New opportunities and challenges for hybrid data and model driven bioprocess optimization and scale-up / 生物工程学报

Currently, biomanufacturing technology and industry are receiving worldwide attention. However, there are still great challenges on bioprocess optimization and scale-up, including: lacing the process detection methods, which makes it difficult to meet the requirement of monitoring of key indicators and parameters; poor understanding of cell metabolism, which arouses problems to rationally achieve process optimization and regulation; the reactor environment is very different across the scales, resulting in low efficiency of stepwise scale-up. Considering the above key issues that need to be resolved, here we summarize the key technological innovations of the whole chain of fermentation process, i.e., real-time detection-dynamic regulation-rational scale-up, through case analysis. In the future, bioprocess design will be guided by a full lifecycle in-silico model integrating cellular physiology (spatiotemporal multiscale metabolic models) and fluid dynamics (CFD models). This will promote computer-aided design and development, accelerate the realization of large-scale intelligent production and serve to open a new era of green biomanufacturing.

Research progress on the influence of sex differences on the action of psychotropic drugs / 中国临床药理学与治疗学

Epidemiological survey shows that the prevalence, incidence rate and symptoms of mental disorders are affected by sex. A large number of studies have shown that men and women respond differently to psychotropic drugs in clinical application. Moreover, some studies have shown that there are sex specific psychotropic drug use patterns in patients with depression. This paper summarizes the sex differences in pharmacokinetics of psychotropic drugs caused by physiological differences between men and women, and collates the current pharmacodynamic studies of mainstream clinical psychotropic drugs, with special attention to the effect of sex hormones on the therapeutic response of psychotropic drugs. This paper discusses the specific role and necessity of therapeutic drug monitoring in dealing with clinical sex differences in psychotropic drugs, hoping to provide reference for individual rational drug use.

Numerical simulation and optimization of impeller combination used in stirred bioreactor / 生物工程学报

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.

Progress in intelligent control of industrial bioprocess / 生物工程学报

Industrial bioprocess is a complex systematic process and bio-manufacturing can be realized on the basis of understanding the metabolism process of living cells. In this article, the multi-scale optimization principle and practice of industrial fermentation process are reviewed, including multi-scale optimizing theory and equipment, on-line sensing technology for cellular macroscopic metabolism, and correlated analysis of physiological parameters. Furthermore, intelligent control of industrial bioprocess is further addressed, in terms of new sensing technology for intracellular physiological metabolism, big database establishment and data depth calculation, intelligent decision.

Accurate Detemination of Isotopic Abundance of Intracellular Metabolites of Saccharopolysporaerythraea Based on Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry / 分析化学

A method for measuring 13C isotopic abundance of intracellular metabolites of Saccharopolysporaerythraea by ultra-high performance liquid chromatography (UPLC)-triple quadrupole mass spectrometry was established.First, the chromatographic conditions of UPLC were optimized, and then the MS conditions such as unique tube lens voltage, collision energy, and ion pair were optimized.On the bases of length of the parent and daughter ions carbon chains and whether the daughter ions contain 13C atoms, the one-to-one method, one-to-many method and SIM method were established for measuring 13C isotopic abundance.Then these methods were used to measure naturally labeled intracellular metabolite standards and 13C labeled samples, and according to the gap between the experimental value and the theoretical value, the best method was established for each metabolite of different characteristics.The results showed that one-to-one method was most effective for measuring the metabolites of daughter ions not containing 13C atoms represented by sugar phosphates, one-to-many method was the best for measuring the metabolites of both parent and daughter ions containing 13C short carbon chains represented by carboxylic acids, SIM method could play a role in measuring the metabolites of both parent and daughter ions containing 13C long carbon chains represented by coenzyme A.This method had a good measurement precision and could be applied to the measurement of Saccharopolysporaerythraea intracellular metabolites, which contributed to the consequent study of metabolic mechanism and the efficient expression of erythromycin.

The progress of radiotherapy in triple negative breast cancer / 实用肿瘤学杂志

The triple negative breast cancer is one of importance in clinical subtypes of breast cancer, which is easy to recur and metastasis, and its prognosis is very poor.Radiotherapy, as an effective method for breast cancer,can reduce the risk of local recurrence.This article elaborates its characteristics,the progress of ra-diotherapy in the breast conserving surgery and modified radical mastectomy in triple negative breast cancer,when is the appropriate time for radiotherapy and radiotherapy sensitization,and hope that it will be helpful to the treat-ments.

