Objective To investigate the epidemiological status and molecular characteristics of diarrheagenic Escherichia coli(DEC)infection in Xiangshan County,providing a basis for its infection surveillance and outbreak control.Methods The real-time fluores-cence quantitative PCR(qRT-PCR)was used to detect virulence genes in isolated E.coli strains.The identified DEC strains were per-formed whole genome sequencing(WGS).The sequencing data were utilized for bacterial resistance gene analysis,multilocus sequence typing(MLST),and core genome multilocus sequence typing(cgMLST).Results A total of 29 DEC strains were isolated,with a detection rate of 9.8%.Enteroaggregative E.coli(EAEC)and enterotoxigenic E.coli(ETEC)were the main strains,accounting for 51.7%and 31.1%,respectively,while enteropathogenic E.coli(EPEC)had a relatively low proportion(17.2%).Drug resistance gene analysis showed that these strains were generally resistant to multiple antibiotics such as fluoroquinolones and carbapenems.MLST typing identified 19 sequence types(STs),including one novel ST type(STcpEC1).Among them,ST117,ST2178,ST31,and ST48 were dominant types.ETEC strains were mainly concentrated in the clonal complex CC-10,while the molecular typing of EAEC and EPEC strains was more dispersed.cgMLST analysis further confirmed the genetic characteristics of different DEC strains.Conclusion DEC infections in Xiangshan County are mainly caused by EAEC,ETEC,and EPEC.These strains have abundant resistance genes.Among them,the molecular typing of EAEC and EPEC is relatively dispersed,while ETEC is concentrated in the clonal complex CC-10.

Objective To investigate the clinical application value of serum exosomal miR-552 and miR-221 in the diagnosis and prog-nosis of gastric cancer.Methods 133 patients diagnosed with gastric cancer in the Department of Gastrointestinal Surgery,Suqian First People's Hospital from January to December 2019 were selected as the gastric cancer group,108 patients with chronic gastritis as the benign lesion group,and 94 healthy individuals undergoing physical examination as the healthy control group.Their blood samples were collected,and serum exosomes were extracted.The expression levels of miR-552 and miR-221 in serum exosomes were detected by real-time fluorescence quantitative PCR(qRT-PCR).The receiver operating characteristics(ROC)curve was used to assess the di-agnostic efficacy of miR-552 and miR-221 alone and in combination for gastric cancer.The correlations of serum exosomal miR-552 and miR-221 levels with the clinicopathological parameters of gastric cancer patients were further analyzed,and the survival curve was used to evaluate the prognosis of gastric cancer patients.Results The expression levels of serum exosomal miR-552 and miR-221 in gastric cancer patients,benign lesion patients,and healthy controls showed statistically significant differences(FmiR-552=296.271,FmiR-221=249.638,P＜0.01)and a decreasing trend.The analysis results of the ROC curve showed that the combined detection of serum exosom-al miR-552 and miR-221 for the diagnosis of gastric cancer had an area under the ROC curve(AUCROC)of 0.901,a sensitivity of 89.50%,and a specificity of 96.90%,all significantly higher than the detection of miR-552(sensitivity of 88.7%and specificity of 87.2%)and miR-221(sensitivity of 83.5%and specificity of 85.1%)alone(P＜0.05).The expression levels of serum exosomal miR-552 and miR-221 were related to tumor size,TNM staging,invasion depth,and lymph node metastasis(P＜0.05).The analysis results of the survival curve revealed that gastric cancer patients with high expression of serum exosomal miR-552 and miR-221 had sig-nificantly shorter survival time than those with low expression(χ2miR-552=30.657,χ2miR-221=38.251,P＜0.05).Conclusion Serum exo-somal miR-552 and miR-221 have high diagnostic efficacy for gastric cancer and may serve as novel biological markers for the diagnosis and prognosis evaluation of gastric cancer.

