Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×10⁴ were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 10⁵, and 3D5B7 reached 10⁷. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Horses ; Animals ; Mice ; Encephalitis Virus, Western Equine ; Ascites ; Immunosuppressive Agents ; Antibodies, Monoclonal ; Immunoglobulin M

Objective To establish a method for uranium aerosol sample collection, dry ashing treatment, and laboratory laser-fluorescence measurement in the workplace of uranium processing and fuel fabrication facilities. Methods Through optimization experiments, the effects of sampling flow, sample pH value, and test temperature on uranium aerosol concentration results were studied, and the detection limit, precision, and recovery rate of the method were tested. Results Under the optimal test conditions, the detection limit of the method was 0.025 ng/mL; the minimum detectable concentration of 1 m³ of aerosol samples was 1.25 × 10⁻³ μg/m³; the relative standard deviation (RSD) of the measurement results was less than 5%; the recovery rate was between 96% and 104%. Conclusion The detection limit, precision, and accuracy of the method meet the testing requirements for uranium aerosol samples in the workplace of uranium processing and fuel fabrication facilities.

Objective:To explore and analyze differential expressed genes in malignant pleural mesothelioma (MPM) by bioinformatics method, and to study their prognostic value in MPM and their potential role in immunotherapy.Methods:In January 2022, the dataset GSE51024 was downloaded from the GEO database, and MPM (55 cases) and normal tissue (41 cases) samples were obtained. Using R software and HMDD and miRNet database, MPM-related differential genes were screened and co-expressed genes were identified. Co-expressed genes were enriched and functionally annotated, and protein-protein interaction (PPI) networks were constructed and key genes were identified using the STRING database and Cytoscape software. TRRUST and GEPIA databases were used to predict transcription factors of key genes and to analyze prognosis and survival. The correlation between key genes and the degree of infiltration of immune cells was analyzed using TIMER.Results:A total of 435 co-expressed genes were obtained, which were mainly concentrated in the extracellular matrix tissue and the signaling pathways of cell adhesion molecules. Combined with PPI and TRRUST database, seven key MPM prognostic genes were identified. Among them, cyclin 20 (CDC20) , cell cycle checkpoint kinase 1 (CHEK1) , enhancer of Zeste homolog 2 (EZH2) , ribonucleotide reductase subunit M2 (RRM2) , topoisomerase 2A (TOP2A) , ubiquitin like plant homeodomain and ring finger domain 1 (UHRF1) were significantly up-regulated in MPM, while cyclin A1 (CCNA1) was significantly down-regulated. The expressions of CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 genes were significantly associated with MPM overall survival ( P<0.05) . The expressions of CDC20, CHEK1, EZH2, RRM2 and TOP2A genes were positively correlated with B cells and dendritic cells ( P<0.05) , and negatively correlated with neutrophils ( P<0.05) . Conclusion:CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 may be potential prognostic markers in MPM patients, and their expressions may be related to MPM tumor immunity.

Objective:To explore and analyze differential expressed genes in malignant pleural mesothelioma (MPM) by bioinformatics method, and to study their prognostic value in MPM and their potential role in immunotherapy.Methods:In January 2022, the dataset GSE51024 was downloaded from the GEO database, and MPM (55 cases) and normal tissue (41 cases) samples were obtained. Using R software and HMDD and miRNet database, MPM-related differential genes were screened and co-expressed genes were identified. Co-expressed genes were enriched and functionally annotated, and protein-protein interaction (PPI) networks were constructed and key genes were identified using the STRING database and Cytoscape software. TRRUST and GEPIA databases were used to predict transcription factors of key genes and to analyze prognosis and survival. The correlation between key genes and the degree of infiltration of immune cells was analyzed using TIMER.Results:A total of 435 co-expressed genes were obtained, which were mainly concentrated in the extracellular matrix tissue and the signaling pathways of cell adhesion molecules. Combined with PPI and TRRUST database, seven key MPM prognostic genes were identified. Among them, cyclin 20 (CDC20) , cell cycle checkpoint kinase 1 (CHEK1) , enhancer of Zeste homolog 2 (EZH2) , ribonucleotide reductase subunit M2 (RRM2) , topoisomerase 2A (TOP2A) , ubiquitin like plant homeodomain and ring finger domain 1 (UHRF1) were significantly up-regulated in MPM, while cyclin A1 (CCNA1) was significantly down-regulated. The expressions of CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 genes were significantly associated with MPM overall survival ( P<0.05) . The expressions of CDC20, CHEK1, EZH2, RRM2 and TOP2A genes were positively correlated with B cells and dendritic cells ( P<0.05) , and negatively correlated with neutrophils ( P<0.05) . Conclusion:CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 may be potential prognostic markers in MPM patients, and their expressions may be related to MPM tumor immunity.

