Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.

Objective To investigate the clinical application value of the combined detection of fecal syndecan 2(SDC2)and tissue factor pathway inhibitor 2(TFPI2)gene methylation in the diagnosis of colorectal cancer and advanced adenoma.Methods 124 pa-tients with colorectal lesions,including 43 colorectal cancer,52 advanced adenoma,and 29 non-advanced adenoma,and 46 patients with other gastrointestinal diseases such as colorectal inflammatory diseases,ulcers,etc.,who visited 5 hospitals in Zhengzhou from 2020 to 2023 were enrolled as the disease group.In addition,78 healthy individuals who underwent physical examinations in the same period were selected as the healthy control group.Their fecal samples were collected before undergoing colonoscopy,and the methyla-tion levels of SDC2 and TFPI2 genes were detected using the quantitative methylation-specific PCR(qMSP).Results The positive rates of the combined detection of SDC2 and TFPI2 gene methylation in the colorectal cancer group and advanced adenoma group(95.34％and 88.46％)were significantly higher than those in the non-advanced adenoma group(48.27％,x2=18.75/13.63,P＜0.05)and healthy controls(3.84％,x2=96.38/91.54,P＜0.05).In the colorectal cancer group,the postoperative positive rate of the combined detection in 34 patients with positive methylation of SDC2 and TFPI2(5.88％)was significantly lower than the preoperative positive rate(95.34％,x2=54.74,P＜0.05).Moreover,the mean Ct values of methylation of SDC2 and TFPI2 genes(32.38±3.72 and 31.85±3.21)in the colorectal cancer group were significantly lower than that in the advanced adenoma group(35.07±3.09 and 34.96±2.78,t=3.84 and 5.05,P＜0.05).The analysis results of the ROC curve showed that the sensitivities of SDC2,TFPI2,and their combination detection in the diagnosis of colorectal cancer were 86.40％,88.37％,and 89.35％,respectively,while in differenti-ating advanced adenoma they were 73.52％,66.73％,and 80.15％,respectively.The combined detection of SDC2 and TFPI2 in differ-entiating colorectal cancer and advanced adenoma achieved the highest diagnostic efficacy(AUCROC=0.889 and 0.738,respectively).Conclusion The combined detection of methylation of SDC2 and TFPI2 genes in fecal samples has high diagnostic value for colorectal cancer and advanced adenomas,and also has certain clinical application value for the progression and postoperative monitoring of the disease.

Objective To investigate the diagnostic value of plasma heparin-binding protein(HBP)in the bloodstream infection of tumor patients.Methods The tumor patients treated at Ruijin Hospital,Shanghai Jiao Tong University School of Medicine from Janu-ary 2023 to December 2023 were selected and further divided into the following three groups:124 patients with positive blood cultures(bloodstream infection group),95 with negative blood cultures but positive cultures from other body fluids(culture-positive group),and 100 with negative bacterial cultures(culture-negative group).The levels of HBP,procalcitonin(PCT),C-reactive protein(CRP),interleukin 6(IL-6),white blood cell(WBC),and neutrophil(NE)in the three groups were detected.The level differences of each indicator among three groups were compared.The differences in the expression levels of HBP in the bloodstream infection group with different pathogens such as Gram-negative bacteria,Gram-positive bacteria,and fungi were also compared.The Logistic regression analysis was used to evaluate the predictive efficacy of each indicator for the risk of bloodstream infection.The receiver operating char-acteristics(ROC)curve was used to assess the diagnostic value of each indicator alone and in combination for the bloodstream infection of tumor patients.Results The expression level of HBP in the bloodstream infection group(32.14[14.01,110.40]ng/mL)was sig-nificantly higher than that in the culture-positive group(23.00[5.90,54.87]ng/mL,t=5.39,P＜0.05),and both of them were sig-nificantly higher than that in the culture-negative group(6.80[5.90,16.05]ng/mL,Z=47.248,P＜0.05).HBP had a certain clini-cal predictive value for the occurrence of bloodstream infection in tumor patients(OR=1.017,95％CI:1.008～1.027,P＜0.001).In addition,the levels of HBP in the Gram-negative bacterial infection group(78.04[21.60,157.20]ng/mL)were significantly higher than that in the Gram-positive bacterial infection group(51.00[18.45,106.50]ng/mL,t=2.01,P＜0.05),and both of them were significantly higher than that in the fungal infection group(5.90[5.90,14.17]ng/mL,Z=3.40,P＜0.05).The area under the ROC curve(AUCROC),sensitivity,and specificity of HBP for the diagnosis of bloodstream infection in tumor patients were 0.717,67.37％,and 67.26％,respectively,which were superior to those of PCT(0.700,77.17％,and 52.14％),CRP(0.667,63.16％,and 62.39％),and IL-6(0.629,60.47％,and 60.47％).The AUCROC,sensitivity,and specificity of the combination of HBP,CRP,PCT,and IL-6 were 0.739,70.24％,and 68.42％,respectively,which were better than those of each indicator alone.Conclusion The detection of HBP has high clinical application value in the diagnosis of bloodstream infections of tumor patients and may serve as a potential biomarker for the bloodstream infection of tumor patients.

