Objective To analyze the virulence genes and antibiotics resistance of Escherichia coli(E.coli)isolates cause urinary tract infection(USI)in children,and further understand the epidemiological characteristics of E.coli isolates in children with USI.Methods Children with urinary system infection admitted to Quanzhou Maternity and Child Health Care Hospital&Children's Hospital from January to December 2021 were collected.E.coli was isolated from urine,and the drug sensitivity of the E.coli to 15 commonly used clinical antibiotics was detected by instruments.Among them,33 strains of E.coli that showed Extended-spectrum beta-lactamase(ESBL)production were increased,genomic DNA was extracted,and the whole genome sequence of the strains was analyzed by high-throughput sequencing technology.Results In the gene sequence analysis of 33 strains of E.coli,86 virulence genes were obtained,and the bacterial structural virulence genes accounted for the most(43/86),including capsular,fimbriae and cell-membrane system,followed by functional virulence genes and toxigenic genes,while fimH,fdeC and terC virulence genes had the highest detection rates.In the analysis of drug resistance,Cefazolin(96.97%)was the most resistant,and carbapenems were also found to be resistant.In addition,39 inactivation genes,18 target alteration genes and 51 e?ux system genes were obtained.The most information related to antibiotic e?ux(1 273/1 815),mainly RND and MFS superfamily e?ux pumps.There were 19～43 drug resistance genes in each E.coli genome.A variety of β-lactamase resistance genes were found,in which CTX-M/EC/TEM gene family dominated.Conclusion In the urine isolation of ESBL-producing E.coli in USI children,the β-lactamase should continue to be monitored.Virulence genes fimH,fdeC,terC and acrAB/emrAB/mdtABC-TolC e?ux pump gene detected higherratio,should be more attention.

Objective To investigate the clinical prognosis value of the positivity rate of oligoclonal bands(OCB)and immunoglobulin G(IgG)level of cerebrospinal fluid(CSF)in severe encephalitis.Methods A total of 699 cases of encephalitis patients admitted to the Department of Neurology of the First Affiliated Hospital of Air Force Military Medical University,and Xijing 986 Hospital from January 2016 to October 2020 were enrolled.According to the severity of their diseases,these patients were divided into a mild(n=360)group and a severe(n=339)group.CSF and serum samples were collected from the patient at the time of admission,and the differences in cerebrocyte count,glucose contem,glucose content,chlorine content,IgG of CSF and OCB of CSF and serum were compared.According to the GOS score of patients with severe encephalitis at discharge,the patients were divided into good prognosis group(n=259)and poor prognosis group(n=80),and multivariate Logistic regression analysis was used to analyze factors that affected the prognosis of severe encephalitis patients,and the correlation between the OCB and IgG of CSF and prognosis of patients with severe encephalitis.The predictive value of CSF IgG for the prognosis of patients with severe encephalitis was tested,and receiver operating characteristic(ROC)curve was plotted.Results Compared to patients with mild encephalitis,patients with severe encephalitis had a higher proportion of fever,pulmonary infection,status epilepticus,and mechanical ventilation,and were more likely to be complicated by stroke and hydrocephalus,and the differences were statistically significant(χ2=5.319～245.179,all P<0.05).There were significant differences in the positive rate of cerebrocyte count,chlorine content,IgG content and OCB in cerebrospinal fluid between the two groups(Z=-3.623,-4.875,-3.518,χ2=6.581,all P<0.05).CSF OCB and CSF IgG were independent risk factors for poor prognosis in patients with severe encephalitis(Wald χ2=7.295,0.001,all P<0.05).A restrictive cubic spline plot showed a linear relationship between CSF IgG and poor prognosis.The AUC(95%CI)of CSF IgG was 0.754(0.632～0.876).Conclusion The CSF IgG content and positive rate of CSF OCB in patients with severe encephalitis with poor prognosis are higher than those in patients with good prognosis,and detecting these two indicators has certain reference value for the prognosis prediction of patients with severe encephalitis.