Objective To investigate the clinical significance and application value of circulating tumor cells(CTCs)detected by fluo-rescence in situ hybridization(FISH)in the diagnosis of breast cancer.Methods A total of 114 breast cancer patients and 30 patients with benign breast lesions admitted to the Department of Thyroid and Breast Surgery,Yixing Hospital Affiliated to Jiangsu University from January 2014 to June 2022 were enrolled.Their peripheral blood cells were enriched by the immunomagnetic bead negative enrich-ment method,and the levels of CTCs were detected by FISH technology.The diagnostic efficacy of CTCs in breast cancer was evaluated by the receiver operating characteristics(ROC)curve,and the correlations of the levels of CTCs with clinicopathological parameters of breast cancer patients were analyzed by the chi-square analysis.Results The detection results of FISH showed that 75 of 114 breast cancer patients were positive for CTCs and 39 were negative.The evaluation results of the ROC curve for the diagnostic efficacy of CTCs in breast cancer showed that when the cut-off value was 2.5 CTCs/mL,its sensitivity and specificity were 52.0%and 89.7%,respec-tively,and the area under the ROC curve(AUCROC)was the best(0.68).There were significant differences in the positive rates of CTCs among different TMN stages and molecular types of breast cancer patients(P＜0.05),but not among different vascular invasion types and immunohistochemical indicators of breast cancer patients(P＞0.05).Conclusion The positive of CTCs detected by FISH may indicate different TNM staging and poor molecular typing.The CTCs is expected to become an important biomarker affecting the clinical decision-making and prognosis evaluation in the treatment of early breast cancer.

Objective To investigate the relationship between the expression levels of lymphocyte activation gene-3(LAG-3)and pen-traxin-3(PTX3)and the disease severity of the patients with allergic rhinitis(AR).Methods A total of 128 AR patients visited the Department of Otolaryngology,Chengdu Integrated TCM and Western Medicine Hospital from January 2021 to June 2023 were retro-spectively selected as the research subjects(AR group).According to the severity of the condition,the patients were further divided into the mild group(n=40),moderate group(n=42),and severe group(n=46).In addition,80 healthy volunteers who underwent physical examinations in our hospital were selected as the control group.The clinical data such as the gender,age,body mass index(BMI),allergens,and disease onset time of all subjects were recorded and analyzed,and all patients were scored using the Score For Allergic Rhinitis(SFAR).The enzyme linked immunosorbent assay(ELISA)was used to detect the expression levels of serum LAG-3 and PTX3 in all subjects.The correlation between the expression levels of serum LAG-3 and PTX3 was analyzed by the Pearson correla-tion analysis.The receiver operating characteristic(ROC)curve was used to evaluate the diagnostic value of serum LAG-3 and PTX3 levels,both individually and in combination,for AR.Results The expression levels of serum LAG-3 in the AR group(468.74±104.32 μg/L)were significantly lower than that in the control group(691.53±184.65 μg/L,t=7.795,P<0.05),while those of ser-um PTX3 in the AR group(24.83±7.54 ng/L)were significantly higher than that in the control group(17.34±5.37 ng/L,t=7.793,P<0.05).The expression levels of serum LAG-3 in the AR patients with positive immunoglobulin(IgE)and SFAR score≥7 were 442.46±92.37 μg/L and 448.27±103.24 μg/L,respectively,which were significantly lower than that in the AR patients with negative IgE(497.61±115.32 μg/L)and SFAR score<7(498.66±112.76 μg/L,P<0.05).While the expression levels of serum PTX3 in the AR patients with positive IgE and SFAR score≥7 were 28.24±8.17 ng/L and 26.43±8.73 ng/L,respectively,which were significantly higher than that in the AR patients with negative IgE(21.08±6.25 ng/L)and SFAR score<7(22.51±6.89 ng/L,P<0.05).Com-pared with the mild group,the expression levels of serum LAG-3 in both the moderate and severe groups were reduced,and that of ser-um LAG-3 in the severe group was significantly lower than that in the moderate group.While the expression levels of serum PTX3 showed an opposite trend,and the levels of serum PTX3 in the severe group were significantly higher than that in the moderate group(P<0.05).The Pearson correlation results showed a significant negative correlation between serum LAG-3 and PTX3 levels in AR pa-tients(r=-0.402,P=0.000).The analysis of the ROC curve showed that the area under the ROC curve(AUCROC),sensitivity,and specificity of the combination of serum LAG-3 and PTX3 in the diagnosis of AR were 0.881,89.80％,and 73.80％,respectively,which were better than that of serum LAG-3 and PTX3 alone.Conclusion The combined detection of serum LAG-3 and PTX3 for the diagnosis of AR has higher clinical application value and may be used to evaluate the severity of the disease.