Objective To investigate the clinical diagnostic value of plasma RAS association domain family 1A(RASSF1A)gene methylation combined with tumor marker detection in non-small cell lung cancer(NSCLC).Methods A total of 98 NSCLC patients admitted to Qinhuangdao First Hospital from June 2023 to March 2024 were selected as the NSCLC group,and 95 patients who under-went pulmonary examinations during the same period but were not diagnosed with NSCLC were selected as the disease control group.Their general clinical data were collected.The correlations among plasma RASSF1A gene methylation,tumor markers,and clinicopatho-logical features were analyzed.The effects of RASSF1A gene methylation and tumor markers on the diagnosis of NSCLC were analyzed by the multivariate Logistic regression.A predictive model was constructed,and its effectiveness was evaluated by the receiver operating characteristics(ROC)curve and goodness of fit test.Results There were significant differences(P＜0.05)in smoking history,neu-ron specific enolase(NSE),carcinoembryonic antigen(CEA),cytokeratin 19 fragment antigen 21-1(CYFRA21-1),and RASSF1A methylation levels between the NSCLC group and non-NSCLC group.There were also significant differences(P＜0.05)in RASSF1A methylation levels,NSE,CEA,and CYFR21-1 levels among NSCLC patients with different clinical characteristics such as tumor diam-eter,differentiation degree,and growth type.The results of multivariate Logistic analysis showed that RASSF1A methylation levels(OR=1.071,95％CI:1.042-1.100),NSE(OR=1.168,95％CI:1.132-1.204),CEA(OR=1.154,95％CI:1.121-1.187),and CYFR21-1(OR=1.089,95％CI:1.023-1.195)were all independent risk factors for the diagnosis of NSCLC.A model predicting the occurrence of NSCLC was constructed using the principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),and the ROC curve analysis results showed that the area under the ROC curve(AUCROC),sensitivity,and specificity of the prediction model for the RASSF1A methylation level combined with NSE,CEA,and CYFR21-1 were 0.922(95％CI:0.896-0.948),0.897,and 0.851,respectively,which were higher than those of the RASSF1A methylation level,NSE,CEA,and CYFR21-1 alone.Conclusion The prediction model of plasma RASSF1A gene methylation level combined with tumor markers has high diagnostic value for NSCLC and can be used for the clinical diagnosis of NSCLC.

Objective To investigate the distribution of Streptococcus,sensitivity to commonly used antibiotics,and prognosis of the patients with bloodstream infections,and provide evidence for early diagnosis and selection of antibiotics.Methods The Streptococci i-solated from the blood samples of the First Affiliated Hospital of Nanjing Medical University from 2017 to 2022 were collected and di-vided into α-and β-hemolytic Streptococci according to the type of hemolysis.Their sensitivity to commonly used antibiotics was ana-lyzed.The differences in clinical data and prognosis between the two groups were also compared.Results A total of 314 strains of Streptococcus were isolated from 305 patients,including 270 strains of α-hemolytic Streptococcus and 44 β-hemolytic Streptococcus.The results of the drug sensitivity test showed that the proportion of Streptococcus strains with a diameter of 17-19 mm in the inhibition zone of vancomycin increased year by year.The positive alarm time of blood culture for β-hemolytic Streptococcus was significantly shorter than that for α-hemolytic Streptococcus(P＜0.001).Using 10.5 hours as the diagnostic threshold could assist in distinguishing blood-stream infections caused by α-and β-hemolytic Streptococcus.The overall mortality rate of patients with streptococcal bloodstream infec-tion was 15.1％,and there was no statistically significant difference in the mortality rate between the two groups(P=0.813).Conclu-sion The main Streptococcus causing bloodstream infections in our hospital is α-hemolytic Streptococcus.There are differences in the antimicrobial sensitivity and clinical characteristics among patients with different streptococcal bloodstream infections.The positive a-larm time of blood culture can assist in distinguishing the type of streptococcal bloodstream infections.In addition,the diameter of the inhibition zone of vancomycin against Streptococcus has drifted towards the breakpoint of drug resistance.