Objective:To investigate five kinds of tuberculin skin test (TST), tubercle bacillus-antibody(TB-Ab), interferon-γ release assay(IGRA), tubercle bacillus-DNA (TB-DNA) and collection of bacterial centrifugal smears methods, the application value of combined detection in improving the diagnostic efficiency of pneumoconiosis complicated with tuberculosis.Methods:A total of 150 eligible patients with pneumoconiosis from January 2018 to January 2019 in Zhangjiakou Pulmonary Hospital were selected as the research subjects, and all of them underwent TST, TB-Ab, IGRA, TB-DNA and bacterial centrifugal smear detection. Compared the positive rates of five detection methods in pneumoconiosis and its different stages, and compare the proportion of tuberculosis infection and tuberculosis in different stages of pneumoconiosis.Results:Among the 150 patients with pneumoconiosis, 41 cases (27.33%) were with pneumoconiosis complicated with tuberculosis infection, 24 cases (16.00%) with pneumoconiosis complicated with clinically diagnosed pulmonary tuberculosis, 21 cases (14.00%) with pneumoconiosis complicated with confirmed pulmonary tuberculosis, and 45 cases (30.00%) with pneumoconiosis complicated with pulmonary tuberculosis; with the improvement of pneumoconiosis stage, the proportion of pneumoconiosis combined with tuberculosis infection and pulmonary tuberculosis increased significantly ( P<0.05). Compared with TB-Ab, TB-loop-mediated isothermal amplification(LAMP), and interlayered cup collection centrifuge smear method, the overall positive rate of IGRA detection and pneumoconiosis stage Ⅲ were higher ( P<0.05), but there was no significant difference compared with TST detection ( P>0.05). The positive rate of combined detection was higher, but there was no significant difference compared with IGRA detection ( P>0.05). With the increase of pneumoconiosis stage, the positive reaction intensity of TST decreased, and the positive value of TB-Ab and IGRA increased. Conclusions:The combined detection of TST, TB-Ab, IGRA, TB-DNA and bacterial centrifugal smear method can significantly improve the diagnostic efficiency of pneumoconiosis combined with tuberculosis.

Objective:To investigate the effect of abnormal D-dimer level on the prognosis of patients with frontal lobe injury, and to provide a new idea for clinical diagnosis, treatment and prognosis evaluation of frontal lobe injury.Methods:A retrospective analysis was performed on 71 patients with traumatic brain injury (TBI) who were admitted to the Surgical Care Unit of the First Affiliated Hospital of Zhejiang University School of Medicine from November 1, 2020 to February 1, 2021. According to whether TBI involved in the frontal lobe, the patients were divided into the experimental group ( n = 44) and the control group ( n = 27). Clinical data including sex, age, body mass index (BMI), length of hospital stay, ISS trauma score, GCS coma score, qSOFA score, GOS prognostic score and coagulation index test results within 24 h of admission of the two groups were recorded, and the difference of each index between the two groups was compared. Results:① D-dimer levels were significantly higher in the experimental group than in the control group ( P<0.01); ② The incidence of complications in the experimental group was 40.9%, of which the incidence of lower limb vein thrombosis was 18.2%, and the incidence of complications in the control group was 18.5%, of which the incidence of lower limb vein thrombosis was 3.7%; there were statistical differences between the two groups ( P<0.05); ③ The average hospital stay of patients was 10.96 days in the control group, and 15.50 days in the experimental group with a statistically significant difference between the two groups ( P<0.05); ④ The 28-day mortality rate of patients in the experimental group was 18.2% and 7.4% in the control group. There was no significant difference in 28-day mortality between the two groups ( P>0.05); ⑤ The level of D-dimer was negatively correlated with GOS prognostic score in patients with frontal lobe injury ( r=-0.501, P<0.01). Conclusions:Patients with frontal lobe injury have increased D-dimer levels, increased incidence of complications, and longer hospital stay. There is a significant negative correlation between D-dimer level and prognosis in patients with frontal lobe injury. D-dimer can be used as a sensitive indicator to evaluate the prognosis of patients with frontal lobe injury. The higher the D-dimer level, the worse the prognosis of patients.

