Objective To explore the related risk factors for thyroid dysfunction(irTD)after immuno-therapy in patients with non-small cell lung cancer(NSCLC)and to construct a predictive model.Methods A retrospective analysis was conducted on 197 NSCLC patients who received immunotherapy at Chaohu Hospital,Anhui Medical University between January 2019 and June 2024.The patients were divided into a training set(n=137)and a validation set(n=60)in a 7∶3 ratio.Risk factors were screened through Lasso and logistic regression,and a dynamic nomogram model was constructed.The model's performance was evaluated using ROC curve,calibration curve,decision curve analysis(DCA),and clinical impact curve(CIC).Results Multivariate analysis showed that Gender(OR=0.172,95%CI:0.047～0.623),M stage(OR=2.919,95%CI:1.063～8.015),ln(SII)(OR=0.167,95%CI:0.066～0.423),ALC(OR=3.395,95%CI:1.493～7.716),and TSH(OR=1.464,95%CI:1.126～1.904)were independent risk factors for the occurrence of irTD.The model formula based on these factors is:logit(P)=9.261-1.760×Gender+1.071×M stage-1.787×ln(SII)+1.222×ALC+0.381×TSH.The model achieved an AUC of 0.832(95%CI:0.719～0.945)in the validation set,which was similar to the results in the training set.The calibration curve demonstrated good consistency between the predicted probability and actual observed values.DCA and CIC confirmed that the model has good clinical applicability.Conclusion This study identifies key risk factors for the occurrence of irTD after immunotherapy in NSCLC patients.The dynamic nomogram model developed(web calculator:https://lingchun.shinyapps.io/dynnomapp/)can effectively identify high-risk populations for irTD,providing a reliable tool for clinical decision-making.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective To explore the related risk factors for thyroid dysfunction(irTD)after immuno-therapy in patients with non-small cell lung cancer(NSCLC)and to construct a predictive model.Methods A retrospective analysis was conducted on 197 NSCLC patients who received immunotherapy at Chaohu Hospital,Anhui Medical University between January 2019 and June 2024.The patients were divided into a training set(n=137)and a validation set(n=60)in a 7∶3 ratio.Risk factors were screened through Lasso and logistic regression,and a dynamic nomogram model was constructed.The model's performance was evaluated using ROC curve,calibration curve,decision curve analysis(DCA),and clinical impact curve(CIC).Results Multivariate analysis showed that Gender(OR=0.172,95%CI:0.047～0.623),M stage(OR=2.919,95%CI:1.063～8.015),ln(SII)(OR=0.167,95%CI:0.066～0.423),ALC(OR=3.395,95%CI:1.493～7.716),and TSH(OR=1.464,95%CI:1.126～1.904)were independent risk factors for the occurrence of irTD.The model formula based on these factors is:logit(P)=9.261-1.760×Gender+1.071×M stage-1.787×ln(SII)+1.222×ALC+0.381×TSH.The model achieved an AUC of 0.832(95%CI:0.719～0.945)in the validation set,which was similar to the results in the training set.The calibration curve demonstrated good consistency between the predicted probability and actual observed values.DCA and CIC confirmed that the model has good clinical applicability.Conclusion This study identifies key risk factors for the occurrence of irTD after immunotherapy in NSCLC patients.The dynamic nomogram model developed(web calculator:https://lingchun.shinyapps.io/dynnomapp/)can effectively identify high-risk populations for irTD,providing a reliable tool for clinical decision-making.

Objective:To explore the related prognostic factors in patients with aortic dissection (AD) after surgery or interventional therapy, and to construct a multi-factor Cox regression analysis nomogram prediction model.Methods:The clinical data of patients diagnosed with AD in the emergency department of Affiliated Hospital of Jining Medical University of Shandong Province from January 2019 to December 2021 were collected to analyze retrospectively. The survival time was followed up, the survival curve was drawn by Kaplan-Meier method, the difference of survival rate among different Stanford types was tested by Log-Rank test, and Cox regression univariate and multivariate survival analysis was used to screen the prognostic factors. According to the results of multivariate analysis, the death risk nomogram model after AD was established, the differentiation of the model was evaluated by C-index, the accuracy of the model was evaluated by calibration curve, and the clinical benefit of the model was evaluated by decision curve analysis (DCA).Results:A total of 256 patients with AD were included, the mortality rate was 4.45% (37/256), and the overall survival rates at 3 months, 6 months and 12 months after operation were 90.23%, 87.50% and 85.55%, respectively. There was no significant difference in postoperative survival rate among AD patients with different Stanford types (χ 2=2.30, P=0.13). Cox multivariate regression analysis showed that history of hypertension ( HR=3.791, 95% CI 1.150-12.501, P=0.029), number of branch vessels involved ( HR=1.210, 95% CI 1.029-1.422, P=0.021), preoperative left ventricular ejection fraction (LVEF) ( HR=0.936, 95% CI 0.880-0.996, P=0.038) and perioperative complications ( HR=4.024, 95% CI 1.839-8.807, P<0.001) were independent prognostic factors in patients with AD. Furthermore, the predictive nomogram model of 3-month, 6-month and 12-month survival rate in patients with AD was constructed, and the C-index was 0.778. The calibration curve indicated that the accuracy of the model was good, and the clinical benefit of the DCA model was good. Conclusions:Hypertension history, the number of branch vessels involved, preoperative LVEF and perioperative complications are independent risk factors of postoperative death in patients with AD. The nomogram prediction model based on the above factors can be used to evaluate the postoperative survival of patients with AD.

