Objective:To establish a rapid quantitative PCR (qPCR) technique for Mycobacterium marinum skin infections, and to analyze its clinical diagnostic efficiency. Methods:DNA was extracted from Mycobacterium marinum colonies and serially diluted (10 -1 to 10 -8). Twelve pairs of previously reported primers and probes, as well as 6 pairs of newly designed primers and probes in this study, were used for qPCR amplification to identify the most sensitive primers and probes for the detection of Mycobacterium marinum. Skin lesion tissues were collected from 72 patients with confirmed Mycobacterium marinum infections (experimental group) and 68 with other mycobacterial infections (control group) at Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences in 2021. These skin tissues were subjected to qPCR amplification, interferon-gamma release assay (IGRA), acid-fast staining, and tissue culture to evaluate the diagnostic efficacy. Results:The newly designed primers and probes targeting the mycobacterial enhanced infection locus 2 (Mel2) demonstrated the highest sensitivity, with a detection limit of 0.86 copies/μl (cycle threshold value = 37) ; the qPCR amplification with the Mel2 primers/probes did not yield positive results when used for the detection of other mycobacteria (including Mycobacterium leprae and Staphylococcus spp) . Among the 72 patients in the experimental group, 44 were positive for qPCR with a sensitivity of 61.1% (95% CI: 49.6% - 71.5%), and 47 were positive for culture with a sensitivity of 65.2% (95% CI: 53.8% - 75.3%) ; all the 68 controls were negative for both qPCR and culture, with their specificities both being 100%. Among 65 patients subjected to IGRA, 31 were positive with a sensitivity of 47.7% (95% CI: 36.0% - 59.6%), while 16 out of 25 controls were negative for IGRA with a specificity of 64.0% (95% CI: 44.5% - 79.8%). Among 58 patients subjected to acid-fast staining, 37 were positive with a sensitivity of 63.8% (95% CI: 50.9% - 74.9%), and 52 out of 66 controls were negative for acid-fast staining with a specificity of 78.8% (95% CI: 67.5% - 86.9%). The combination of qPCR and culture resulted in a sensitivity of 93% and a specificity of 100% for the detection of Mycobacterium marinum. Conclusion:In this study, a highly sensitive qPCR assay was developed for the detection of Mycobacterium marinum, and its combination with culture could further improve the detection sensitivity.

Background::Somatic copy number variations (SCNVs) in the CDKN2A gene are among the most frequent events in the dysplasia-carcinoma sequence of esophageal squamous cell carcinoma. However, whether CDKN2A SCNVs are useful biomarkers for the risk stratification and management of patients with esophageal squamous cell dysplasia (ESCdys) is unknown. This study aimed to investigate the characteristics and prognostic value of CDKN2A SCNVs in patients with mild or moderate (m/M) ESCdys. Methods::This study conducted a prospective multicenter study of 205 patients with a baseline diagnosis of m/M ESCdys in five high-risk regions of China (Ci County, Hebei Province; Yanting, Sichuan Province; Linzhou, Henan Province; Yangzhong, Jiangsu Province; and Feicheng, Shandong Province) from 2005 to 2019. Genomic DNA was extracted from paraffin biopsy samples and paired peripheral white blood cells from patients, and a quantitative polymerase chain reaction assay, P16-Light, was used to detect CDKN2A copy number. The cumulative regression and progression rates of ESCdys were evaluated using competing risk models. Results::A total of 205 patients with baseline m/M ESCdys were enrolled. The proportion of ESCdys regression was significantly lower in the CDKN2A deletion cohort than in the diploid and amplification cohorts (18.8% [13/69] vs. 35.0% [28/80] vs. 51.8% [29/56], P <0.001). In the univariable competing risk analysis, the cumulative regression rate was statistically significantly lower ( P = 0.008), while the cumulative progression rate was higher ( P = 0.017) in ESCdys patients with CDKN2A deletion than in those without CDKN2A deletion. CDKN2A deletion was also an independent predictor of prognosis in ESCdys ( P = 0.004) in the multivariable analysis. Conclusion::The results indicated that CDKN2A SCNVs are associated with the prognosis of ESCdys and may serve as potential biomarkers for risk stratification.