Objective To differentiate acute aortic dissection(AAD)from acute myocardial infarction(AMI)using commonly used blood biomarkers and develop and validate a diagnostic model.Methods A total of 350 chest pain patients presenting to Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,from January to December,were selected,including 200 AAD patients and 150 AMI patients.The patients were randomly divided into training and validation set in a 4∶3 ratio.Nineteen blood biomarker values were collected from all patients,and the least a bsolute shrinkage and selection operator(LASSO)regression model was applied to the training set to identify the optimal predictors for constructing a differential diagnostic model between AAD and AMI.The diagnostic efficacy of the model was assessed using receiver operating characteristic(ROC)curves.Results Compared with the AMI group,patients in the AAD group exhibited significantly higher levels of monocyte count(MONO),neutrophil count(NEU),high-sensitivity C-reactive protein(hs-CRP),high-density lipoprotein(HDL),prothrombin time(PT)and D-dimer(D-D)(U=17 160～26 593,all P<0.05),whereas levels of lymphocyte count(LYM),triacylglycerol(TG),activated partial thromboplastin time(APTT),thrombin time(TT),fibrinogen(FIB),high-sensitivity cardiac troponin I(hs-cTnI),N-terminal pro-brain natriuretic peptide(NT-proBNP),aspartate aminotransferase(AST)and creatine kinase(CK)were significantly lower(U=8 943～13 063,all P<0.05).LASSO regression analysis identified D-D,hs-cTnI,NEU,LYM,hs-CRP and APTT as optimal predictors in the training set(U=2 433～8 657,all P<0.05).The constructed diagnostic model was as follows:Y=0.101(D-D)+0.065(NEU)+0.003(hs-CRP)-0.200(LYM)-0.004(APTT)-0.000 046 6(hs-cTnI)-0.006,with an optimal cutoffvalue of 0.093.The AUC(95%CI)was 0.937(0.90～0.97,P<0.01)in the training set,0.90(0.85～0.95,P<0.05)in the validation set,and 0.925(0.90～0.95,P<0.01)in the overall dataset,with a diagnostic sensitivity of 87.9%and specificity of 92.3%.Conclusion The diagnostic model based on D-D,hs-cTnI,NEU,LYM,hs-CRP,and APTT provides a valuable reference for the differential diagnosis of AAD and AMI.

Objective To investigate the predictive value of serum human epididymis protein 4(HE4)levels in primary Sj?gren's syndrome(pSS)patients for renal injury.Methods A retrospective analysis of 77 pSS patients admitted to the Second People's Hospital of Yichang from September 2021 to August 2023 was performed,including 43 cases of renal injury group 34 instances of non-renal injury group,and 54 healthy physical examination subjects(HCs)as control group.Fasting peripheral venous blood(4ml)was collected to detect the serum levels of HE4,Cys-C,TNF-α,CR,C3,C4,immunoglobulin,Anti-SSA,Anti-SSB and other indicators,and analyzed the value of HE4 in the early diagnosis of kidney injury in pSS patients.Results Compared with HCs,the pSS patients had increased levels of HE4(120.02±103.86 pmol/L vs 57.5±16.52 pmol/L),Cys-C(1.30±0.81mg/L vs 0.87±014 mg/L),and the differences were statistically significant(t=4.382,3.860,all P<0.05).The serum levels of HE4,CR and TNF-α in the renal damage group were higher than those in the non-renal damage group,and the differences were statistically significant(t/χ2=2.552～4.371,all P<0.05).Pearson correlation analysis showed that,the levels of serum HE4,Cys-C,CR and TNF-α were all positively correlated with renal damage in pSS(r=0.287～0.546,all P<0.05).Logistic regression analysis showed that elevated serum HE4 level might be an independent risk factor for inducing renal damage in pSS(Wald χ2=11.932,P<0.05).Receiver operating characteristic(ROC)curve analysis showed that the best cut-offvalue of serum HE4 for the diagnosis of pSS renal damage was 70.46pmol/L,the maximum Youden index was 0.625,AUC(95%CI)=0.876(0.799～0.954).Conclusion The serum HE4 level in patients with pSS is positively correlated with renal injury and has predictive value for the occurrence of renal injury.

