HIV-1 gp41 has been successfully anchored on the cell surface of yeast Saccharomyces cerevisiae by yeast cell-surface display systems using His-tag for the detection of protein expression. Gp41 activity has been detected by gp41 monoclonal antibody. The vector for gp41 yeast display has been constructed as follows: the gene-encoding gp41 was amplified by PCR using pMD18T-gp41 as a template, and then inserted into shuttle vector pICAS-His by restriction enzyme digestion. Next, the vectors were introduced into Saccharomyces cerevisiae MT8-1. After cultivation, recombinant cells were immunofluorescence labelled. The bright green cells were observed by the microscopy indicating the proteins have been displayed on the cell surface successfully, flow cytometry convinced that gp41 has been folded correctly on the cell surface. Then different concentrations of initial glucose were used to enhance the expression of protein. gp41 has been expressed by 82.46% yeast cells as the concentration of glucose was 1%. Protein expression was depressed when the concentration was increased.
Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; HIV Envelope Protein gp41 ; biosynthesis ; genetics ; Humans ; Protein Folding ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; cytology ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Surface Properties

Objective To evaluate the inter-fraction setup error during the treatment with megavoltage computed tomography (MVCT) and provide theoretical basis for clinical target volume-planning target volume (CTV-PTV) margins for nasopharyngeal carcinoma (NPC) patients treated with tomotherapy.Methods Thirty-seven consecutive NPC patients treated with tomotherapy were prospectively enrolled for the study between February 2015 and September 2015.For each patient,one MVCT scan was obtained after conventional positioning,online correction and tomotherapy delivery daily,and the scan was registered to the planning CT to determine inter-fraction setup error.The expanding margin for PTV (MPTV) was calculated with the recipe:MPTV =2.5∑ + 0.76 (∑:systematic error;6:random error).Results The average absolute errors of the inter-fraction were (2.102 ± 0.040 6) mm,(1.490 ± 0.034 8) mm,(1.306 ± 0.335) mm and (1.392 ± 0.038 4) ° in the three dimensions.Gradual increases in both inter-fraction three-dimensional displacement were observed with time and treatment (P ＜ 0.05).The total MPTV ac counting for inter-error were 3.467 5 mm,2.979 5 mm and 2.888 5 mm.Conclusions Tomotherapy irradiation technology personalized MPTV should be adopted for the design of tomotherapy plan.Displacement increased as a function of time.

Objective:To evaluate the dose-response relationship of alfentanil inhibiting gag reflex when combined with propofol in elderly patients undergoing painless gastroscopy.Methods:Patients of American Society of Anesthesiologists physical statusⅠor Ⅱ, aged ≥60 yr, scheduled for elective painless gastroscopy, were selected. Propofol 1.5 mg/kg combined with alfentanil was given intravenously in all the patients. The dose of alfentanil was determined by the Dixon up-and-down method. The initial dose of alfentanil was set at 5 μg/kg. The dose of alfentanil in the next patient was determined according to the development of gag reflex, and the ratio between the two successive doses was 1.1. The median effective dose (ED 50) and 95% confidence interval of alfentanil-induced inhibition of gag reflex when combined with propofol in elderly patients undergoing painless gastroscopy were calculated using the by up-and-down sequential allocation. Results:The ED 50 (95% confidence interval) of alfentanil-induced inhibition of gag reflex when combined with propofol 1.5 mg/kg was 2.8 (2.4-3.2) μg/kg in elderly patients undergoing painless gastroscopy. Conclusion:When combined with propofol 1.5 mg/kg, the ED 50 of alfentanil inhibiting gag reflex is 2.8 μg/kg in elderly patients undergoing painless gastroscopy.