Progress in omics research of Aspergillus niger / 生物工程学报

Aspergillus niger, as an important industrial fermentation strain, is widely applied in the production of organic acids and industrial enzymes. With the development of diverse omics technologies, the data of genome, transcriptome, proteome and metabolome of A. niger are increasing continuously, which declared the coming era of big data for the research in fermentation process of A. niger. The data analysis from single omics and the comparison of multi-omics, to the integrations of multi-omics based on the genome-scale metabolic network model largely extends the intensive and systematic understanding of the efficient production mechanism of A. niger. It also provides possibilities for the reasonable global optimization of strain performance by genetic modification and process regulation. We reviewed and summarized progress in omics research of A. niger, and proposed the development direction of omics research on this cell factory.

Effect of glucagon-like peptide-1 receptor agonists on growth and energy metabolism in human medullary thyroid cancer cells / 中国癌症杂志

Background and purpose:This study aimed to investigate the effect of glucagon-like peptide-1 receptor agonists on proliferation, secretion of calcitonin and energy metabolism of medullary thyroid cancer (MTC) cell.Methods:The MTC cell line (TT) was culturedin vitro. After treatment with exenatide and liraglutide (0, 1, 10 and 100 nmol/L) for 24, 48 and 72 h, the proliferation of TT was analyzed by CCK-8 kit, the calcitonin was measured by calcitonin assay kits, and the energy metabolism of TT was measured by Seahorse XF instrument.Results:When compared with control group, neither exenatide nor liraglutide had effects on proliferation of TT (P>0.05); the calcitonin levels did not change signiifcantly after treatment with GLP-1 receptor agonists (P>0.05). Exenatide and liraglutide did not alter glycolysis and mitochondrial respiration in TT cells in a dose- and time-dependent manner.Conclusion:GLP-1 receptor agonists have no effect on the development of TT. Further collection of the safety data of exenatide and liraglutide on thyroid is still needed.

Development and application of morphological analysis method in Aspergillus niger fermentation / 生物工程学报

Filamentous fungi are widely used in industrial fermentation. Particular fungal morphology acts as a critical index for a successful fermentation. To break the bottleneck of morphological analysis, we have developed a reliable method for fungal morphological analysis. By this method, we can prepare hundreds of pellet samples simultaneously and obtain quantitative morphological information at large scale quickly. This method can largely increase the accuracy and reliability of morphological analysis result. Based on that, the studies of Aspergillus niger morphology under different oxygen supply conditions and shear rate conditions were carried out. As a result, the morphological responding patterns of A. niger morphology to these conditions were quantitatively demonstrated, which laid a solid foundation for the further scale-up.

Construction and application of black-box model for glucoamylase production by Aspergillus niger / 生物工程学报

Carbon-limited continuous culture was used to study the relationship between the growth of Aspergillus niger and the production of glucoamylase. The result showed that when the specific growth rate was lower than 0.068 h(-1), the production of glucoamylase was growth-associated, when the specific growth rate was higher than 0.068 h(-1), the production of glucoamylase was not growth-associated. Based on the result of continuous culture, the Monod dynamics model of glucose consumption of A. niger was constructed, Combining Herbert-Pirt equation of glucose and oxygen consumption with Luedeking-Piret equation of enzyme production, the black-box model of Aspergillus niger for enzyme production was established. The exponential fed-batch culture was designed to control the specific growth rate at 0.05 h(-1) by using this model and the highest yield for glucoamylase production by A. niger reached 0.127 g glucoamylase/g glucose. The black-box model constructed in this study successfully described the glucoamylase production by A. niger and the result of the model fitted the measured value well. The black-box model could guide the design and optimization of glucoamylase production by A. niger.

Optimization and application of chemically defined medium for 13C metabolic flux analysis of Streptomyces rimosus M4018 / 生物工程学报

The aim of this study is to develop a synthetic medium suitable for 13C metabolic flux analysis (13C-MFA) of Streptomyces rimosus. The cell growth rate and oxytetracycline production by S. rimosus M4018 were compared when M4018 cells were growth on the optimized chemically defined media with organic nitrogen sources or inorganic nitrogen sources. First, a synthetic medium contained KNO3 as the main nitrogen source was screened, then optimized by a response surface method. Using this new medium, the oxytetracycline yield was increased from 75.2 to 145.6 mg/L. Furthermore, based on the 13C-MFA, we identified that Entner-Doudoroff pathway does not exist in S. rimosus cells cultured in a chemically defined medium with feed of 100% 1-13C labeled glucose. This study is helpful for subsequent 13C-MFA application of S. rimosus.

