Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering / 生物工程学报

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

Optimization of retinin expression and the application with wax emulsion in nanocoatings / 生物工程学报

Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30 ℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.

Application of CBL and PBL based on SMART principle in nursing practice teaching in radiotherapy / 中华医学教育探索杂志

Objective:To apply CBL combined with PBL based on SMART (specific, measurable, attainable, relevant and time-based) principle in nursing practice teaching in radiotherapy.Methods:A total of 100 nurses who performed nursing practice in the Department of Radiotherapy in Guangdong Provincial Hospital of Traditional Chinese Medicine from May 2016 to May 2020 were selected as the research objects. They were divided into a control group and a study group according to their admissions, with 50 people in each group. The study group used CBL combined with PBL teaching based on SMART principle, and the control group used traditional practice teaching. After the clinical practice, the two groups were assessed on theoretical and clinical practice skills, and the two groups' teaching satisfaction and teaching effects were evaluated through seminars and questionnaire surveys. SPSS 22.0 was used for t test and chi-square test. Results:The theoretical and clinical practice performance assessment scores of the practical nurses in the study group were higher than those in the control group, with statistically significant differences ( P<0.001). The teaching satisfaction rate of the practice nurses in the control group was 62.00% (31/50), and that of the practice nurses in the study group was 96.00% (48/50), with significant differences ( P<0.001). In terms of improving independent learning ability, information acquisition and problem analysis ability, improving clinical thinking ability, mobilizing learning enthusiasm, enhancing teamwork ability, nurse-patient communication ability, and recognition of innovation ability, the teaching satisfaction of the research group was higher than that of the control group. Conclusion:The application of SMART principle in teaching has the advantages of clear goals and quantifiable evaluation. The combination of CBL and PBL based on SMART principle can help to improve the mastery of theoretical and practical skills of radiotherapy practice nurses, and achieve satisfactory teaching results.

Quantification of ambrisentan in human plasma by LC-MS/MS and its pharmacokinetic application / 中国临床药理学与治疗学

AIM: To establish an LC-MS/MS method to determine the concentration of ambrisentan in human plasma and apply it to the study of human pharmacokinetics. METHODS: After extracting ambrisentan and internal standard from human plasma by liquid-liquid extraction, chromatographic separation was performed on a Waters Symmetry C

Metabolic engineering of Escherichia coli for production of hydroxytyrosol / 生物工程学报

Hydroxytyrosol is an important fine chemical and is widely used in food and medicine as a natural antioxidant. Production of hydroxytyrosol through synthetic biology is of important significance. Here we cloned and functionally characterized a hydroxylase encoding gene HpaBC from Escherichia coli BL21, and both subunits of this enzyme can be successfully expressed to convert the tyrosol into hydroxytyrosol. A HpaBC gene integration expression cassette under the tac promoter was constructed, and integrated into the genome of a tyrosol hyper-producing E. coli YMG5A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1H1. Shake flask fermentation showed that strain YMGRD1H1 can directly use glucose to produce hydroxytyrosol, reaching a titer of 1.81 g/L, and nearly no by-products were detected. A titer of 2.95 g/L was achieved in a fed-batch fermentation conducted in a 5 L fermenter, which is the highest titer for the de novo synthesis of hydroxytyrosol from glucose reported to date. Production of hydroxytyrosol by engineered E. coli lays a foundation for further construction of hydroxytyrosol cell factories with industrial application potential, adding another example for microbial manufacturing of aromatic compounds.

Characterization of a sucrose phosphorylase from Leuconostoc mesenterides for the synthesis of α-arbutin / 生物工程学报

Sucrose phosphorylase (SPase) gene from Leuconostoc mesenteroides ATCC 12291 was synthesised after codon optimization, and inserted into pET-28a plasmid to generate pET-28a-spase. The recombinant strain Escherichia coli BL21 (DE3)/pET-28a-spase was induced for Spase expression. The recombinant protein Spase was purified and characterized. The specific enzyme activity of SPase was 213.98 U/mg, the purification ratio was 1.47-fold, and the enzyme activity recovery rate was 87.80%. The optimal temperature and the optimal pH of the SPase were identified to be 45 °C and 6.5 respectively, and Km, Vmax and kcat of the SPase for sucrose was 128.8 mmol/L, 2.167 μmol/(mL·min), and 39 237.86 min-1. The recombinant SPase was used for α-arbutin production from hydroquinone and the reaction process was evaluated. The optimal conditions for synthesis of α-arbutin by SPase were 40 g/L hydroquinone, 5:1 molar ratio of sucrose and hydroquinone, and 250 U/mL recombinant SPase at pH 7.0 and 30 °C for 24 h in the dark, and then 500 U/mL glucoamylase was added at 40°C for 2.5 h. Under the optimized process, the yield of α-arbutin reached 98 g/L, and the hydroquinone conversion rate was close to 99%. In summary, the recombinant SPase was cloned and characterized, and its application for α-arbutin production was feasible.

Activation of low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 by heterologous activators / 生物工程学报

As self-subunit swapping chaperones or metallochaperones, the activators assist nitrile hydratases to take up metal ions and they are essential for active expression of nitrile hydratases. Compared with nitrile hydratases, the activators have a low sequence identity. Study of the activation characteristics and the relationships between structures and functions of the activators is of great significance for understanding the maturation mechanism of nitrile hydratase. We co-expressed low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 with four heterologous activators respectively and determined their activation abilities. Then we made sequence analysis and structure modelling, and studied the functions of the important domains of the activators. Results showed that all four heterologous activators could activate L-NHase, however, the specific activities of L-NHases were different after activation. L-NHase showed the highest specific activity after being activated by activator A, which was 97.79% of that of the original enzyme, but the specific activity of L-NHase after being activated by activator G was only 23.94% of that of the original enzyme. Activator E and activator G had conserved domains (TIGR03889), and deletion of their partial sequences resulted in a substantial loss of activation abilities for both activators. Replacing the N-terminal sequence of activator G with the N-terminal sequence of activator E, and adding the C-terminal sequence of activator E to the C-terminus of activator G could increase the specific activity of L-NHase by 178.40%. The activation by nitrile hydratase activators was universal and specific, and the conserved domains of activators were critical for activation, while the N-terminal domain and C-terminal domain also had important effects on activation.