Objective To investigate the expression level and clinical application value of circular RNA hsa_circ_0001766 in gastric cancer(GC)tissues and serum of patients with GC.Methods The PCR amplification products from GES-1 cells of normal gastric mucosa were analyzed for the cyclic sites of hsa_circ_0001766 by the Sanger sequencing technique.The expression levels of hsa_circ_0001766 in GES-1 cells treated or untreated with RNase R(RNase R)were detected by real-time fluorescence quantitative PCR(qRT-PCR).The expression levels of hsa_circ_0001766 in GC and adjacent tissues of 73 GC patients and serum samples of 34 GC patients were also detected by qRT-PCR.The receiver operating characteristics(ROC)curve was used to evaluate the clinical diag-nostic value of hsa_circ_0001766 in GC.The correlations between hsa_circ_0001766 and clinicopathological parameters in GC patients were also analyzed.Results There was cyclic sites in hsa_circ_0001766 and RNase R had no significant impact on the expression lev-el of hsa_circ_0001766 in GES-1 cells(t=1.678,P=0.169).Compared with adjacent tissues,the expression levels of hsa_circ_0001766 in GC tissues were significantly up-regulated(U=1 360,P<0.001).Compared with healthy controls,the expres-sion levels of hsa_circ_0001766 in serum of GC patients were significantly up-regulated(U=375,P<0.001).The area under the ROC curve(AUCROC),sensitivity,and specificity of hsa_circ_0001766 in GC tissues for diagnosing GC were 0.759(95％CI:0.682-0.837,P<0.000 1),79.45％,and 68.49％,respectively.The AUCROC,sensitivity,and specificity of serum hsa_circ_0001766 in diagnosing GC were 0.773(95％CI:0.662-0.884,P<0.000 1),73.53％,and 72.12％,respectively.The expression levels of hsa_circ_0001766 in GC tissues were correlated with lymph node metastasis(x2=5.509,P=0.019)and TNM staging(x2=5.161,P=0.023).The 3-year survival rate of GC patients with high expression of hsa_circ_0001766 in GC tissues was significantly lower than that with low ex-pression(x2=3.700,P=0.037).Conclusion The hsa_circ_0001766 in GC tissues and serum of GC patients is highly expressed,and its change in the expression level is of great significance for the diagnosis and prognostic evaluation of GC patients.

Objective To investigate the role and clinical significance of Calpain activity and 145 kDa cleavage fragments of αⅡ-spec-trin breakdown products(SBDP145)in systemic lupus erythematosus(SLE).Methods 124 patients with confirmed SLE and de-tailed clinical data were collected as the SLE group,and 75 healthy individuals who underwent physical examination during the same period were selected as the control group.Their serum samples were collected,and serum SBDP145 levels and Calpain activity were de-tected by the enzyme-linked immunosorbent assay(ELISA)and substrate-based method,respectively.The Mann-Whitney U test,Spearman rank correlation,univariate and multivariate Logistic regression,and receiver operating characteristic(ROC)curve were used to evaluate the clinical value of Calpain activity and SBDP145 in the diagnosis and assessment of SLE.Results Compared with healthy controls,serum Calpain activity([17.000[8.5000,33.500]RFU vs 7.500[4.000,12.500]RFU)and SBDP145 levels(5.283[3.532,9.463]ng/mL vs 1.472[0.994,2.212]ng/mL)in SLE patients were significantly increased(Z=-9.229,P<0.001;Z=-6.881,P<0.001).Furthermore,the Calpain activity was significantly correlated with SLE disease activity index(SLEDAI-2K)score(r=0.349,P<0.001).Logistic regression analysis showed that the increased Calpain activity and SBDP145 levels were significantly associated with the occurrence of SLE(OR=1.164,95％CI:1.016-1.334,P=0.029;OR=3.822,95％CI:1.928-7.574,P<0.001).The ROC curve analysis showed that the area under the ROC curve(AUCROC)of serum Calpain activity in distin-guishing SLE and healthy controls was 0.762(95％CI:0.697-0.827).When the cut-off value was 13.75 RFU,its sensitivity and speci-ficity were 63.1％and 81.1％,respectively.The AUCROC of serum SBDP145 levels in distinguishing SLE and healthy controls was 0.891(95％CI:0.845-0.936).When the cut-off value was 2.377 ng/mL,its sensitivity and specificity were 88.7％and 80.0％,re-spectively.The AUCROC,sensitivity,and specificity of combining the two with traditional SLE clinical indicators,including complement C3,erythrocyte sedimentation rate,and anti-dsDNA antibodies,in the diagnosis of SLE reached 0.986(95％CI:0.972-1.000),93.3％,and 96.0％,respectively.The Spearman correlation analysis results showed that the organ damage index(SDI)score of SLE patients was positively correlated with serum Calpain activity and SBDP145 levels(r=0.342,P<0.001;r=0.250,P=0.005).Con-clusion The elevated Calpain activity and SBDP145 levels are closely related to the occurrence and development of SLE,suggesting that they may be served as potential biomarkers for the diagnosis and assessment of SLE patients.

