For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.
Animals ; Biomarkers ; Cricetinae ; Epithelial Cells ; virology ; Gene Deletion ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombination, Genetic ; Vaccinia ; virology ; Vaccinia virus ; genetics ; physiology ; Virus Replication

Objective:To investigate the correlation between preeclampsia and thyroid dysfunction in pregnancy.Methods:107 early pregnant women with preeclampsia admitted to our hospital from Jan. 2017 to Jan. 2020 were all enrolled (observation group) . The observation group were in 6-34 gestational weeks, with an average parity of (1.67±0.35) times. In addition, 100 cases of normal pregnant women in the same period were selected as the control group. The control group were in 6-34 gestational weeks, with an average parity of (1.61±0.31) times. The two groups were compared at early, mid and late pregnancy in terms of thyroid function, and the correlation of preeclampsia and abnormal thyroid function was analyzed.Results:1. TSH levels in the observation group were (1.92±1.24) , (2.07±0.82) , and (2.30±1.23) mU/L in the first trimester, second trimester and third trimester, respectively, showing an upward trend. The difference between the two groups was statistically significant ( P=0.024) . FT4 levels of the observation group in the first trimester, second trimester and third trimester were (0.80±0.26) , (0.60±0.34) and (0.59±0.32) pmol/L, respectively, showing a decreased trend and the difference was statistically significant compared with those of the control group ( P=0.012) . 2. The incidence of hypothyroidism, TPOAb positive and reduced free tetraiodothyronine in the observation group was significantly higher than that in the control group ( P=0.001, 0.023, 0.005) . There was no significant difference in the incidence of hyperthyroidism or subclinical hypothyroidism between the two groups ( P=0.169, 0.846) . 3. Correlation analysis showed that preeclampsia was related to hypothyroidism, normal thyroid function with TPOAb positive and reduced free tetraiodothyronine ( P=0.000, 0.000, 0.000) . Conclusions:There are changes in thyroid function in pregnant women with preeclampsia. Hypothyroidism, positive TPOAb and reduced free tetraiodothyronine are closely related to the onset of preeclampsia.

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R² = 0.033; P = 0.018), followed by acute kidney injury (AKI; R² = 0.032; P = 0.011) and plasma MIP-1β level (R² = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1β level were associated with patients' lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements.
Acute Kidney Injury/complications* ; Bacteria/classification* ; Chemokine CCL4/blood* ; Community-Acquired Infections/microbiology* ; Humans ; Lung ; Microbiota/genetics* ; Pneumonia, Bacterial/diagnosis* ; Prognosis ; RNA, Ribosomal, 16S/genetics*