Objective: In order to illustrate the hypoxia-induced changes of neural cells in inflammatory response, oxidative stress, and energy metabolism process and to compare the sensitivity of neural cells’ responses to hypoxia. Methods: Different types of neural cells (BV2, N9, Gl261, HT22) were treated with hypoxia (0.1% O2, 5% CO2) for 0-24 hours. Cell proliferation was detected by Cell Counting Kit-8 method and cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay. Total RNA was extracted by Trizol reagent, and the inflammation, oxidative stress, and energy metabolism-related genes expression were measured by quantitative real-time PCR and Western blot. The ROS production was detected by flow cytometer with fluorescence probe. Results: Hypoxia stimulation decreased cell proliferation and cell viability. The hypoxia-induced changes of microglial cells (BV2 and N9) were mainly involved in inflammatory response and glucose metabolism process. The changes of astrocytes Gl261 and neural cell HT22 were mainly involved in glucose metabolism process. Hypoxia stimulation significantly increased oxidative stress in microglia and astrocytes. Conclusion: Different types of neural cells have different degrees of sensitivity in response to hypoxic stimulation. In terms of energy metabolism and inflammatory response, microglia are more sensitive to hypoxia treatment, which is manifested as a significant up-regulation of glycolytic enzymes and inflammation genes, whereas microglia and astrocytes are more sensitive to hypoxia treatment in terms of oxidative stress, which is indicated by their quick response and significant increase of ROS production.
To investigate whether carrimycin (CAM) affects the occurrence and development of melanoma by regulating the polarization of macrophages, and the following related cell biology assays were used to examine its function. Methods: The effect of CAM on macrophage polarization was detected by real-time quantitative polymerase chain reaction (Q-RT-PCR) and Western blot. Flow cytometry and Cell Counting Kit-8 were used to detect the effect of CAM on mouse macrophages in vitro and in vivo phagocytosis and proliferation. Cell line-derived xenograft model was constructed via B16-F10 to evaluate the anti-tumor effect of CAM on melanoma. Results: In the mRNA level, CAM could up-regulate the levels of TNF-α and iNOS in M1 and down-regulate the level of Arg-1 in M2. In the protein level, CAM can increase the expression of p-STAT1 and decrease the expression of p-STAT3. In the cell line-derived xenograft model, these data shown that the occurrence and CAM development of melanoma was inhibited after CAM treatment, the tumor inhibition rate was 41.6%, and promoted the increase of the number of M1 macrophages (P<0.05). Conclusion: CAM promotes the increase in the number of M1 macrophages in vivo and inhibits the progression of melanoma, suggesting that CAM may achieve anti-tumor effects by inducing the polarization of macrophages to M1.
Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-glucosidase (GAA) deficiency, which is characterized by glycogen accumulation in the heart, skeletal muscle, and central nervous system (CNS). AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease. In this study, AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice, and the changes of GAA protease activity, glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated. Methods: Codon optimized GAA gene (coGAA) was carried by AAV9 vector, and the AAV vector was packaged by baculovirus production process. Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×1013, 3.0×1013, 1.2×1014 vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×1013 vg/kg. After reaching the end point of the experiment, the mice were euthanized, GAA protease activity was determined by fluorescence spectrophotometry, glycogen accumulation was observed by PAS staining, and pathological changes were detected by HE staining. Results: Five weeks after administration, GAA protein was widely expressed in all tissues of adult model mice, with higher expression levels in heart and liver, and lower expression levels in brain and spinal cord. After rAAV9-coGAA treatment, glycogen content in myocardium, skeletal muscle and brain decreased, and vacuolar degeneration in myocardium and skeletal muscle decreased significantly. After treatment, the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice. The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased, but the pathological improvement of skeletal muscle was limited. Conclusion: A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity, reduce glycogen accumulation and improve pathology in Pompe model mice. The therapeutic effect was dose-dependent, and the injection also had certain therapeutic effect on aged animals. This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.
Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ees value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ees value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.