Objective To establish an efficient and stable one-step real-time quantitative PCR(qRT-PCR)method for detecting mi-croRNA-like small RNAs(milRNAs)encoded by novel Bunyavirus and evaluate its clinical application value.Methods Three kinds of milRNAs encoded by novel Bunyavirus,including sRNA S-1480,sRNA M-692,and sRNA L-4706,were selected based on prelimi-nary screening.The specific qRT-PCR primers and reaction systems for them were designed and optimized.The sensitivity,specificity,and clinical applicability of the method were further evaluated by standard curve drawing,primer concentration/sequence optimization,fluorescent dye optimization,product sequencing verification,and serum specimen detection of 20 patients infected with novel Bunya-virus and 20 healthy controls.Results A one-step qRT-PCR detection system was successfully established.The standard curve con-structed with standard substance showed a good linear relationship in the range of 10 fmol/L to 100 nmol/L(R2>0.98).The detection limits for three kinds of milRNAs were 10 fmol/L.The analysis results of the receiver operating characteristics(ROC)curve showed that sRNA S-1480(AUCROC=0.972 5),sRNA M-692(AUCROC=0.757 5),sRNA L-4706(AUCROC=0.957 5),and their combina-tion(AUCROC=0.995 0)could effectively distinguish the patients infected with novel Bunyavirus from healthy controls.Conclusion The established one-step qRT-PCR detection system exhibits high sensitivity and provides an efficient and reliable alternative for the clinical diagnosis of novel Bunyavirus infection.

Objective To investigate the clinical significance of the combined screening of thyroid stimulating hormone(TSH)and seven candidate pathogenic genes of congenital hypothyroidism(CH)for CH.Methods 16 645 newborns delivered in Nanjing Women and Children's Healthcare Hospital from July 2022 to July 2023 were performed the screening of TSH.Their DNA was extracted from dried blood spots and the chip capture second-generation sequencing technology was used to detect the candidate pathogenic genes,in-cluding dual oxidase 2(DUOX2),dual oxidase maturation factor 2(DUOXA2),prophet of pit-1(PROP1),thyroid-stimulating hor-mone receptor(TSHR),thyroid peroxidase(TPO),thyroglobulin(TG),and paired box 8(PAX8).The sensitivity,specificity,pos-itive predictive value(PPV),and negative predictive value(NPV)of the screening of TSH,candidate genes,and their combination for CH were analyzed.Results A total of 13 CH patients were screened out based on sensitive thyroid stimulating hormone(sTSH)and free thyroxine(FT4),including 3 patients with hyperthyrotropinemia.Among them,11 were screened out by TSH alone,4 were screened out by candidate genes alone,and 2 were screened out by the combination of TSH and candidate genes.The sensitivity,speci-ficity,PPV,and NPV of TSH for screening CH were 84.62％,99.23％,7.91％,and 99.97％,respectively.The sensitivity,specifici-ty,PPV,and NPV of candidate genes for screening CH were 30.77％,99.87％,15.38％,and 99.87％,respectively.The sensitivity,specificity,PPV,and NPV of the combination of TSH and candidate genes for screening CH were 100％,99.09％,7.88％,and 100％,respectively.The primary mutant gene in the samples with positive candidate genes was DUOX2(85.71％),mainly point muta-tions,among which the c.1588A>T variant was the most common(16.67％).PAX8(14.29％)was the second most common variation,and all of the variation point were c.280G>A.No positive samples for the pathogenic variants of DUOXA2,TSHR,PROP1,TPO,and TG were detected.Conclusion The combined screening of TSH and candidate genes helps to improve the screening efficacy of CH.The genetic etiology of CH in Nanjing area may be mainly the variation of DUOX2 and PAX8 genes.

