To investigate the effect and mechanism of cell apoptosis of siRNA knockdown silent information regulator 2 (SIRT2) in Parkinson’s disease model cells induced by 1-methyl-4-phenylpyridinium (MPP+). Immortalized mouse hippocampal neuron HT-22 cells were cultured in vitro and treated with MPP + at different concentrations, and CCK-8 assay was used to detect cell inhibition. The cells were divided into control group, MPP+ optimal concentration group (1 mmol/L MPP+ treatment, injury group), the negative transfection group (based on the control group which was transfected with SIRT2 negative sequence), and the SIRT2-siRNA treatment group (based on the injury group which was transfected with SIRT2-siRNA). The apoptosis of cells in each group was observed, apoptosis-related proteins (Bcl-2, Bax, Caspase-9) and the main proteins mediating fission and fusion of mitochondrial function (Drp1, Fis1, OPA1, Mfn1, Mfn2) were detected by Western blot. Compared with the control group, the cell inhibition rate of MPP+ treatment group increased, and with the concentration increased, the inhibition rate gradually increased (P<0.05). Compared with the SIRT2 siRNA treatment group, the injury group increased the expression of apoptosis and mitochondrial fission factors (Bax, Caspase-9, Drp1, Fis1) and decreased the expression of anti-apoptotic and mitochondrial fusion factors (Bcl-2, Opa1, Mfn1, Mfn2). The expression of SIRT2 significantly increased in a cell model of MPP+-induced Parkinson’s disease, and the inhibition of the SIRT2 was able to decrease apoptosis, promote mitochondrial fusion, inhibit mitochondrial fission and protect neurons.
To explore the expression of miR-5047 in breast cancer cells and its role in breast cancer cell proliferation and migration, and to clarify the role of decitabine (DAC) in the regulation of miR-5047 expression. The expression level of miR-5047 in human breast cancer cell lines and normal breast epithelial cells MCF10A was detected by real-time quantitative PCR (qRT-PCR). Transfect miR-5047 mimic and negative control mimic NC into MDA-MB-231 and MCF7 cells, respectively, and verify the transfection efficiency by qRT-PCR. Plate cloning experiments, MTT experiments, and scratch healing experiments were used to detect the proliferation and migration ability of breast cancer cells, and qRT-PCR and Western blot methods were used to detect the expression of related genes and proteins after over-expression of miR-5047. MDA-MB-231 and MCF-7 cells were treated with DAC at final concentrations of 5 μmol/L and 10 μmol/L, and qRT-PCR was used to detect the effect of DAC on miR-5047 expression under different concentrations and treatment time. At the same time, the effect of DAC on the epithelial mesenchymal transition (EMT) of breast cancer cells was detected by morphological observation and Western blot. Compared with normal breast epithelial cells MCF-10A, the expression of miR-5047 in breast cancer cells was significantly down-regulated. Overexpression of miR-5047 can significantly inhibit the proliferation and migration of breast cancer cells, promote the expression of epithelial cell marker E-cadherin, and inhibit the expression of mesenchymal cell marker Vimentin. The expression of miR-5047 can promote the expression of epithelial cell marker E-cadherin and inhibit the expression of mesenchymal cell marker Vimentin. After treating MDA-MB-231 and MCF7 cells with different concentrations of DAC, the expression of miR-5047 was enhanced, and the effect was most significant when 10 μmol/L DAC was used for 48 h. DAC can induce epithelial transformation of MDA-MB-231 cells. The expression of miR-5047 is significantly down-regulated in breast cancer cell lines. Overexpression of miR-5047 can inhibit the proliferation and migration of breast cancer cells. The low expression of miR-5047 in breast cancer cells can inhibit the proliferation and migration of breast cancer cells. DAC treatment can enhance the expression of miR-5047 in breast cancer cells and induce epithelial transformation of the cells.