Objective:To determine hydrazine quantitatively in workplace air by gas chromatography with large bore capillary column.Methods:In October 2019, hydrazine in the air was adsorbed by acid silica gel tube sampling and desorped using sulfuric acid solution. After derivatization with furfural and extraction, the content of hydrazine was determined by DM-FFAP capillary column gas chromatography with flame ionization detector.Results:The linear regression equation was y=353.8 x+21.2 ( r=0.9998) between 0.1-2.0 μg/ml of target concentration. The detection limit was 0.030 μg/ml. The lower limit of quantification was 0.100 μg/ml. If 15 L air sample was collected, the minimum detection concentration was 0.004 mg/m 3 and the minimum quantitative concentration was 0.013 mg/m 3 respectively. The average desorption efficiency was 86.5%-89.4%. The recovery was 94.4%-97.1%. The relative standard deviation was 1.6%-4.9%. Hydrazine and furfural derivative was 2-furaldehyde hydrazine. Conclusion:The method has symmetrical peak shape of hydrazine derivatives chromatographic peaks, short analysis time, easy operation, and is suitable for the determination of the concentration of hydrazine in the air in the workplace.

Objective:To screen and identify plasma differentially expressed genes and related signal pathway by human gene expression profile array and fluorescent quantitative PCR.Methods:From September 2018 to October 2019, 291 workers from a Mercury-in-glass thermometer factory in Jiangsu Province were selected for an occupational health examination, a total of 60 persons were divided into two groups: high and low mercury exposure groups (30 persons in each group) . Plasma total RNA samples from the high exposure group and the low exposure group (10 cases each) were detected by gene expression microarray, and differentially expressed genes (DEGs) with fold change >2 were selected. DEGs were submitted to David and Metascape for gene function clustering, pathway and protein interaction network analysis. Finally, fluorescence quantitative PCR was performed to verify the changes in the expression levels of key DEGs in the high exposure group and the low exposure group (another 20 cases in each group) .Results:A total of 269 DEGs, of which 203 up regulated and 66 down regulated were identified in the differential expression analysis of gene expression microarray. Bioinformatics analysis suggested that, DEGs were involved in forebrain development, glial cell fate determinants of GO biological process and PID NF-KB, PTEN signal pathway. NFE2L1, SOX8, SOX6 and RNF2 ( P<0.05) were confirmed down regulated in high level group by fluorescent quantitative PCR compared with the low level group (fold changes were 2.10, 11.52, 2.19, and 4.38 respectively) . Conclusion:The plasma NFE2L1, SOX8, SOX6 and RNF2 gene expressions are significantly altered in occupa tional high mercury exposure population. PTEN signaling pathway and fate of glia cells determines the biological process may be closely related to the body injury caused by mercury exposure.

Objective:To establish a rapid method for detecting the activity of 2019-nCoV2.Methods:The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection system was screened after propidium monoazide (PMA) treatment and exposure to heat-inactivated 2019-nCoV samples, and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method . The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant, to evaluate its effect in detecting virus activity.Results:For the PMA-RT-qPCR assay, 200 μmol/L of PMA, 10 min of incubation time, 15 min of exposure time, and the CDC ORF1ab detection system were selected; there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus; the Ct values of PMA-RT-qPCR for virus inactivated by 95 ℃ and chlorine-containing disinfectant were higher than that of control groups at different dilutions; only partial dilutions of 70 ℃ and 56 ℃ heat-inactivated virus had higher Ct values than control groups. Conclusions:The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95 ℃ heat and chlorine disinfectant, and provides an auxiliary means for judging the infectivity of the virus in the sample.