Objective To investigate serum exosomal miRNA spectrum in patients with gestational diabetes mellitus(GDM)and eval-uate its clinical value in the diagnosis of GDM complicated with premature delivery.Methods Serum samples of pregnant women with GDM registered and delivered in our hospital were collected and divided into the premature delivery group and term labor group based on pregnancy outcomes,with 22 cases in each group.Serum exosomal miRNAs were sequenced,and the differentially expressed miRNAs were further verified by fluorescence quantitative PCR.The receiver operating characteristic(ROC)curve was drew according to the verified expression level of miRNAs,and the value of exosomal miRNAs in the diagnosis of GDM complicated with premature de-livery was analyzed.The potential functions of candidate miRNAs were predicted using the Kyoto Encyclopedia of Genes and Genomes(KEGG).Results A total of 94 differentially expressed miRNAs,including 50 up-regulated and 44 down-regulated,were identified in the premature delivery group and term labor group.The verification results of fluorescence quantitative PCR showed that 7 miRNAs had significant difference between the two groups(P<0.05).Moreover,the expression trend was consistent with the sequencing results.The analysis results of the ROC curve showed that the seven miRNAs had good diagnostic efficacy for GDM combined with premature delivery.The areas under the ROC curve(AUCROC)of hsa-miR-16-5p,hsa-miR-25-3p,hsa-miR-30b-5p,and hsa-miR-92a-3p were all more than 0.7.Their sensitivity and specificity were 0.375 and 1.000,0.563 and 0.941,0.563 and 0.824,and 0.765 and 0.647,respectively.The binary logistic regression analysis showed that the AUCROC of the combination of hsa-miR-16-5p,hsa-miR-25-3p,hsa-miR-30b-5p,and hsa-miR-92a-3p for the diagnosis of GDM complicated with premature delivery increased to 0.982,and that its sensi-tivity and specificity were both more than 0.850.These candidate miRNAs were related to the ubiquitin-mediated proteolysis pathway,actin cytoskeleton regulatory pathway,mTOR signaling pathway,and P53 signaling pathway.Conclusion Serum exosomal miRNAs in GDM patients complicated with premature delivery have significant difference,which may be served as potential diagnostic markers.

Objective To investigate the coagulation abnormalities and molecular genetic characteristics of a family with asymptomatic inherited fibrinogen disorders(IFD).Methods The clinical data of a family with IFD,including 5 individuals from two generations,were collected.Their peripheral blood coagulation indicators were detected.The coding sequences of FGA,FGB and FGG genes were amplified by PCR and Sanger sequencing was used to identify the candidate variants,which were further validated in the family mem-bers.The bioinformatic software was used to analyze the pathogenicity and conservation of the missense mutation and its effect on the spatial structure and function of the protein.Results The IFD patients had significantly low fibrinogen antigen(Fg:Ag)concentration and fibrinogen coagulation(Fg:C)activity concentration as well as prolonged thrombin time(TT),while coagulation indicators of the unaffected relatives were normal.The results of Sanger sequencing showed that all IFD patients carried a heterozygous missense variant of c.1001A>C(p.Asn334Thr)in the FGG gene.The bioinformatic analysis suggested that Asn334Thr was a pathogenic variant,while homology analysis indicated that the Asn334 locus was highly conserved in evolution.The analysis of protein spatial structure showed that the Asn334Thr mutation altered hydrogen bonds between amino acids.Conclusion The heterozygous missense variant c.1001A>C(p.Asn334Thr)in the FGG gene may be the pathogenic cause of the proband.The finding enriches the spectrum of FGG gene mutations and provides experimental evidence for the genetic counseling of affected families.