Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.
Biotransformation ; Colletotrichum ; metabolism ; Dehydroepiandrosterone ; chemistry ; Fermentation ; Hydroxylation ; Industrial Microbiology ; Mutation

Objective To investigate the effects of epidermal growth factor receptor(EGFR)interference on activation of astrocytes after intra-cerebral hemorrhage and its mechanism.Methods Rat model of intra-cerebral hemorrhage was induced by injection of Ⅶ collagenase.Sham operation group was set as control.After 7 d,the neurological function score was used to judge whether the model was successful or not.The expression of EGFR and GFAP in brain tissue of rats in intra-cerebral hemorrhage group and sham operation group were detected by SP immunohistochemical method.Astrocytes were isolated from newborn rats' cerebral cortex and cultured in vivo.Effects of EGFR interference on activation of astrocytes were observed.The experimental rats were divided into 4 groups: ① control group was not handled.② CNTF group was added with 20 μg/L CNTF.③ Negative group was added with 20 μg/L CNTF,and EGFR siRNA negative control was transfected.④ EGFR group was added with 20 μg/L CNTF,and EGFR siRNA was transfected.The expressions of EGFR,GFAP,p-JAK1,p-STAT3 were detected by real-time PCR and Western blot.Results The modeling success rate was 89.5％(17/19).The expressions of EGFR and GFAP in brain tissue of rats with intra-cerebral hemorrhage were significantly higher than those in sham operation group(P<0.05).The relative expressions of EGFR mRNA and protein in EGFR group were significantly lower than those in control group,CNTF group and negative group(P<0.05).The relative expressions of GFAP mRNA and protein in the CNTF group and the negative group were significantly higher than those in the controlgroup and the EGFR group(P<0.05),but there was no significant difference between the control group and the EGFR group(P>0.05).The relative expressions of p-JAK1 and p-STAT3 protein in the CNTF group and the negative group were significantly higher than those in the control group and the EGFR group(P<0.05),but there was no significant difference between the control group and the EGFR group(P>0.05).Conclusion The expressions of EGFR and GFAP significantly increase in the rats with intra-cerebral hemorrhage.Interference of EGFR expression can inhibit the activation of astrocytes in rats,which may be related with the inhibition of JAK1 and STAT3 phosphorylation.

Objective To investigate the effects of epidermal growth factor receptor(EGFR)interference on activation of astrocytes after intra-cerebral hemorrhage and its mechanism.Methods Rat model of intra-cerebral hemorrhage was induced by injection of Ⅶ collagenase.Sham operation group was set as control.After 7 d,the neurological function score was used to judge whether the model was successful or not.The expression of EGFR and GFAP in brain tissue of rats in intra-cerebral hemorrhage group and sham operation group were detected by SP immunohistochemical method.Astrocytes were isolated from newborn rats' cerebral cortex and cultured in vivo.Effects of EGFR interference on activation of astrocytes were observed.The experimental rats were divided into 4 groups: ① control group was not handled.② CNTF group was added with 20 μg/L CNTF.③ Negative group was added with 20 μg/L CNTF,and EGFR siRNA negative control was transfected.④ EGFR group was added with 20 μg/L CNTF,and EGFR siRNA was transfected.The expressions of EGFR,GFAP,p-JAK1,p-STAT3 were detected by real-time PCR and Western blot.Results The modeling success rate was 89.5％(17/19).The expressions of EGFR and GFAP in brain tissue of rats with intra-cerebral hemorrhage were significantly higher than those in sham operation group(P<0.05).The relative expressions of EGFR mRNA and protein in EGFR group were significantly lower than those in control group,CNTF group and negative group(P<0.05).The relative expressions of GFAP mRNA and protein in the CNTF group and the negative group were significantly higher than those in the controlgroup and the EGFR group(P<0.05),but there was no significant difference between the control group and the EGFR group(P>0.05).The relative expressions of p-JAK1 and p-STAT3 protein in the CNTF group and the negative group were significantly higher than those in the control group and the EGFR group(P<0.05),but there was no significant difference between the control group and the EGFR group(P>0.05).Conclusion The expressions of EGFR and GFAP significantly increase in the rats with intra-cerebral hemorrhage.Interference of EGFR expression can inhibit the activation of astrocytes in rats,which may be related with the inhibition of JAK1 and STAT3 phosphorylation.