Objective:To explore the epidemiological characteristics and treatment outcomes of patients with inhalation injuries combined with total burn area less than 30% total body surface area (TBSA).Methods:A retrospective observational study was performed on medical records of 266 patients with inhalation injuries combined with total burn area less than 30%TBSA who were admitted to the First Affiliated Hospital of Naval Medical University from January 2008 to December 2016 and met the inclusion criteria. The following statistical data of the patients were collected, including gender, age, injury site, injurious factors of inhalation injury, degree of inhalation injury, combined total burn area, tracheotomy, time of tracheotomy, mechanical ventilation, whether stayed in intensive care unit (ICU) or not, microbial culture results of bronchoalveolar lavage fluid, length of hospital stay, length of ICU stay, mechanical ventilation days, and respiratory tract infections. Single factor and multivariate linear regression analysis were used to screen out the risk factors impacting the length of hospital stay, length of ICU stay, and mechanical ventilation days of patients. Single factor and multivariate logistic regression analysis were used to screen out the risk factors impacting respiratory tract infections of patients.Results:The 266 patients included 190 males and 76 females, with the majority age of above or equal to 21 years and below 65 years (217 patients). The major injury site was confined space. The major factor causing inhalation injury was hot air. Mild and moderate inhalation injuries were more common in patients. The combined total burn area was 9.00% (3.25%, 18.00%) TBSA. In 111 patients who had tracheotomy, most of them received the procedures before being admitted to the First Affiliated Hospital of Naval Medical University. The length of hospital stay of patients was 27 (10, 55) days. The length of ICU stay of 160 patients who were hospitalized in ICU was 15.5 (6.0, 40.0) days. The mechanical ventilation days of 109 patients who were conducted with mechanical ventilation were 6.0 (1.3, 11.5) days. A total of 119 patients were diagnosed with respiratory tract infections, with 548 strains including 35 types of pathogens isolated, mainly Gram-negative bacteria. Single factor linear regression analysis showed that age, injurious factors of inhalation injury, combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy, mechanical ventilation, and respiratory tract infections were the factors impacting the length of hospital stay of patients ( β=-0.198, -0.224, 0.021, 0.127, 0.164, -0.298, 0.357, 0.447, 95% confidence interval (CI)=-0.397--0.001, -0.395--0.053, 0.015-0.028, 0.009-0.263, 0.008-0.319, -0.419--0.176, 0.242-0.471, 0.340-0.555, P<0.1). Multivariate linear regression analysis showed that with mechanical ventilation and respiratory tract infections were the independent risk factors impacting the length of hospital stay of patients ( β=0.146, 0.383, 95% CI=0.022-0.271, 0.261-0.506, P<0.05 or P<0.01). Single factor linear regression analysis showed that injurious factors of inhalation injury, combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy (no tracheotomy and prophylactic tracheotomy), mechanical ventilation, and respiratory tract infections were the factors impacting the length of ICU stay of patients ( β=0.225, 0.008, 0.237, 0.203, -0.408, -0.334, 0.309, 0.523, 95% CI=0.053-0.502, 0.006-0.010, -0.018-0.457, -0.022-0.428, -0.575--0.241, -0.687--0.018, 0.132-0.486, 0.369-0.678, P<0.1). Multivariate linear regression analysis showed that with respiratory tract infections was the independent risk factor impacting the length of ICU stay of patients ( β=0.440, 95% CI=0.278-0.601, P<0.01). Single factor linear regression analysis showed that injury site, injurious factors of inhalation injury (smoke and chemical gas), combined total burn area, degree of inhalation injury (moderate and severe), tracheotomy (no tracheotomy and prophylactic tracheotomy), and respiratory tract infections were the factors impacting mechanical ventilation days of patients ( β=-0.300, 0.545, 0.163, 0.005, 0.487, 0.799, -0.791, -0.736, 0.300, 95% CI=-0.565--0.034, 0.145-0.946, 0.051-1.188, 0.001-0.009, 0.127-0.847, 0.436-1.162, -1.075--0.508, -1.243--0.229, 0.005-0.605, P<0.1). Multivariate linear regression analysis showed that smoke inhalation, severe inhalation injury, and respiratory tract infections were the independent risk factors impacting mechanical ventilation days of patients ( β=0.210, 0.495, 0.263, 95% CI=0.138-0.560, 0.143-0.848, 0.007-0.519, P<0.05 or P<0.01). Single factor logistic regression analysis showed that age, injury site, combined total burn area (10%-19%TBSA and 20%-29%TBSA), degree of inhalation injury (moderate and severe), tracheotomy (prophylactic tracheotomy and no tracheotomy), and mechanical ventilation were the factors impacting respiratory tract infections of patients (odds ratio=1.079, 0.815, 1.400, 1.331, 1.803, 1.958, 0.990, 0.320, 3.094, 95% CI=0.840-1.362, 0.641-1.044, 1.122-1.526, 1.028-1.661, 1.344-2.405, 1.460-2.612, 0.744-1.320, 0.241-0.424, 2.331-4.090, P<0.1). Multivariate logistic regression analysis showed that with mechanical ventilation was the independent risk factor impacting respiratory tract infections of patients (odds ratio=4.300, 95% CI=2.152-8.624, P<0.01). Conclusions:The patients with inhalation injuries combined with total burn area less than 30%TBSA are mainly young and middle-aged males. Smoke inhalation, degree of inhalation injury, with mechanical ventilation and respiratory tract infections are the factors that affect the outcomes of patients with inhalation injuries combined with total burn area less than 30%TBSA. Additionally, prophylactic tracheotomy shows its potential value in reducing respiratory tract infections in patients with moderate or severe inhalation injuries.