Objective To explore the optimal conditions for stable transfer of reporter gene containing enhanced green fluorescent protein(EGFP)into human breast cancer cells MDA-MB-231 cells under different electrotransfection conditions.Methods A PiggyBac(PB)transposon system containing EGFP reporter gene was constructed,and transfection conditions such as waveform,voltage,shock time,shock times,plasmid concentration,cell density,transposon to transposase ratio were controlled during electrotransfection of MDA-MB-231 cells.The transfection efficiency was detected by flow cytometry fluorescein isothiocyanate(FITC)channel.The expression of fluorescent protein was observed under laser scanning confocal microscope,the electrotransfection efficiency and expression of EGFP reporter gene were verified,and the electrotransfer efficiency of MDA-MB-231 cells was analyzed under different electrotransfection conditions.Results The optimal transfection conditions of MDA-MB-231 cells were as follows:voltage 280V,exponential wave,electric shock once,concentration of transposon plasmid about 1 000 ng/μl,mass ratio of transposon to transpotase 1:1,number of cells 2×106,electrotransfection rate up to 60.23%±5.63%.The state of MDA-MB-231 cells was good.Conclusion The electrotransfection conditions of MDA-MB-231 cells were successfully optimized to achieve stable and efficient transfection,which provided efficient electrotransfer experimental parameters for the relevant basic research of MDA-MB-231 cell line.

Objective To analyze and compare the ability of two chemiluminescent immunoassay systems for detecting HBsAg mutant strains.Methods The two chemiluminescent immunoassay systems,I3000 and Liaison XL,were used to detect nine HBsAg mutant strains with known HBsAg mutation sites respectively,and to compare the detection ability of these two systems for the nine HBsAg mutant strains.The clinical serum samples that were negative for HBsAg by the I3000 system were reviewed by the Liaison XL system for HBsAg,qRT-PCR amplification primers were designed for the HBsAg mutant strains with positive results by the Liaison XL system,and fluorescence quantitative PCR and Hepatitis B serologic tests were performed on the clinical samples that had inconsistent negative-positive results between the two aforementioned systems.Results The Liaison XL detected all nine HBsAg mutant strains with known HBsAg mutation sites with positive results,whereas the I3000 detected six of them with positive but three with negative results.Using the Liaison XL system to test 1 520 samples from hospitalized patients who were negative for HBsAg with the I3000 detection system,four positive results were found,including one positive result by the fluorescence quantitative PCR assay,which belonged to the HBsAg mutant of the mutant strain P124L-F/Y143H-D144E-G145R and the patient's test values for HBeAb and HBcAb were high,suggesting that the patient was a minor triple positive.Conclusion Liaison XL can detect the P124L-F/Y143H-D144E-G145R HBsAg mutant in clinical samples,and it is very important to select HBsAg test reagents with strong HBsAg mutant recognition and capture ability.

Objective To investigate the prevalence of specific IgE antibodies against Alternaria alternata and Aspergillus fumigatus in serum samples from clinical allergy patients across selected provinces in China.Methods Data on specific IgE antibodies for Alternaria A.and Aspergillus F.were collected from 20 hospital laboratories in 17 cities spanning 11 provinces.The study analyzed the levels of specific IgE and their variations across different provinces and seasons.Results A total of 27 471 cases of Alternaria A.and 32 843 cases of Aspergillus F.specific IgE data were included.The national average positive rate of Alternaria A.IgE was 10.40%,with the highest rate of 22.68%in Jiangsu and the lowest rate of 2.06%in Guangxi.For Aspergillus F.specific IgE,the average positive rate was 4.24%,with Hubei province having the highest rate(7.25%)and Hunan province the lowest(1.23%).The difference in IgE levels for both Alternaria A.and Aspergillus F.among provinces were statistically significant(H=9 955,16 993,all P<0.0001).Among patients,5.85%had Alternaria A.specific IgE levels at grade 3 or above,while only 0.57%had Aspergillus F.specific IgE levels at this level.When examining seasonal variations using data from Liaoning,Hunan and Anhui provinces,significant seasonal changes were observed for both Alternaria A.and Aspergillus F.IgE antibodies(HAlternaria A=347.6,338.0,401.3,HAspergillus F=196.6,133.7,231.7,all P<0.0001).Conclusion The sensitization to Alternaria A.and Aspergillus F.exhibits distinct geographical characteristics and vary significantly with seasons.Given the relatively high IgE levels associated with Alternaria A.,it should be given adequate clinical attention.