Objective: To understand delay in treatment student tuberculosis (TB) patients in Wuhan from 2011 to 2018, and to provide a reference for prevention and control measures of tuberculosis in schools. Methods: The medical cases of students with tuberculosis in Wuhan recorded by the national tuberculosis management information system during 2011-2018 were analyzed for the delay and change trend of medical treatment of students with tuberculosis, and Logistic regression was used to analyze influencing factors. Results: The median number of student TB patients seeking treatment in Wuhan was 9(3,21) days, and 51.14% of student TB patients were delayed. From 2011 to 2018, the overall delay rate of TB patients in students decreased slowly and then gradually increased, decrea sing from the highest rate (56.4%) in 2011 to the lowest one (45.6%) in 2014, and then gradually increased to 53.8% in 2018, there was no significant difference in the delay rate between the first and the second four years(χ2=2.84, P=0.09); The delay rate of different gender students was the same as that of the whole. The fluctuation of female students was slightly obvious, but the difference was not statistically significant(χ2=0.07, P>0.05); Among the students of different age groups, the delay rate of 13-18 years old was the lowest(χ2=87.23, P<0.01). The delay rate of 19-22 years old and university groups of different school levels showed a slow decline, while that of other age groups and other school level groups showed an overall upward trend. The results of multivariate logistic regression analysis showed that the main factors affecting the delay rate of medical treatment were the remote urban area(OR=1.84, 95%CI=1.56-2.16), the ethnic minorities (OR=2.73, 95%CI=1.46-5.09) and the age of 13-18(OR=0.57, 95%CI=0.33-1.00). Conclusion Delay in treatment of TB patients in Wuhan is not optimistic, with more than half of the students have delayed in treatment, the main factors that affect the high rate of TB patients’ treatment delay are the students in the far urban areas and ethnic minorities. It is very important to take targeted prevention and control measures to guide the students to actively seek medical treatment according to the time, the place and the person.

Objective: To determine the knowledge, attitudes and practices (KAP) regarding tuberculosis prevention and control among college students in Wuhan, China, while analyzing the factors affecting tuberculosis prevention behavior. Methods: Referring to the National KAP Questionnaire for Tuberculosis Prevention and Control among College Students, which was designed by China Center for Disease Control and Prevention, 22 colleges and universities in Wuhan were selected using a judgment sampling method, and a questionnaire survey was carried out through the online questionnaire platform. The formation of tuberculosis prevention and control behavior among this population was analyzed by single factor and multi factor Logistic regression. Results: The total awareness rate of KAP regarding tuberculosis prevention and control among college students in Wuhan was 79.69%; the awareness rate of core knowledge was 79.28%, and the passing rate was 92.46%. The correct attitude holding rate was 89.69%, while the passing rate was 90.56%. In respect to correct behavior, the formation rate was 72.11%, with a passing rate of 96.62%. The multivariate Logistic regression analysis showed that the following factors were more likely to promote tuberculosis prevention and control behaviors that met the target: female college students ( OR =1.86); college students whose father had a primary school education level, junior high school and equivalent, high school and equivalent, and junior high school students ( OR =2.94, 3.05, 3.17, 3.24 ); no history of tuberculosis ( OR =3.32); the passing knowledge of core knowledge ( OR =9.91), and the passing attitude holding score ( OR =7.35)( P <0.05). Conclusion The overall awareness rate of KAP regarding tuberculosis prevention and control among college students in Wuhan is acceptable,the mainly influenced factors are gender, history of tuberculosis, awareness rate of core knowledge about prevention and control, attitudes and cognition, and parents educational background.