Effect of human pancreatic cancer cell supernatant on expression of TIM-3 and function of dendritic cells / 中国病理生理杂志

AIM:To investigate the influence and mechanisms of human pancreatic cancer tumor microenvironments on T-cell immunoglobulin mucin-3 (TIM-3) expression and function of dendritic cells (DCs).METHODS:Tumor-infiltrating dendritic cells (TIDC) and para-carcinoma tissue DCs were isolated by Ficoll-Hypaque density centrifugation from trypsinized pancreatic carcinoma tissues, and the peripheral blood mononuclear cells were isolated from pancre-atic cancer patients or healthy people.The expression of TIM-3 on DCs was detected by flow cytometry.DCs isolated from healthy people peripheral blood mononuclear cells were induced by rhGM-CSF and IL-4.The expression of TIM-3 in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells or human skin fibroblast (Hs27) cells for 48 h, and in the DCs treated with supernatants of Capan-2 cells, anti-VEGF-R2, anti-IL-10 and EP2 re-ceptor blocking peptide were evaluated by flow cytometry.The releases of IFN-βand IL-12 in the culture supernatants of DCs pretreated with monoclonal antibody ( mAb) to TIM-3 or DNase+RNase, followed by stimulation with apoptotic Ca-pan-2 cells, were detected by ELISA.RESULTS:DCs in tumor microenvironments had higher expression of TIM-3 than the DCs from para-carcinoma tissues and pancreatic cancer patient or healthy people peripheral blood ( P<0.01 ) .TIM-3 expression in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells for 48 h was much higher than that in Hs27 cells (P<0.05).Treatment with a combination of anti-VEGF-R2, anti-IL-10 and EP2 receptor blocking peptide largely diminished the upregulation of TIM-3 on the DCs mediated by Capan-2 cell superna-tants (P<0.05).The concentrations of IFN-βand IL-12 in the DCs with high expression level of TIM-3 were lower than those in the DCs with low TIM-3 expression level.Treatment with mAb to TIM-3 resulted in much more IFN-βand IL-12 releases (P<0.01), but DNase+RNase made this effect disappear.CONCLUSION:TIM-3 serves as a negative regula-tor of DCs innate immune responses in the pancreatic cancer microenvironments.The secretion of soluble factors to tumor microenvironment by pancreatic cancer cells, including IL-10, VEGF and PGE2 , may contribute to the regulation of TIM-3 expression.

Vailidation of a LC-MS/MS method for quantification of ibuprofen enantiomers in Beagle dog plasma / 中国药理学通报

Aim To develop a simple,rapid and ac-curate analysis method for determination of chiral ibu-profen in Beagle dog plasma.Method The plasma sample was submitted to liquid - liquid extraction u-sing hexane /isopropanol (95 ∶5,V/V),with ketopro-fen as the internal standard (IS).The separation was accomplished in a Lux 5u Cellulose-3 (250 mm·4.6 mm,5 μm)column,and the mobile phase consisted of methanol and a mixture of 1 mmol·L -1 ammonium acetate-methanol-formic acid (90 ∶1 0 ∶0.2,V/V/V) with the volume ratio of 82 ∶1 8 at a flow rate of 0.8 mL· min -1 .The mass spectrometer consisted of an ESI interface operating at negative ionization mode and the detection was performed using multiple reaction monitoring at the transitions of m /z 205.2 /1 61 .2 for ibuprofen and m /z 253.1 /209.2 for ketoprofen (IS). Method validation included the evaluation of the matrix effect, extraction recovery, linearity, lower LOQ, within-run and between-run precision,stability and di-lution effect.Results The calibration curve was line-ar across the concentration range of 0.2 ~50 mg·L -1 for each ibuprofen enantiomer with a lower LOQ of 0.2 mg·L -1 .The within-run and between-run precision (RSD%)was in the range 1 .01 % ~1 3.1 % for each ibuprofen.The pharmacokinetic parameters for orally single dose of (S +)and racemic ibuprofen in Beagle dogs were as follows: Cmax , T1 /2 , AUC(0-t) were (82.98 ±1 4.83 )mg·L -1 ,(3.21 7 ±0.7298)h, (362.0 ±58.67)h·mg·L -1 for (S +)ibuprofen and 70.62 /74.48 mg·L -1 ,1 .520 /5.432 h ,1 77.8 /649.6 h·mg·L -1 for (R -)/(S +)ibuprofen,re-spectively.Conclusions A simple,rapid,accurate, high sensitivity and repeatability method has been suc-cessfully developed,which can analyze the concentra-tions of (R -)/(S +)ibuprofen in Beagle dog plasma simultaneously.The method could be applied for the investigation of pharmacokinetics of ibuprofen enanti-omers in Beagle dogs.

The conflict and relation between informed consent and medical privacy regulations / 中国医学伦理学

The article conclude the core of the informed consent from the development of the right , the essential of the medical privacy regulations. It analyze the conflict between the informed consent and medical privacy regulations used in the practice of clinical, point out the coherence of the two and the applicable qualifications of each.