Objective:To illustrate the characteristics of the distribution of prophages among the Group A Streptococcus(GAS) by mining the existing whole genome sequencing of the GAS, performing bioinformatic analyses, extracting data about prophages, and analyzing the state of prophages in the genome and genetic composition of some prophages. Methods:It was a retrospective study.Genome assembly sequences of GAS reported in GenBank till May 2020 were collected, and the important background information of these strains was sorted out to create a local genomic database.A phylogenetic tree of the whole genome of GAS was conducted using the bioinformatics software.The core genome was analyzed, and potential prophages and their integrity in the genome were predicted to obtain the characteristics of the distribution of prophages.Genotype types, number of core genes, and number, length and carrying rate of prophages in the database for GAS were analyzed.Results:A database containing the genome sequence of 2 529 GAS strains was established, involving 140 emm genotypes.These strains were isolated from 19 countries from East Asia, Europe, America and Oceania.Stratified by the disease background, these strains were mainly divided into invasive infection, non-invasive infection and immune sequelae.Prophage analysis of 1 798 genomes showed that at least one complete prophage was detected in 1 366 (76.0%) genomes.The number of complete prophages of each strain ranged from 0 to 6, and the length ranged from 32.8 to 62.6 kb, which was mainly 30-40 kb in length.The phiHKUssa, phiHKUvir and phiHKU488 were the most common prophages present in dominant clones circulated in China in recent years, which mainly carried virulence genes like the speC, spd1 and ssa. Conclusions:Prophages are widely distributed in the genome of GAS, which are of great significance in the evolution and expansion of dominating clones and thus reshape the population structure within the emm genotype.The establishment of a local genome database provides important baseline data for molecular epidemiological surveillance.

Objective To understand the prognosis of patients with bone metastases from first-treatment non-small cell lung cancer(NSCLC)of different pathological types after different first-line treatment regimens,in order to provide clinical prediction of disease progression,guidance of treatment,and improvement of prognosis.Methods 403 NSCLC patients with bone metastases who received primary treatment with bone metastases from Chaohu Hospital of Anhui Medical University from 1 January 2019 to 31 December 2023 were selected for univariate analysis using survival analysis(Log-rank test)and multifactorial analysis with Cox regression,and it was found that histopathological subtyping was an independent factor affecting the prognosis of patients(P=0.001,HR=1.952),after which the effect of this factor on prognosis was removed,and the total number of patients was divided into two groups according to histopathological classification:adenocarcinoma group(316)and squamous carcinoma group(87),and then multifactorial analysis was performed again using Cox regression model to analyse the factors affecting survival prognosis.Results In this data analysis,patients with the pathological type of squamous carcinoma had a median overall survival(mOS)of 15 months(95%CI:12.85～17.15)and a median progression-free survival(mPFS)of 9 months(95%CI:7.34～10.67),whereas patients in the adenocarcinoma group had a mOS of 25 months(95%CI:23.26～26.74)and mPFS was 16 months(95%CI:14.43～17.57),a statistically significant difference.The OS of the overall group was affected by multiple factors,including Eastern Cooperative Oncology Group(ECOG)score status(1/＞1)at the time of initial diagnosis,bone metastases to Trunk+limb bones+cranium,number of bone metastases≥4,mutation of ALK target genes,and the use of chemotherapy+targeted therapy as the first line of treatment.therapy;similarly,PFS was also affected by the above factors.In addition,in the adenocarcinoma and squamous carcinoma models based on histopathological classification,chemotherapy+targeted therapy and chemotherapy+immunotherapy were found to be the protective factors for the two groups,and ALK target gene mutation was only a protective factor for the adenocarcinoma group.Conclusion This study further confirmed the prognostic factors influencing the prognosis of patients with bone metastases from primary NSCLC,which provides an important reference for clinical treatment.