Objective: To analyze the epidemiological characteristics and assoicated factors of tuberculosis (TB) aggregation in schools in Wuhan from 2017 to 2020, and to provide scientific basis for school based TB prevention and control strategies. Methods: Questionnaire star was used to collect data on tuberculosis prevention and control in various schools in Wuhan. Multivariate logistic regression analysis was used to analyze the influencing factors of tuberculosis aggregation in schools. Results: A total of 37 outbreaks of tuberculosis aggregation in schools were reported in Wuhan from 2017 to 2020, which involved 28 different schools, including 24 colleges and universities and 4 senior high schools, 176 cases were reported, among which 39 were positive for pathogens and 17 cases of rifampicin resistant, and the median duration of single cluster epidemic was 48 (28, 368) days, universities were more prone to cluster outbreaks than middle schools ( χ 2=75.27, P <0.01), the incidence in male was higher than that of in female in cluster outbreak ( χ 2=22.82, P =0.00). The results of multivariate Logistic regression analysis showed that boarding school ( OR =5.12), TB screening at school entry ( OR =3.27), etiology tracking and registrationin school ( OR =7.28), treatment and isolation of sick students on time ( OR =9.12), whether the dormitories and classrooms were often ventilated ( OR =4.97), and whether the management of school suspension and return was strictly implemented ( OR =4.68) were associated with the occurrence of TB cluster outbreak( P <0.05). Conclusion Schools should actively strive for policies and funding, strictly implement TB screening and physical examination for freshmen, as well as the management of contact tracing and registeration, targeted TB health education, guidance for teachers and students for cleaning, disinfection and hand hygiene, timely treatment and reporting of suspected symptoms, to prevent the occurrence of TB clustering outbreaks.

Objective: To analyze the epidemiological characteristics of pulmonary tuberculosis among students in Wuhan from 2011 to 2020, and to provide a basis for the scientific development of interventions and strategies. Methods: Epidemiological distribution, time of onset and treatment, as well as treatment outcomes of student tuberculosis in Wuhan from 2011 to 2020 registered in the national tuberculosis information management system were described. Results: During 2011-2020, 4 337 student tuberculosis patients in Wuhan were registered. The average annual incidence rate was 22.44/10 million, and the annual decreasing rate of 7.86% The incidence of male and female patients was 1.76:1, and the incidence rate of male was higher than that of female( χ 2=184.18, P <0.01). Most of patients aged 19-22 years old, accounting for 47.89%; Tuberculosis reports were highest during March to May, and September to November, and lowest during January to February, and July to August. Student patients were mainly concentrated in Hongshan District, Jiangxia District and Wuchang District, where schools were more distributed in Hongshan District, Dongxihu District, Wuchang District and Xinzhou District. The median duration from tuberculosis onset to treatment was 9(3, 21) days, which varied significantly by region, age, nationality, and patient residence ( Z =-9.25, 47.14, 9.88,43.96, P <0.01). The treatment and outcome of student tuberculosis patients were varied significantly by year and nationality( P <0.05). Conclusion The incidence of student tuberculosis in Wuhan City showed a slow downward trend. Most of student tuberculosis are college and high school students. Time and place of case detection are relatively fixed. The time of treatment and the outcome of treatment vary significantly. Tuberculosis prevention and control strategies should be formulated according to the local conditions according to the tuberculosis distribution characteristics, as well as enhancing surveillance, health promotion, active discovery and supervision management of tuberculosis in school settings.