Objective: Cellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research and industrial processes. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-glycans and O-glycans, which in turn can unpredictably affect activity and secretion. Understanding the roles of glycosylation in the function of cellulase is important for further improvement of the enzyme technology for biomass conversion. Methods: The N-glycans defective mutants PoCel7A* and TaCel7A*, removal of the N-glycans on the catalytic domain of cellobiohydrolases from Penicillium oxalicum (PoCel7A) and Trichoderma atroviride (TaCel7A), were constructed using site directed mutagenesis. The extracellular protein concentration and enzymatic activity of the recombinant strains, expressing of PoCel7A *, TaCel7A or TaCel7A*, were determined to investigate whether the removal of N-glycosylation affects the activity and secretion of cellobiohydrolases. Results: The mutant PoCel7A* homologous expression in P. oxalicum, the removal of Asn137 glycosylation site at PoCel7A hardly affected the activity and secretion of Cel7A. However, after the Asn287 glycosylation site of TaCel7A was removed (TaCel7A *) and heterologous expression in P. oxalicum, the pNPCase, FPase and Avicelase activity of mutant decreased by 21.2%, 15.2% and 17.6%, respectively. Furthermore, compared to the parental strain, the expression levels of the UPR marker genes pdi1, bip1 and hac1 were significantly induced in recombinant strains, indicating increased demand for protein folding and transport capacity in recombinant strains. Conclusion: These results indicate that differential roles of N-glycan modifications in contributing to the function of Cel7A and highlight the potential of improving the activity and secretion of Cel7A by tuning proper interactions between glycans and functional residues.
Objective: To establish and evaluate a quantitative detection method for thrombomodulin (TM) based on a plate chemiluminescence immunoassay (CLIA) platform. Methods: The analysis system was composed of microplate coated with streptavidin, serum, the paired antibody coupled biotin and horseradish peroxidase. The double antibody sandwich mode was adopted to test quantitatively TM antigen. Moreover,the reaction conditions of TM were optimized and the analytical performance was evaluated. Results: The working concentration of biotinylated antibody and enzyme-labeled antibody was 0.5 μg/mL and 0.75 μg/mL, respectively. The incubation time was 15 min; the minimum detection limit was 0.2 TU/mL and the range of the detection method was 1~200 TU/mL. Inter-assay and intra-assay precision (CV) were less than 8%. The established method had good stability during 10 days at 37 ℃. 207 clinical plasma measured values had a high correlation with Sysmex measured values (R2>0.96). Conclusion: This study has successfully established a quantitative detection method of TM plate chemiluminescence, which has good performance indicators and can meet the needs of clinical detection.
Objective: The natural synthesis pathway of isopentenyl pyrophosphate, the precursor of isoprene compounds in microorganisms, is subject to strict metabolic regulation, which limits the efficient biosynthesis of isoprene compounds, and the use of the artificial isopentenol utilization pathway (IUP) is independent of the biological endogenous metabolic pathways. By introducing IUP into microorganisms, a large amount of isopentenol pyrophosphate can be synthesized, thereby promoting the large amount of synthesis of isoprene compounds. Methods: Introducing the artificial IUP into the oleaginous yeast Yarrowia lipolytica to strengthen the isopentenyl pyrophosphate biosynthesis and promote the efficient accumulation of β-carotene. Results: The tertiary structures of two key proteins involved in IUP, ScCK (Choline kinase from Saccharomyces cerevisiae) and AtIPK (isopentenyl phosphate kinase from Arabidopsis thaliana), are predicted to be acidic hydrophilic proteins without transmembrane domain and signal peptide. Both proteins have loose and unstable structural features and contain a variety of post-translational features. Modification sites and multiple interacting proteins are involved in phosphorylation. The β-carotene synthesis pathway was constructed by introducing carRP and carB, and strengthening the expression of thmgR and ggs1 in Yarrowia lipolytica by using homologous recombination technique, and 2.68 mg/L β-carotene was accumulated in the engineered strain. The ura on the genome were removed by the Cre-loxP system, and then IUP was integrated into the genome of this engineered strain. The yield of β-carotene was achieved at the levels of 410.2 mg/L in the engineered strain, which was nearly 200 folds greater than that from the original engineered strain. The fermentation conditions were as follows: cells were incubated for 96 h in the medium containing 20 mmol/L isoprenol and the ratio of carbon to nitrogen was set as 4/3 in the medium. Conclusion: IUP can promote the efficient accumulation of β-carotene in Y. lipolytica. This study provides a new strategy for the efficient biosynthesis of β-carotene and other isoprene compounds by using IUP.
Early diagnosis, early detection, and early treatment is an important strategy for improving the survival rate of patients. However, the current immunological reagents based on enzyme-linked immunosorbent assay and chemiluminescence are mostly limited to 10-14~10-12 mol/L, which cannot meet the needs for early diagnosis. On the contrary, single molecule detection allows to detect very low amount of biomarkers(~ 10-18 mol/ L) through limiting the immune complex into extremely small space(below nL). The results are produced by counting positive spots.The key for this technology is to create an array of small space. After decades of development, the detection range has been successfully limited to zL (10-21 L) through physical isolation,nanopores, or high-resolution microscope. For now, the SiMoA based on microarray has become the gold standard for single-molecule immunoassays. And the Quanterix’s HD-1 analyzer based on SiMoA has been applied for clinical practice, while the technology based microdroplets are mainly used for laboratory research. However, the later provide an avenue for point of care testing (POCT). Herein, this review will focus on the progress of single-molecule detection based on microarrays and microdroplets. It will provide a theoretical basis for the development of ultra-high sensitivity detection methods and promote this technology into clinical applica-tions.