Objective:To establish a nucleic acid detection and genotyping method for Mycoplasma pneumoniae ( Mp) based on nucleic acid in clinical samples. Methods:Through genomic comparison, the specific target sequences of Genotype 1 and Genotype 2 Mp strains were selected to design synthetic primers and probes, and a PCR detection and classification method for Mp dual fluorescent probe was established, and the specificity, accuracy, detection limit and repeatability of the method were evaluated. The established fluorescence PCR method was used to detect the nucleic acid of clinical specimens and compared with the reported fluorescent PCR methods. Results:The nucleic acid of 18 pathogens, including other species of Mycoplasma and common respiratory bacteria and viruses, which were closely related to the Mp species, were detected, and the results showed that there was no cross-reactivity. The accuracy of detection and typing of 90 Mp nucleic acid was 100%. The detection limits of Genotype 1 and Genotype 2 Mp samples were 1.0 copy/μl, and the experimental coefficient of variation of repeatability within groups and between groups was less than 2.5%. In the detection of 88 nucleic acid of clinical specimens, the Kappa value was 0.675 and the P value was 0.267 compared with the reported real-time PCR method, showing a high degree of agreement. Conclusions:The method for detecting and genotyping Mp in this study has high sensitivity, specificity, and accuracy, which can be applied to the monitoring and prevention and control of Mp in the disease control system of provinces and cities at all levels in China. This method promotes the improvement of the Mp prevention and control system in China, strengthens the surveillance ability, and is of great significance for the early warning and prediction of Mp.

Objective:To investigate the survival status of patients with acute aortic dissection (AD) treated by different methods, and evaluate the factors affecting the survival of AD patients.Methods:According to the retrospective research method, the patients diagnosed with AD in the Affiliated Hospital of Jining Medical University from January 1, 2019 to December 31, 2020 were collected. The treatment data and follow-up of patients were counted, the survival of patients was analyzed by Kaplan-Meier survival curve, and the factors affecting the survival of patients were analyzed by Cox regression model.Results:A total of 251 patients were included in this study, including 169 patients in the surgical treatment group and 82 patients in the conservative treatment group. The in-hospital fatality rate in the surgical treatment group and the conservative treatment group were 8.88%(15/169) and 43.90%(36/82), respectively, and the difference were statistically significant ( P<0.05). The survival time of surgical treatment group and conservative treatment group was (328.08±8.17)d and (194.43±19.80)d, respectively, and the difference was statistically significant ( P<0.05). Surgery ( RR=5.424, 95% CI: 2.821-10.428, P<0.05), acute myocardial infarction ( RR=0.448, 95% CI: 0.221-0.906, P<0.05), and shock ( RR=0.266, 95% CI: 0.102-0.693, P<0.05) and stroke ( RR=0.277, 95% CI: 0.127-0.604, P<0.05) were the factors affecting 1-year survival in AD patients. Conclusions:Active surgical treatment is recommended for AD patients with surgical indication as soon as possible.

Objective:To investigate the effects of survival status in Stanford type A aortic dissection (TAAD) patients over 70 years old by different treatment methods.Methods:The clinical data of 151 TAAD patients over 70 years old from January 2012 to January 2022 in the Affiliated Hospital of Jining Medical University were retrospectively analyzed. Among them, 60 patients were treated with surgery (surgical treatment group), and 91 patients with conservative method (conservative treatment group). The duration of hospitalization and complications (including pericardial effusion, acute myocardial infarction, pericardial tamponade, shock, stroke, mesenteric ischemia and acute renal failure) were recorded. The patients were followed up within 30 d after the onset of illness. The survival status was recorded. The multivariate Cox regression analysis was used to analyze the independent risk factors of death in TAAD patients over 70 years old.Results:The duration of hospitalization in surgical treatment group was significantly longer than that in conservative treatment group: 14.00 (7.00, 19.75) d vs. 5.00 (2.00, 10.00) d. The incidences of pericardial tamponade, shock and acute renal failure: 1.7% (1/60) vs. 13.2% (12/91), 8.3% (5/60) vs. 24.2% (22/91) and 0 vs. 9.9% (9/91), with statistical differences ( P<0.01 or <0.05), but no statistical differences in the incidences of pericardial effusion, acute myocardial infarction, stroke, mesenteric ischemia between two groups ( P>0.05). Patient follow-up for 30 d, the mortality rate in surgical treatment group was significantly lower than that in conservative treatment group: 15.0% (9/60) vs. 46.2% (42/91), with statistical difference ( χ2 = 15.69, P<0.01). The multivariate Cox regression analysis result showed that conservative treatment, female, increased aortic root diameter and concomitant stroke were the independent risk factors of death in TAAD patients over 70 years old ( RR = 2.311, 2.135, 1.051 and 3.737; 95% CI 1.056 to 5.057, 1.083 to 4.212, 1.004 to 1.100 and 1.393 to 10.026; P<0.05 or <0.01). Conclusions:The early surgical treatment is recommended in TAAD patients over 70 years old with surgical indications.