Objective:To assess the prevalence of metabolic syndrome(MS) and its relationship with hyperuricemia(HUA) in perimenopausal women in Anning city, Yunnan province.Methods:This is a cross-sectional survey. In May 2021, a multi-stage stratified sampling method was used to collect demographics and clinical data [ethnicity, living community, height, weight, waist circumference, blood pressure, fasting plasma glucose, triglycerides(TG), serum uric acid, high density lipoprotein-cholesterol(HDL-C), alanine transaminase(ALT), etc] in a total of 6 721 perimenopausal women aged 45-60 years.Results:A total of 6 721 perimenopausal women were included in this study. The prevalences of MS and HUA were 14.05%(95% CI 13.22%-14.88%) and 6.46%(95% CI 5.88%-7.07%), respectively. The average age, HDL-C, urea, direct bilirubin, and albumin levels in the perimenstrual HUA population were lower than those in the non-HUA population while the levels of TG, ALT, heart rate, body mass index(BMI), and creatinine were higher(all P<0.05). The prevalence of HUA in perimenopausal women with ethnic minorities and family history of chronic diseases was higher than that in Han nationality and without family history of chronic diseases. The prevalence of MS in perimenopausal women was increased with the increase of serum uric acid( Z=-15.313 8, P<0.001). Multivariate logistic regression model showed that HUA was positively correlated with MS( OR=1.526, 95% CI 1.192-1.954) after adjusting for covariates such as BMI and ethnicity, and the incidence of MS in perimenopausal women in HUA group was 1.526 folds higher than that in non-hyperuricemia group. Conclusion:HUA is highly positively correlated with MS in perimenopausal women. The management of uric acid level in perimenopausal women should be strengthened.

As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.
CRISPR-Cas Systems/genetics* ; Corynebacterium glutamicum/metabolism* ; Gene Editing ; Plasmids ; RNA, Guide/metabolism*

Objective: To evaluate the value of two oral mucosal contouring methods for predicting acute radiation-induced oral mucositis (A-ROM) in nasopharyngeal carcinoma (NPC) patients. Methods: A total of 150 AJCC 7^th stage Ⅱ-IVB NPCs receiving radical tomotherapy (TOMO) in Zhejiang Cancer Hospital from 2017 to 2019 were included in this prospective observational study. Oral cavity contour (OCC) and mucosal surface contour (MSC) were applied to delineate the oral mucosal structure. A-ROM grade was prospectively assessed and recorded weekly according to RTOG scoring criteria. The prediction value of two methods for A-ROM was statistically compared. Results: The incidence rate of ≥3 grade A-ROM was 33.3%. In univariate analysis, V₅, V₁₀, V₁₅, V₄₅, V₅₀, V₅₅, V₆₀, V₆₅ and V₇₀ of OCC and V₅, V₁₀, V₅₀, V₅₅, V₆₀, V₆₅, V₇₀ and D_mean of MSC were significantly correlated with the risk of ≥3 grade A-ROM (all P<0.05). In binary logistic regression analysis, gender and smoking were significantly associated with the incidence of ≥3 grade A-ROM by using OCC (male vs. female: OR=0.141, 95%CI=0.037-0.538, P=0.004; smoking vs.non-smoking: OR=5.109, 95%CI=1.413-18.470, P=0.013). For MSC, gender, smoking, N stage and MSC- V₅₅ were the independent predictors (male vs. female: OR=0.129, 95%CI=0.032-0.519, P=0.004; smoking vs.non-smoking: OR=4.448, 95%CI=1.224-16.164, P=0.023; N stage: OR=2.291, 95%CI=1.268-4.137, P=0.006; MSC-V₅₅: OR=1.432, 95%CI=1.008-2.033, P=0.045). The cutoff value of MSC-V₅₅ was 7.70%, the area under ROC curve was 0.754, the sensitivity and specificity were 0.680 and 0.740, retrospectively (all P<0.001). Conclusions Compared with OCC, MSC yields a higher prediction accuracy for the severity of A-ROM in nasopharyngeal carcinoma patients receiving TOMO treatment.

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size ( < 0.01) and a shorter lifespan ( < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis ( < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment ( < 0.05). CONCLUSIONS Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.
Animals ; Antineoplastic Agents ; Antioxidants ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; Cyclic N-Oxides ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Mice ; Spin Labels