Objective:To establish a nomograph prediction model of early critical changes in children with febrile convulsion, and to provide guidance for the prevention and nursing of children with febrile convulsion.Methods:Convenient sampling method was adopted to select 384 children with febrile convulsion in Anhui Children ′s Hospital from January 2018 to April 2021 as the research objects. Based on pews, the children with febrile convulsion were divided into 334 cases of non risk group and 50 cases of risk group. Binary Logistic regression analysis were used to determine the independent risk factors affecting the early critical changes of children with febrile convulsion. A nomogram was drawn based on the independent risk factors. The discrimination and consistency of the model were verified by model ROC curve and Hosmer Lemeshow goodness of fit.The external validation of model prediction efficiency were verified by validation data. Results:Binary Logistic regression analysis showed that age, respiratory rhythm disorder, unconsciousness, breath rate, heart rate, neutrophil-to-lymphocyte ratio (NLR), red blood cell distribution width (RDW), duration of first convulsion and mean body temperature after first convulsion were influence factors for early critical changes in children with febrile convulsion ( P<0.05). The C-index of the model was 0.974 (95% CI 0.954-0.993), and the C-index of the external validation of the model was 0.922 (95% CI 0.880-0.966). The results of H-L fitting test showed that the difference was not statistically significant( χ2=0.29, P>0.05). Conclusions:The early critical changes of children with febrile convulsion may be affected by respiratory rhythm disorder, confusion of consciousness, breath rate, heart rate, NLR, RDW, duration of the first convulsion, mean temperature after the first convulsion and other factors. Pediatric emergency department should collect corresponding intervention measures for children with febrile convulsion according to the establishment of prediction model to prevent their early deterioration.

ObjectiveTo investigate the correlation between systemic immune-inflammation index (SII) and prognosis in patients with hepatic alveolar echinococcosis. MethodsA retrospective analysis was performed for the clinical data of 242 patients who were admitted to Department of Hepatopancreatobiliary Surgery, Qinghai University Affiliated Hospital, from January 2015 to December 2018 and underwent surgery for hepatic alveolar echinococcosis, and SII was calculated. The chi-square test was used for comparison of categorical data between two groups, and a Spearman correlation analysis was performed. The receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value of SII; the Kaplan-Meier method was used to plot survival curves and analyze overall survival time in the two groups, and the log-rank test was used for comparison of survival rates between the two groups; univariate and multivariate Cox regression analyses were used to identify the influencing factors for the prognosis of patients with hepatic alveolar echinococcosis. ResultsThe Spearman correlation analysis showed that SII was positively correlated with the postoperative fatality rate of patients with hepatic alveolar echinococcosis (r=0.267, P＜0.001). The ROC curve showed that the optimal cut-off value of SII before surgery was 758.92, and based on this, 242 patients with hepatic alveolar echinococcosis were divided into low SII (SII ≤758.92) group with 126 patients and high SII (SII ＞758.92) group with 116 patients. The low SII group had 1-, 3-, and 5-year survival rates of 98.20%, 88.47%, and 6610%, respectively, and the high SII group had 1-, 3-, and 5-year survival rates of 90.80%, 53.05%, and 27.40%, respectively. The low SII group had a cumulative survival rate of ＞50% and a mean survival time of 55.584 months (95% confidence interval［CI］: 53550-57.617), while the high SII group had a cumulative survival rate of ＜50%, a mean survival time of 39.384 months (95% CI: 35.070-43.698), and a median survival time of 43 months (95% CI: 34.694-51.306). The low SII group had a significantly better survival rate than the high SII group, and there was a significant difference in overall survival rate between the two groups (χ2=46.979, P＜005). The univariate analysis showed that SII ＞758.92 (hazard ratio ［HR］=5.907, 95% CI: 3.386-10.306, P=0.001) was an influencing factor for the overall survival time of patients with hepatic alveolar echinococcosis, and the multivariate Cox regression analysis showed that preoperative peripheral blood SII (HR=3.507, 95% CI: 1.911-6.435, P=0.001) was an independent risk factor for the overall survival rate of patients with hepatic alveolar echinococcosis. ConclusionPreoperative SII level is clearly correlated with the prognosis of patients with hepatic alveolar echinococcosis and can thus be used as a clinical indicator to evaluate the prognosis of patients. The higher the peripheral blood SII before surgery, the worse the prognosis of patients.