Engineering MEP pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control / 生物工程学报

The pathway of 2-methyl-D-erythritol-4-phosphate (MEP) is the exclusive isoprenoid precursor biosynthetic pathway in Escherichia coli, with a higher theoretical yield than mevalonate (MVA) pathway. However, due to lack of information about the regulation of MEP pathway, only engineering MEP pathway in E. coli achieved limited improvement of heterologous isoprenoid production. We used exogenous MEP pathway genes to improve MEP pathway in E. coli and optimized the glucose feeding to release the potential of MEP pathway. The results demonstrate that co-expression of dxs2 from Streptomyces avermitilis and idi from Bacillus subtilis can increase amorphadiene production with 12.2-fold compared with the wild-type strain in shake flask fermentation. Then we established a high-cell density fermentation process for the engineered strain, and found that the phase from 24 to 72 h is important for product biosynthesis. The optimization of glucose feeding rate during 24 to 72 h significantly improved product accumulation, which was improved from 2.5 to 4.85 g/L, within the same process time. Considering the attenuation of strain metabolism after 72 h, this study further modulated the glucose feeding rate during exponential phase to control strain growth and the amorphadiene yield eventually reached to 6.1 g/L. These results provided useful information to develop engineered E. coli for isoprenoid production through MEP pathway engineering.

Accurate Determination of 13 C Isotopic Abundances of Free Intracellular Amino acids with Low Concentration by GC-MS-Selective Ion Monitoring Method / 分析化学

13 C isotopic abundance of intracellular free amino acid with a characteristic of fast- turnover can quickly reflect changes in intracellular metabolic state. But the concentration of intracellular free amino acid is low, the existed 13 C isotope detection method based on GC-MS can not satisfy the requirement with full scan mode. In this study, the selected ion monitoring method was used to detect accuracy higher likelihood of analysis of 13 C isotopic abundance of free intracellular amino acid. First, in the full scan mode we analyzed of the fracture law of different amino acids, found the feature corresponding to each amino acid fragments, and established 16 kinds of free intracellular amino acids characteristic fragment library. Then using this characteristic fragment library, only specific m/z signal was detected in sample analysis, which realized the selected ion monitoring and improved the quality of signal. The results of amino acid standards showed that the signal-to-noise ratio, measurement precision and accuracy were improved by 17, 2. 0 and 3. 8 times compared with the full scan mode. In the analysis of coenzyme Q10 producing strains of samples, this method was successfully used to detect isotopic abundance of 8 kinds of free intracellular amino acids. This method plays an important role in the detection of 13 C isotopic abundance of the intracellular free amino acid in cell metabolism research.

Influence of methanol concentration on purification recovery of consensus interferon-a produced by Pichia pastoris / 生物工程学报

We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-a (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in clFN expression compared to using 0.25% methanol. However, the purification recovery rate of cIFN at 0.25% methanol was 3.75-fold higher than that at 0.75% methanol. To seek the reason, we analyzed the stability of clFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that cIFN formed a lot of aggregates in media when the induction was controlled at 0.75% methanol, and two different aggregate forms were found: disulfide bond covalent aggregates and non-covalent aggregates. However, these aggregates almost disappeared when the methanol concentration was controlled at 0.25%, at the same time, cIFN bioactivity of supernatant increased almost 4.48-fold. Finally, 0.73g monomer cIFN was obtained after purification from 1 liter supernatant at 0.25% methanol induction, showed a 2.84-fold increase compare to the induction at 0.75% methanol.

Metabolic characterization of rat sertoli cell in vitro culture / 生物工程学报

Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat's testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine, amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2-4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.

STUDIES ON CONDITIONS OF CELL HIGH DENSITY CULTIVATION FOR GENETICALLY ENGINEERED METHYLOTROPHIC PICHIA PASTORIS / 微生物学通报

Recently the methylotrophic Pichia pastoris,with many advantages such as high expression,stable genetics and protein secretion,has been used extensively as a host expressing heterologous proteins.The optimal seed medium among seven media MM,MD,MGY,BMGY,YMPD,YMPGy and MGyB is BMGY with addition of 4mL/L PTM1 The high density synthesized medium in shake flask culture is as follows: Glycerol 4%(NH 4) 2SO 4 10g/L,CaSO 4 0.93g/L,K 2SO 4 18 2g/L,MgSO 4?7H 2O 14 9g/L,add 0.1mol/L PBS(pH=6 0) to 1 0 liter.The harvested yeast cell optical density (OD 600 ) reached 65 or more after 26 hours cultivation.By analysis of SDS\|PAGE,the results of Pichia pastoris culture by methanol inducement for 72 hours showed that the expression of recombinant human serum albumin had been achieved by methanol inducement after 12 hours,and the mount of targeted protein reached the summit after 24 hours inducement by methanol.The high density synthesized medium in shake flask culture in this experiment,which is similar to the mediums of batch fermentation and batch\|fed fermentation,is benefit to direct the P.pastoris fermentation in the fermentor.