Objective To investigate the value of derived neutrophil to lymphocyte ratio(dNLR)as a pre-dictive biomarker for immunotherapy response in advanced non-small cell lung cancer(NSCLC).Methods A total of 92 patients with advanced NSCLC who received anti-programmed cell death receptor(PD-1)combined therapy in the hospital from August 2018 to December 2019 were selected as the research objects.Peripheral blood samples were collected within 24 h before immunotherapy,complete blood cell count was measured,and dNLR was calculated.Patients with advanced NSCLC were treated with PD-1 inhibitors or combination regi-mens,and the response to immunotherapy was evaluated by objective response rate(ORR)and disease control rate(DCR).The receiver operating characteristic(ROC)curve was used to analyze the predictive value of dN-LR for the diagnosis and response to immunotherapy in advanced NSCLC.Multivariate Logistic regression model was used to analyze the relationship between dNLR and immunotherapy response in advanced NSCLC.Kaplan-Meier survival curve and Log-Rank test were used to analyze the overall survival(OS),progression-free survival(PFS)and disease-specific survival(DSS)of the low dNLR group and the high dNLR group.Re-sults The ORR and DCR of advanced NSCLC patients after immunotherapy were 32.61％and 65.22％,re-spectively,and the PFS and OS were 17.0(8.5,25.5)and 24.0(12.7,36.1)months,respectively.The dNLR of DCR group was lower than that of non-DCR group(P＜0.001).The dNLR of ORR group was lower than that of non-ORR group(P＜0.001).The area under the curve of dNLR for predicting DCR or ORR was 0.897(95％CI 0.829-0.965)and 0.874(95％CI 0.795-0.953),respectively.Multivariate Logistic regres-sion analysis showed that dNLR≥2.28 increased the risk of non-response to immunotherapy,and this inde-pendent relationship still existed after further adjustment for objective confounding factors(P＜0.05).Sur-vival curve results showed that patients with high dNLR had significantly shorter PFS,OS,and DSS(P＜0.05).Multivariate Cox regression analysis showed that high dNLR was an independent factor affecting the poor prognosis of patients with advanced NSCLC(P＜0.05).Conclusion High dNLR advanced NSCLC pa-tients are more difficult to benefit from immune therapy,and prognosis is worse.dNLR is promising as a pre-dictive biomarker for immunotherapy response in advanced NSCLC.

Objective To predict the micro-level energy release rate in the rat femoral cortical bone and investigate the variation in the micro-level energy release rate with age.Methods Based on previous experimental data and numerical simulation of fracture modes for cortical bone,load-displacement curves and fracture modes measured by simulation and experiment were compared,and the micro-level energy release rates of rat femoral cortical bone at different months were predicted by back-calculation.Results It was predicted that the micro-level energy release rate of rat femoral cortical bone at 1-,3-,5-,7-,9-,11-,and 15-month age was 0.08-0.12,0.12-0.14,0.15-0.19,0.25-0.28,0.23-0.25,0.19-0.22,and 0.13-0.16 N/mm,respectively.Conclusions The decrease in the microlevel energy release rate with increasing age led to a decreasing failure load,indicating that the microlevel energy release rate is one of the main factors determining fracture occurrence;however,no significant decrease was observed at the time of fracture,indicating that the microlevel energy release rate was not linearly proportional to the fracture time.These results can help explain the mechanism of cortical bone fractures at the clinical level.

Objective To determine the genotype and clustering characteristics of multidrug-resistant (MDR) mycobacterium tuberculosis in Wuhan City. Methods A total of 149 mycobacterium tuberculosis strains from MDR tuberculosis (MDR-TB) patients were genotyped by DTM-PCR, mycobacterial interspersed repetitive unit–variable number tandem repeat typing (MIRU-VNTR) and sequencing of drug-resistant associated genes. The Hunter-Gaston discriminatory index (HGDI), clustering rate and recent minimum estimated infection rate were calculated. Results The cumulative HGDI of all MIRU loci was 0.9944. Gene locus with the highest HGDI（0.6467）was QUB11b. Combing MIRU-VNTR and DTM-PCR, 111 unique genotypes and 11 clustered genotypes（38 strains）from 149 isolates were identified. The clustering rate was 25.50%, and the recent minimum infection rate was estimated to be 18.12%. After further genotyping by sequencing of drug-resistant associated genes, the results showed that 17 (44.74%) strains in each cluster shared the same mutation sites, while the other strains had different mutation sites. Conclusion The combination of DTM-PCR, 24 locus MIRU-VNTR typing and drug resistance gene sequencing demonstrates a relatively high discriminatory power, which is suitable for the genotyping of multi drug resistant mycobacterium tuberculosis in Wuhan. The epidemic of MDR-TB in Wuhan is mainly caused by reactivation, and the recent transmission rate is not high.