Objective To establish a model to predict the severity of patients with COVID-19 associated diarrhea by analyzing the differences of laboratory detection indicators in different grades of patients with COVID-19 associated diarrhea.Methods A total of 649 COVID-19 patients combined with diarrhea hospitalized in Wuhan Huoshenshan Hospital from February 2020 to April 2020 were retrospectively selected,and the patients with obvious causes of diarrhea had been excluded.They were further divided into the common group(n=282),severe group(n=314),and critical group(n=53),and the differences in clinical and laboratory indicators among the three groups were compared.The XGBoost model was established,and its diagnostic efficacy in predicting the severity of patients with COVID-19 associated diarrhea was evaluated by the ROC curve.Results There were statistically significant differences in blood routine test,liver function,electrolytes,fecal occult blood and other laboratory indicators among the three groups of COVID-19 associ-ated diarrhea(P＜0.05).The white blood cell count,absolute value and percentage of neutrophils,and levels of serum lactate dehydro-genase(LDH),α-hydroxybutyrate dehydrogenase(α-HBDH),γ-glutamyl transpeptidase(GGT),B-type natriuretic peptide,and blord glucose(Glu)in the critical group were significantly higher than those in the common group and severe group(P＜0.05),while the percentages of lymphocytes,monocytes,eosinophils,and basophils,and chloride concentration were significantly lower than those in the common group and severe group(P＜0.05).The results of the ROC curve showed that the prediction model constructed by eight indicators,including C-reactive protein(CRP),LDH,interleukin-6(IL-6),Glu,PT％activity,chloride(Cl-),D-dimer(DD),and procalcitonin(PCT),had significant predictive value for critical patients(AUCROC=0.939),but no obvious predictive value for the patients in the common group(AUCROC=0.630)and severe group(AUCROC=0.553).Conclusion The COVID-19 patients com-bined with diarrhea have a higher probability of developing severe or critical conditions compared with those without diarrhea.The indi-cators such as CRP,LDH,IL-6,Glu,PT％activity,Cl-,DD,and PCT have significant predictive value on whether the COVID-19 patients combined with diarrhea turn to critical illness.

Objective To prepare a variety of quality control(QC)materials for SARS-CoV-2 variants as an addition to the conven-tional SARS-CoV-2 nucleic acid QC products for the laboratory detection of mutant strains by optimizing the preparation and purification process of MS2 phage virus-like particle(VLP)technique,and evaluate their performances.Methods The typical mutation sequence fragments or full length S genes were designed and synthesized according to the genomic information of SARS-CoV-2 variants.Then,they were inserted into the downstream of maturase gene,coat protein and the pac-site of MS2 phage to construct a series of recombi-nant expression vectors.After induced by the prokaryotic expression system,the VLP products were purified through the polyethylenei-mine precipitation,ultrafiltration,nuclease digestion,and gel filtration chromatography.The obtained VLP were validated by the nucle-ic acid electrophoresis,protein electrophoresis,protein concentration determination,and fluorescence PCR,and their performances such as nucleic acid residue and stability were also evaluated.Results A total of 10 kinds of VLP containing the targeted sequences of the gene to be tested were prepared.The length of the foreign sequence wrapped in them ranged from 297 bp to 3 822 bp,which could be combined into a variety of QC materials for the mutation detection of different SARS-CoV-2 variants.The prepared VLP QC materials could not be effectively amplified without nucleic acid extraction or reverse transcription steps during the routine nucleic acid detection.The simulated QC samples remained stable after repeated freeze-thaw cycles.They could be stored stably for 2 months at 25 ℃ and 4 weeks at 37 ℃.Conclusion The established preparation and combined purification process of VLP QC materials can encapsulate vari-ous exogenous nucleic acid sequences with different lengths into the viral coat protein to form VLP,with high production efficiency.The VLP QC products prepared by the above process have stable performance and almost no residual exogenous nucleic acid,which can ef-fectively meet clinical requirements and ensure the quality of laboratory testing.

