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 assess the effect of viral macrophage inflammatory protein (vMIP) -Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) , and to explore the mechanisms. Methods: A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells, and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-Ⅱ gene on the APOBEC3G expression in 293T cells. Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN) -α for 36 hours, and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment. Some 293T cells transfected with vMIP-Ⅱ plasmids were treated with 75 μmol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μmol/L U0126 (an ERK signaling pathway inhibitor) separately; after 24 hours, total protein was extracted from 293T cells, and Western blot analysis was conducted to determine the expression of APOBEC3G. A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene, and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G, sequences with the lengths of 1 560, 960, 720, 480, 420, 360, 330 and 240 bp, and the regulatory element-free region (NEG) of APOBEC3G, separately. Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group), or the recombinant plasmid and empty plasmid (control group). Subsequently, the activity of the APOBEC3G promoter was evaluated, and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP-Ⅱ was analyzed. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference (LSD) -t test. Results: The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013, 1.472 ± 0.013 respectively) than in the control group (1, 0.364 ± 0.030 respectively; t = 6.22, 6.54 respectively, both P < 0.05) . The APOBEC3G expression significantly differed among the empty plasmid group, vMIP-Ⅱ plasmid group, empty plasmid + IFN-α group and vMIP-Ⅱ plasmid + IFN-α group (1, 2.030 ± 0.108, 2.700 ± 0.081 and 2.600 ± 0.099 respectively; F = 67.026, P < 0.001) , but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t = 3.46, P > 0.05) . The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group, vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group (0.617 ± 0.025, 0.179 ± 0.061, 0.359 ± 0.012 respectively; F = 70.019, P < 0.001) , and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t = 9.66, 11.836 respectively, both P < 0.01) . Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS, 1 560-, 960-, 720-, 480-, 420-, 360-, 330-, 240-bp or NEG sequences (F = 81.092, P < 0.001) , and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group. Conclusion vMIP-Ⅱ upregulates the expression of APOBEC3G, likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.

Objective To observe the clinical characteristics of bacterial pneumonia children,and the changes of the serum levels of white blood cells(WBC),C-reactive protein(CRP) and procalcitonin(PCT) before and after anti-bacterial therapy,and to explore the predictive value in early diagnosis and therapy.Methods 130 bacterial pneumonia children were enrolled prospectively as pneumonia group.The clinical data were collected and the serum CRP,PCT and WBC were detected before anti-bacterial therapy (within 24h after admission) and after anti-bacterial therapy (the seventh day after admission).34 healthy children were enrolled as control group.The general clinical characteristics of the children in the pneumonia group were observed.The levels of serum CRP,PCT and WBC between the pneumonia group and the control group were compared.The levels of serum CRP,PCT and WBC before and after anti-bacterial therapy in the bacterial pneumonia children were compared.The clinical value of PCT,CRP and WBC in early predicting bacterial pneumonia was identified.Results Compared with the control group,the sex and age of the bacterial pneumonia children demonstrated no statistically significant differences (t =1.012,P =0.395;x2 =0.003,P =0.959).The mean course of the disease before admission was (5.34 ± 1.27) d,with mean temperature of (38.27 ± 0.96) ℃,and hospital days of (8.92 ± 3.93) d.35 cases were cured,and 95 cases were improved,with no death.The serum levels of CRP,PCT and WBC in pneumonia children on admission were (12.24 ±6.35) mg/L,(0.18 ± 0.15) ng/mL and (14.25 ± 7.59) 109/L,respectively,which were higher than those of the control group,the differences were statistically significant (t =4.650,5.867,2.548,all P ＜ 0.05).The serum levels of CRP,PCT and WBC in pneumonia children before anti-bacterial therapy were higher than after bacterial therapy,showed statistically significant differences(t =8.165,7.232,5.112,all P ＜ 0.05).The area under the curve (AUC)of the PCT,CRP and WBC in early predicting bacterial pneumonia were 0.928,0.834 and 0.718 respectively by the relative operating characteristic (ROC) curve analysis (P ＜ 0.05).The sensitivity and specificity of PCT in predicting bacterial pneumonia were higher than CRP and WBC.Conclusion The statistics efficacy of PCT in early predicting children bacterial infection was obviously higher than CRP and PCT.The combined detection of CRP,PCT and WBC was benefit to improve the diagnostic sensitivity and specificity of children with bacterial pneumonia.