AIM: To investigate the relationship between serum soluble growth stimulation expressed gene 2 protein(sST2) level and coronary artery complex lesions and their severity. METHODS: A total of 430 patients, who were sequentially admitted to hospital for selective coronary artery angiography, were divided into control group (non-coronary heart disease group, 136 patients), simple lesions group of coronary heart disease (86 patients), complex lesions group (208 patients). To quantitative evaluate the complexity of coronary artery lesions, Syntax scores were further performed on patients in complex lesions groups, including 139 patients in the low-risk group, 36 patients in the medium-risk group, and 33 patients in the high-risk group. The serum soluble ST2 level of each group of patients was tested by means of ELISA. Spearman correlation analysis was used for the correlation between the level of soluble ST2 and the severity of coronary complex lesions. RESULTS: In 430 subjects, the soluble ST2 level of all patients with coronary heart disease (including simple lesions and complex lesions) was significantly higher than that of the control group [(3 449±1 250) vs. (2 743±961) pg/mL, P<0.001]; the sST2 levels of patients in the coronary artery simple lesions group, complex lesions low-risk group, medium-risk group and high-risk group were (3 200±1 406), (3 338±1 064), (3 728±1 228) and (4 261±1 235) pg/mL respectively, and the differences of sST2 levels among above four groups were statistically significant (P<0.001). Logistic regression analysis showed that sST2 was independently associated with coronary heart disease (OR=1.001, P<0.001) and sST2 was independently associated with the severity of coronary artery complex lesions (OR=1.001, P<0.001). Spearman-related analysis shows that the expression levels of sST2 are positively related to the severity of coronary artery lesions (rs: 0.543, P<0.001). The ROC curve showed that the area under the curve of sST2 for complex coronary lesions was AUC=0.726. CONCLUSION: Serum soluble ST2 level may be an important predictor of complicated coronary artery disease.

Objective:To establish an index system of population based SARS-CoV-2 nucleic acid screening, and provide reference to determine the screening coverage appropriately.Methods:The literature review and brain storming sessions were used to develop the basic frame and index system of population based SARS-CoV-2 nucleic acid screening. Based on Delphi method and Analytic Hierarchy Process, 21 domestic experts were selected for two rounds of consultation to determine the index system of population based SARS-CoV-2 nucleic acid screening and its weight.Results:The positive indexes of experts in two rounds of consultations were both 100%. The experts' authority coefficients ( Cr) were 0.88±0.08 and 0.89±0.07, respectively. And the range of coefficient of variation ( CV) were (0.08, 0.24), (0.09, 0.25). The Kendall's W coordination coefficients were 0.34 and 0.22 respectively, which were statistically significant. The index system of population based SARS-CoV-2 nucleic acid screening was established, which had 4 first-level indexes, 11 second-level indexes and 58 third-level indexes. Besides, the weight of each index was determined. Conclusion:The index system of population based SARS-CoV-2 nucleic acid screening has been established, which can provide scientific reference for the health administration to determine the coverage of population based SARS-CoV-2 nucleic acid screening when local COVID-19 epidemic occurs.

Objective:To investigate the induction of specific T lymphocyte by bispecific monoclonal antibody in pancreatic cancer and its killing effects on KIF20A positive pancreatic cancer PANC1 cell line.Methods:CD 3/KIF20A bispecific monoclonal antibody was prepared and concentrated by chemical cross-linking method and purified by Sephrose-25 gel chromatography. Peripheral blood samples of healthy volunteers were collected, and monocytes were isolated using lymphocyte separation solution, and then cultured as dendritic cells (DC) and T cells respectively, and then co-cultured as DC-T cells. Meanwhile vitamin C was used to treat DC-T cells (vcDC-T cells). The levels of IFN-γ, IL-2, IL-4 and IL-12 in the supernatants and T cell subsets were detected by flow cytometry. About 1×10 5 T cells, DC-T cells, and vcDC-T cells with 10, 50, 100 and 300 ng CD 3/KIF20A antibody loaded were cocultured with PANC1 cells (20∶1) for 2, 6 and 10 hours to determine the highest killing rate dosage of CD 3/KIF20A antibody loaded cells. DC-T cells and DC-T cells, vcDC-T cells loaded with the highest killing rate dosage of CD 3/KIF20A antibody were cocultured with PANC1 cells (20∶1) for 2, 6, 8, 10 and 12 hours. The aggregation effect of effector cells on target cells was observed under inverted microscope, the killing rate of tumor cells was detected by LDH method. Results:The molecular weight of CD 3/KIF20A antibody was 130 000 measured and validated by SDS gel electrophoresis. The ratio of CD 8+ CD 28+ and CD40L subsets of vcDC-T cells was increased [(47.6±15.8)% vs (38.2±7.6)%, (52.1±4.9)% vs (44.7±3.2)% ] compared with that of DC-T cells, the ratio of negative regulatory cells (Treg) was decreased [(4.3±0.8)% vs (8.3±1.1)%]; the release of IL-2, IFN-γ and IL-12 was increased [(201.2±17.3) ng/L, (163.4±13.1)ng/L, (303.3±22.6)ng/L vs 221.8±17.6)ng/L, (190.4±11.7)ng/L vs (80.3±8.6)ng/L]. All the differences were statistically significant ( P<0.01). 100 ng CD 3/KIF20A loaded T cells were observed under microscope, which obviously targeted KIF20A + pancreatic cancer PANC1 cells and had a strongest killing power. At the killing cells to targeting cells ratio of 20∶1 with 4-hour coculture, the killing rate of CD 3/KIF20A-vcDC-T cells on PANC1 cells was (88.6±2.6)%, which was significantly higher than (68.4±3.4)% and (39.2±2.1)% in the CD3/KIF20A-DC-T group and (39.2±2.1)% in the DC-T group, increasing by 20% and at lease 45%, respectively. Conclusions:DC-T cells loaded with CD 3/KIF20A antibody can significantly increase the killing rate of KIF20A positive pancreatic cancer PANC1 cells, and vitamin C intervention can further enhance the killing ability of antibody loaded T cells.