Objective To investigate the role of long non coding RNA(lncRNA)Gm14461 in promoting trigeminal neuralgia(TN)and its potential molecular mechanisms.Methods A TN mouse model was induced by surgery for chronic compression injury of the infraorbital nerve.Short hairpin RNA(shRNA)of Gm14461 was injected into TN mice,and the expression level of Gm14461 and mechanical pain threshold(MWT)in the trigeminal ganglia of the mice were measured.A TN cell model was established in vitro using tumor necrosis factor-α(TNF-α)to induce trigeminal ganglia,and the trigeminal ganglia were treated with sh-Gm14461 and cleavage stimulation factor subunit 2(CSTF2)overexpression vector(OE-CSTF2).RNA immunoprecipitation(RIP)analysis of CSTF2 binding to Gm14461,calcitonin gene-related peptide(CGRP),purine receptors P2X3 and P2X7 mRNA.Results Compared with the sham surgery group,the expression of Gm14461 in the trigeminal ganglia of TN group mice was significantly increased(t=35.450),and the MWT value of mice decreased in a time-dependent manner(t=8.427～28.190),with statistical significance(all P<0.05).The expression levels of inflammatory factors TNF-α,IL-1 β and IL-6,as well as pain related proteins CGRP,P2X3,and P2X7,were significantly increased in the trigeminal ganglia of TN mice,and the differences were statistically significant(t=31.750～50.240,all P<0.05).Knocking down Gm14461 significantly improved MWT(t=6.019～26.548),reduced inflammatory factors(t=43.959,26.416,25.431),and pain related protein levels(t=38.709,43.257,45.807)in TN mice,with statistically significant differences(all P<0.05).Compared with the control group,TNF-α induced upregulation of Gm14461 expression in the trigeminal ganglia(t=27.710),significantly increased levels of inflammatory factors and pain related protein expression(t=23.076～29.508),and the differences were statistically significant(all P<0.05).After knocking down Gm14461,the expression of Gm14461 was significantly reduced(t=3.641),and the levels of inflammatory factors and pain related proteins in the trigeminal ganglia induced by TNF-α were significantly reduced(t=20.560～25.136),with statistical significance(all P<0.05).Gm14461 upregulates CSTF2 protein levels by binding to CSTF2,and upregulates CSTF2 protein by binding to mRNA of CGRP,P2X3,and P2X7 and promoting their expression.Overexpression of CSTF2 can reverse the inhibitory effect of Gm14461 knockdown on TNF-α-induced inflammation and pain related protein expression in trigeminal ganglia(TGs)(t=18.870～24.399,all P<0.05).Conclusion Gm14461 upregulates the expression of CGRP,P2X3,and P2X7 by recruiting CSTF2,promotes inflammatory response,thereby reducing the mechanical pain threshold of TN mice and promoting pain.

