G>A of CFTR gene (nucleotide 2770 in coding region changed from guanine to adenine) in children was detected by gene sequencing, thus resulting in amino acid change p. D924N.It is extremely rare to report that CFTR gene mutation causes hereditary pancreatitis, and there is no literature report on c. 2770G>A site.This case is reported as follows, hoping to provide reference and inspiration for pediatricians.

Clinical data, nutrition support and 6-year follow-up results of a patient with Tricho-hepato-enteric syndrome (THES) diagnosed in the Children′s Hospital of Nanjing Medical University in December 2013 were analyzed.The patient suffered severe malnutrition, growth retardation, hypophosphatemia, hypoglobulinemia, high nutritional risk status and significant intestinal dysfunction.The genetic testing revealed SKIV2 L gene variation in this case of THES.During the acute exacerbation of diarrhea, enteral nutrition and periodical short-term parenteral nutrition were given as nutrition support.Deep-hydrolyzed formula by oral and low-fat, low-residue, high-quality-protein diet was given during the remission phase.At last, the diarrhea and nutritional status of the patient improved gradually.The growth and development, including neuromotor development of the case also caught up to children with the same age, and he was capable of schooling.It is suggested that rational individualized nutrition support can significantly improve intestinal function and nutritional status of children with THES, which overcome the dangerous period, improve the quality of life and prolong the survival time.

Objective:To investigate the changes of eosinophil(EOS) in feces of children with allergic enteritis caused by milk protein, and to provide basis for diagnosis and treatment of allergic enteritis.Methods:From July 2018 to June 2019, 70 children with milk protein allergic enteritis (allergic group), 50 children with non allergic common diarrhea (non-allergic group) and 50 children with healthy physical examination (healthy control group) were selected from Children′s Hospital of Nanjing Medical University.White blood cell(WBC), hemoglobin(Hb), blood platelet(PLT), EOS and the percentage of EOS(EOS%) of the 3 groups were counted by automatic hematology analyzer.The EOS in the feces of the 3 groups and the allergic group after treatment were counted by Gomori staining, and the correlation between EOS in the feces and milk protein allergic enteritis was analyzed.Results:For allergic group, EOS count in peripheral blood was(0.71±0.74)×10 9/L, EOS% was (7.56±5.96)%, and PLT was (382.96±85.98)×10 9/L.For non-allergic group, EOS was (0.31±0.18)×10 9/L, EOS% was (3.53±2.26)%, PLT was(315.12±69.81)×10 9/L.For healthy control group, EOS was(0.31±0.15)×10 9/L, EOS% was (3.66±1.65)%, and the PLT was(307.56±85.20)×10 9/L.The number of EOS, EOS% and PLT in allergic group were higher than those in non-allergic group and healthy control group ( F=13.606, 19.055, 16.074, all P<0.05). The number of EOS in feces of allergic group[(10.75 ±17.17)/ 50 fields at ×1 000]was significantly higher than that of non-allergic group[(1.16±3.09)/50 fields at ×1 000], the difference was statistically significant ( F=17.25, P<0.05), and there was no EOS in feces of healthy control group.After 4 weeks of avoidance of milk, EOS in feces decreased significantly in allergic group [(2.26±5.32)/50 fields at ×1 000], the difference was statistically significant ( t=5.822, P<0.05), while EOS in blood decreased [(0.56±0.47)×10 9/L], the difference was not statistically significant ( t=1.981, P>0.05). Conclusions:EOS in fecal of children with milk protein allergic enteritis increased obviously and decreased after treatment, is helpful to guide the clinical diagnosis and may become a new indicator of efficacy monitoring.