Yeast has been well used as a typical microbial platform to make fermented fine chemicals. However, various stress conditions and abnormal metabolic milieu severely restrict the production costs and benefits. One effective way to resolve such bottlenecks is to engineer transcription factors (TFs) to enhance strain tolerance and production efficiency through remodeling the transcript levels of different stress resistant genes. The value of TFs engineering is reviewed from two aspects: novel strategies for the enhancement of both tolerance and yield by TFs engineering are examined. In addition, the applications of artificial transcription factors (ATFs)-based fabricating in metabolic fluxes optimization and quantitative evaluation are discussed. Lastly, we discuss challenges and potential solutions in exploiting TFs engineering for bio-based economic products.
The tRNA are important molecules of the translation process in the central dogma of life. The type and abundance of tRNA have a huge impact on the synthesis of proteins. In recent years, many inspirations have been obtained through the analysis of the structure and function of microbial tRNA and the process of tRNA modification. Moreover, the study of genetic codon expansion has realized the incorporation of unnatural amino acids into specific positions to obtain new functional proteins. At the same time, the codon recoding work carried out through chemical synthesis of microbial genomes will release more codons and tRNAs for more genetic codon expansion research. This work reviewed the latest application research progress of microbial tRNA and codon system in synthetic biology, and discussed the future development trend.
Halides are a kind of compounds which catalyzed by halogenase to the addition of halogen in secondary metabolites, have unique physiological and biochemical functions. Flavin-dependent halogenases have good regional selectivity and stability, and similar FAD binding sites with slight difference in substrates made them very important in industrial application. Therefore, the studies on its structure and synthetic pathway as well as random mutagenesis and directed modification of protein engineering are very important for its industry application. In this paper, the structural characteristics and engineering modification of flavin-dependent halogenases with high regional selectivity are reviewed, and it is of guiding significance for the application of engineering-modification flavin-dependent halides in industrial production.
Bioactive compounds have a broad range of applications in food, feed, cosmetic, nutraceutical, and pharmaceutical industries. Thus, the studies of bioactive compounds have been gaining great attention from science and the public in recent decades. Given the advantages of fast growth, high content of bioactive compounds, easy to be cultured and scaled up, and effective biorefinery, marine diatoms were considered to be the most promising producers of natural bioactive compounds. Although a large number of studies have been carried out to enhance the production of bioactive compounds from marine diatoms, a commercial production scale has seldom been realized due to several intrinsic shortcomings such as old culture technology and high production cost. This review aims to provide an overview of bioactive compounds from marine diatoms and their high production strategies. The path forward for further commercial production of the bioactive compounds from marine diatoms with respect to 3 different opportunities is also discussed, which will in turn accelerate the development of microalgae industry and provide support for human health and high-quality life.
Special yeast, as a kind of biological resource different from traditional yeast, has a wide range of industrial application prospects. By retrieving data from the incoPat database, this article takes the patents of industrial applications of special yeast as the research object. From the perspective of patent analysis, the development trend of technology innovation, technology distribution, main application institutions, research hotspots and application advantages of different kinds of global special yeast industry have been reviewed. Data has shown that the global patent publication amount for special yeast industrial technology presented an increasing trend from 2001 to 2020, and reached a peak in 2019. The patent protection areas concentrated in the US,the P. R. China, South Korea, Japan, Europe and other countries or regions, and the R&D focuses of domestic and foreign applicants differed. Foreign industrial biotechnology companies represented by DSM, Namur DuPont and Novozyme focused on the intellectual property rights of a basic technology of a special yeast. The domestic research institutions represented by Jiangnan University laid more stress on the development of Pichia pastoris technological applications. Finally, the differences in the innovation levels of various kinds of special yeasts from multiple technical branches in recent years have been analyzed, aiming to provide reference for decision-making in scientific research and academic works in the industrial application field of special yeasts in China.
All major economies of the world have adopted biopharmaceutical industry as a strategic sector to prioritise. Biopharmaceutical industry has gradually become the new growth engine for the global economy. The United Kingdom ranks No.1 in Europe by the size of its biopharmaceutical industry. This article elaborates the role played by its local ecosystem in fostering the growth of the UK’s biopharmaceutical industry. We hope this article can serve as a reference for those who are involved in the innovation and venture building in China’s biopharmaceutical industry.