Objective To investigate the mechanism of astragalus polysaccharide(APS)in inhibiting the proliferation and metastasis of osteosarcoma cells.Methods Cell counting kit-8(CCK8)assay was used to detect the inhibitory effect of APS on the proliferation of osteosarcoma cell lines U2OS,HOS,MG63 and osteoblast cell line hFOB1.19,the cell line with the most significant inhibitory effect of APS on osteosarcoma cells was selected for subsequent experiments.Osteosarcoma cells were divided into control group(NC group),APS group,APS+sh-SP1 co treatment group with transfected SP1 knockdown plasmid(APS+sh-SP1),and APS+oe SP1 co treatment group with transfected SP1 overexpression plasmid(APS+oe SP1 group).Western blotting was used to detect the expression of SP1 protein in each group.CCK8 assay was used to detect the proliferation ability of each group.Transwell assay was used to detect the metastatic ability of cells in each group.TOP/FOP Flash assay was used to detect the activity of Wnt/β-catenin signaling pathway.The protein expressions of Wnt3a,β-catenin,CyclinD1,cMYC,matrix metalloproteinase 2(MMP2)and Snail were detected by western blotting.Result After 48h of APS treatment,the cell proliferation inhibition rates of osteosarcoma cells U2OS,HOS,MG63 and osteoblast cells hFOB1.19 were 62.93%±4.79%,20.66%±1.10%,39.31%±3.20%and 5.97%±0.72%,respectively.Compared with osteoblast hFOB1.19,APS significantly inhibited the proliferation of osteosarcoma cells,and the difference was statistically significant(F=208.400,P<0.001),and the inhibitory effect on osteosarcoma U2OS cells was the most significant(t=20.380,P<0.001).Compared with NC group,SP1 protein expression,cell proliferation ability,number of transmembrane cells,Wnt/β-catenin signaling pathway activity in cells,Wnt/β-catenin signaling pathway key proteins Wnt3a,β-catenin,downstream proliferation-related protein CyclinD1,cMYC,and downstream metastasis related proteins MMP2 and Snail in the APS group were decreased,and the differences were statistically significant(t=9.740～90.780,all P<0.05).Compared with the APS group,the expression of SP1 protein,cell proliferation ability,the number of transmembrane cells,the activity of Wnt/β-catenin signaling pathway and the expression of Wnt/β-catenin signaling pathway related proteins in the APS+sh-SP1 group were further decreased,and the differences were statistically significant(t=3.032～12.940,all P<0.05).Compared with the APS group,the expression of SP1 protein,cell proliferation ability,the number of transmembrane cells,the activity of Wnt/β-catenin signaling pathway and the expression of Wnt/β-catenin signaling pathway related proteins in the APS+oe-SP1 group were increased,and the differences were statistically significant(t=3.350～22.450,all P<0.05).Conclusion APS inhibits the proliferation and metastasis of osteosarcoma cells by targeting SP1/Wnt/β-catenin signaling axis.

Objective To evaluate the application of ELISA randomized quality control,and continuously improve the laboratory testing capacity and quality assurance,in order to gradually improve the application of randomized quality control to the daily testing of ELISA.Methods Collected the quality control data of KEHUA HBsAg,compared the difference between randomized quality control data and immobilized quality control data.Group comparison of randomization quality control between rows and columns.The randomized quality control data were analyzed retrospectively and the quality control chart was established by using the randomized quality control data.Analyzed and compared the lost-control situation of randomized quality control and immobilized quality control.Results Randomized quality control S/CO value(2.831±0.343)and immobilized quality control S/CO value(2.651±0.260)in the same microplate,the difference between two was statistically significant(t=5.970,P＜0.05).The differences between randomized quality control and immobilized quality control in columns 2 to 8 were statistically significant(t=2.285～5.536,all P<0.05).There were no statistically significant differences between randomized quality control and immobilized quality control in column 9 to 12(t=0.031～1.605,all P>0.05).There was no statistically significant difference in randomization quality control among all lines(F=0.858,P＞0.05).The randomized quality control data was used to establish a quality control chart.Within the time range of the collected data,the randomized quality control was out of control for 6 times,all were greater than+3s,and the loss of control rate was 4.72%(6/127).Fixed position quality control lost control 9 times during the same period,all of which were greater than+3s,with a loss of control rate of 0.61%(9/1 481).Conclusion The randomized quality control has a greater possibility to reflect the factors affecting all the samples on the microporous plate.Random quality control can be used to find possible systematic errors in testing.Randomized quality control can gradually be fully applied to daily indoor quality control,but the loss of control rate and coefficient of variation may increase.