[Abstract] Chemokines are small secreted proteins produced by cancer and stromal cells. Chemokine receptors are also expressed on the surface of tumor cells and stromal cells. Chemokines bind to their homologous receptors to regulate tumor growth directly and indirectly, including direct regulation of tumor proliferation and metastasis by activating signal pathway, indirect regulation of tumor through acting on vascular endothelial cells and regulating immune response by coordinating the migration and localization of immune cells in tissues. Chemokines can be divided into four categories: CXC, CC, CX3C and C, among which CXC and CC are the most studied subtypes. In view of the fact that CXC chemokines and their receptors play a wide range of roles in malignant tumors and are closely related to the immune system, they are expected to become potential therapeutic targets, to improve tumor immune response by combining with immune checkpoint inhibitors to act in tumor microenvironment (TME). This paper reviews the research progress on chemokine/chemokine receptor axis of CXC subtypes, including the basic biological characteristics of tumor-promoting axis CXCR2/CXCLs, CXCR4/CXCL12 and tumor-suppressing axis CXCR3/CXCL9-11, their direct effect on tumor, indirect effect on TME, targeted therapy and prognostic significance of the receptors and ligands contained in these three axes.

Objective: To express the NS6 nonstructural protein of human norovirus (NoV) in Escherichia coli, and to detect its enzymatic activity after purification. Methods: Human NoV NS6 gene was cloned into the prokaryotic expression vector pDE1 and then was transformed into E. coli BL21 for expression. NS6 protein was purified by Ni-NTA affinity chromatography and Superdex 200 pg column. The activity of NS6 protein was determined by digestion of fusion protein 15VP1-6P at 37 ℃. Results: Human NoV NS6 protein was stably and highly expressed in E. coli. After purification, the expressed product reached a purity of more than 95%, and the relative molecular weight of NS6 protein was about 23×10³ Da. NS6 protein could cleave the fusion protein containing rhinovirus 3C cleavage site. Conclusions The nonstructural protein NS6 of human NoV was successfully expressed in Escherichia coli, which laid a foundation for further study on the pathogenesis of human NoV.

Objective: To evaluate the clinical value of high-risk factors in combination with stratification method in predicting the clinical prognosis of patients diagnosed with N_2-3M₀ nasopharyngeal carcinoma (NPC). Methods: A total of 226 N_2-3M₀ NPC patients who underwent IMRT from November 2013 to May 2015 were enrolled in this study. The relationship between tumor volume, cervical metastatic lymph node characteristics (necrosis and fusion) and T and N staging was analyzed. The high-risk factors that affected the survival were identified. The value of high-risk factors combined with stratification method in predicting the clinical prognosis was assessed. Results: N₃ staging, V_n≥47.15cm³ and lymph node fusion (LNF) were the high-risk factors for distant metastasis in patients with stage N_2-3M₀ NPC. All patients were classified into the low-risk, medium-risk, high-risk and extremely high-risk groups according to high-risk factors. For patients in the low-risk, medium-risk, high-risk and extremely high-risk groups, the 3-year overall survival rates were 84.2%, 76.7%, 58.7% and 36.4%(all P<0.001), 87.3%, 85.2%, 54.5% and 12.1% for the distant metastasis-free survival (DMFS) rates (all P<0.001), 76.8%, 74.3%, 49.2% and 12.1%for the progression-free survival (PFS) rates (all P<0.001), and 89.2%, 88.5%, 91.5% and 88.3% for the loco-regional recurrence-free survival (LRRFS) rates (P=0.914), respectively. The risk stratification method showed the best curve separation for DMFS compared to the V_n, N staging and LNF classification groups (all P<0.05). Conclusion High-risk factors in combination with stratification method has the highest clinical value in predicting the clinical prognosis of N_2-3M₀ NPC patients.