Objective To retrospectively analyze the correlation between inflammatory markers in complete blood counts(CBC)and influenza A virus infection in patients visited the outpatient department of Nanjing First Hospital.Methods The suspected influenza A virus infection patients visited the outpatient department of Nanjing First Hospital from February to March 2023 were collected and di-vided into the positive and negative groups based on the detection results of influenza A virus antigen.The differences in inflammatory markers in CBC between the two groups were compared.The Logistic regression analysis was used to evaluate the correlation between inflammatory markers and influenza A virus infection.Meanwhile,the restricted cubic spline(RCS)and age subgroup(≤6 years,7-12 years,13-17 years,and ≥18 years)analysis were also performed.Results A total of 1 487 outpatients were included,of which 404(27.2％)were positive for influenza A virus antigen.The Logistic regression analysis showed that white blood cell count(WBC),lymphocyte percentage(LY％),and lymphocyte count(LYM)were significantly negatively correlated with influenza A virus infection(P＜0.01),while the neutrophil percentage(NE％),monocyte/lymphocyte ratio(MLR),and neutrophil/lymphocyte ratio(NLR)were significantly positively correlated with influenza A virus infection(P＜0.01).The RCS analysis exhibited the same trend.The age subgroup analysis showed that when the age was greater than 6 years,LYM was significantly negatively correlated with influenza A virus infection(P＜0.01).When the age was greater than 12 years,MLR was significantly positively correlated with influenza A virus infec-tion(P＜0.01).Conclusion The WBC,LY％,LYM,NE％,MLR,and NLR in CBC parameters are important indicators associated with the occurrence of Influenza A virus infection,especially LYM in patients aged over 6 years and MLR in patients aged over 12 years,which may serve as biomarkers for the diagnosis and differential diagnosis of influenza A virus infection.

Objective To investigate the causal relationship between psychiatric and psychological disorders(depression,bipolar dis-order,and schizophrenia)and non-alcoholic fatty liver disease(NAFLD).Methods The two-sample Mendelian randomization(MR)method was adopted,with depression,bipolar disorder,and schizophrenia as exposure variables and NAFLD as the outcome variable.The single nucleotide polymorphisms(SNPs)independently associated with exposure variables were obtained from the summary data of the genome-wide association study(GWAS)as instrumental variables for MR analysis.The analysis results of the inverse-variance weighted(IVW)were used as the primary outcome indicators,while those of the MR Egger regression method,weighted median,and weighted mode as supplementary results.The Cochran's Q test,MR-Egger intercept,and"leave-one-out"method were used for sensi-tivity analysis.Results The results of IVW analysis showed that depression was positively correlated with the incidence risk of NAFLD(OR=1.21,95％CI:1.01-1.44,P＜0.05),while bipolar disorder was negatively correlated with the incidence risk of NAFLD(OR=0.91,95％CI:0.84-1.00,P＜0.05).No causal relationship was found between schizophrenia and NAFLD.The heterogeneity and sen-sitivity analysis supported the robustness of the results of the study.Conclusion Depression and bipolar disorder are causally associat-ed with the incidence of NAFLD.Depression is associated with an increased risk of NAFLD,while bipolar disorder is associated with a reduced risk of NAFLD.

Objective To evaluate the reliability of Vitek 2 AST-N335 card for determining the susceptibility of Acinetobacter bauman-nii(AB)to cefoperazone/sulbactam.Methods A total of 318 non-repeated clinical isolates of AB collected in 2023 were tested for antimicrobial susceptibility to cefoperazone/sulbactam using broth microdilution(BMD),the AST-N335 card,and the Kirby-Bauer(K-B)disk diffusion method.Using BMD as the reference method,the reliability of AST-N335 card was assessed,and the accuracy of K-B disk diffusion method as the confirmatory test was validated.Results Compared with BMD,the susceptibility testing of 318 AB strains to cefoperazone/sulbactam using the AST-N335 card showed categorical agreement(CA)of 87.8％(279/318),very major er-ror(VME)of 6.0％(19/318),major error(ME)of 0％(0/318),and minor error(mE)of 1.9％(6/318),which fall outside of the acceptable error range.In contrast,the K-B method achieved CA of 99.4％(316/318),VME of 0％,ME of 0.3％(1/318),and mE of 0.3％(1/318),all within acceptable limits.Of these,the errors with AST-N335 card occurred within the minimum inhibitory concentration(MIC)range of 8-32 μg/mL.Using BMD as the reference method,further analysis was performed on the 171 AB strains with AST-N335 card MIC values of 8-32 μg/mL for cefoperazone/sulbactam.It was revealed that at MIC of 32 μg/mL,the CA was 0％;at MIC of 16 μg/mL,CA was 5.3％(1/19)and VME rate was 84.2％(16/19),both of which substantially exceeded accepta-ble error ranges.At MIC of 8 μg/mL,the CA was 94.9％(131/138)and VME was 2.2％(3/138),both approaching the acceptable ranges.Conclusion The results obtained with Vitek 2 AST-N335 card in determining for cefoperazone/sulbactam are unreliable when the MIC values fall within the range of 8-32 μg/mL,which leads to an underestimation of the resistance rate to cefoperazone/sulbac-tam.This issue requires urgent attention in both laboratories and clinical practice.The K-B disk diffusion method could serve as a sup-plementary verification approach in routine laboratories.