Brewer’s yeast is the key to beer brewing and can directly influence the beer quality. In the beer brewing process, brewer’s yeast is passed down from generation to generation and preserved several times, causing problems such as the decline of fermentation performance of good strains, resulting in incomplete fermentation and affecting the flavor quality of the final beer. Therefore, this study took 8 lager-type brewer’s yeast strains were selected as the starting strains in this study, and 80 isolated strains were obtained through plate isolation and purification, and finally 8 brewer’s yeast strains with excellent fermentation performance were gained through preliminary sieving and rescreening in triangular flask fermentation and pilot fermentation experiments in fermenter. Among them, 6 yeast strains can be used to brew beer with diacetyl content lower than 0.1 mg/L; 3 yeast strains with fermentation degree higher than 70% are suitable for brewing dry beer; 1 yeast strain with fermentation degree lower than 50% is suitable for brewing low alcohol beer. In terms of flavor: 1 yeast strain was used to brew beer with an alcohol to ester ratio of 3.3, with a more prominent beer ester aroma; another yeast strain was used to brew beer with an alcohol to ester ratio of 4.5, with a higher alcohol content in beer. The fermentation characteristics of 8 yeast strains having been purified and bred are obvious,which are especially easy for practical application in craft breweries.
Objective: The cell lines secreting specific monoclonal antibodies (McAbs) were prepared by using Fusarium solani, one of the pathogenic fungi causing root rot of Fritillaria thunbergii, and the colloidal gold immunochromatographic test strip based on McAbs was developed to provide scientific basis for detecting root rot of F. thunbergii. Methods: Hybridoma technology was used to obtain cell lines that could secrete specific McAbs against F. solani using the whole protein extract of F. solani as the antigen. The specificity, titer, sensitivity and binding protein of McAbs were detected by indirect ELISA and Western blot. Colloidal gold particles were prepared by trisodium citrate reduction method and McAbs were labeled to prepare colloidal gold immunochromatographic strip. Results: Three cell lines secreting specific McAbs against F. solani were obtained, which were named as FsA3, FsG6 and FsD4. The detection sensitivity of FsA3 was 24.41 ng / mL, and that of both FsG6 and FsD4 was 12.21 ng / mL. FsA3, FsG6 and FsD4 had strong reactions to F. solani, and had no cross-reaction to Alternaria tenuissima, A. alternata, Botrytis cinerea, F. equiseti, F. incarnatum, F. oxysporum, Phoma sp., and Phomopsis oblonga. The colloidal gold immunochromatographic strip based on FsG6 showed only a quality control line when detecting the tissue culture seedlings of F. thunbergii. When 100 ng F. solani antigen or the samples of F. thunbergii infested with root rot disease were detected, there were visible quality control lines and test lines. Conclusion: The specificity and sensitivity of the McAbs and test strip are sufficient to detect F. solani isolated from diseased strains of F. thunbergii, which provides the technical support for the rapid detection of root rot of F. thunbergii in the field.
Breast cancer is a common malignant tumor mostly occurring in women. Triple-negative breast cancer (TNBC) is highly malignant and has a complex pathogenesis, which is the worst prognosis type of breast cancer classification. However, the sensitivity of its early screening and diagnosis is still at a low level. Therefore, it is urgent to achieve its early diagnosis and treatment by applying highly specific molecular probes for detection of tumor markers. Aptamer is a class of oligonucleotide screened by SELEX (systematic evolution of ligands by exponential enrichment) technology in a synthetic library of random single-chain nucleic acid sequences. With efficient molecular recognition ability, it has become the most potential bio-targeting molecule and has a wide application prospect in tumor diagnosis and treatment. Currently, several aptamers targeting TNBC cells have been obtained by screening. Here, the new progress of screening TNBC related aptamers based on SELEX and its derivatives, as well as the application of aptamers in the diagnosis and treatment of TNBC are reviewed, to provide a reference for related research.
Salmonella typhimurium is a zoonotic intestinal pathogen, which can cause intestinal inflammation. Effectors are mainly secreted through the type III secretion system(T3SS) encoded by its pathogenic islands (SPIs) to invade host cells and regulate cell signaling pathways, including pro-inflammatory effectors and anti-inflammatory factors. Pro-inflammatory effectors are released to cause inflammation when Salmonella typhimurium invades intestinal epithelial cells. In order to prevent excessive damage of host cells by pro-inflammatory effectors affecting the survival and reproduction of bacteria, Salmonella typhimurium can produce a series of anti-inflammatory factors to regulate intracellular signaling pathways, co-produce with the host and eventually spread throughout the body to cause severe infection. This paper provides an overview of the mechanism by which Salmonella typhimurium utilizes T3SS effectors to invade host cells and regulate cell signaling pathways.
Most of the enzyme-catalyzed reactions in organisms require the participation of cofactors. The balance of cofactors is essential to maintain normal cellular metabolism, and the imbalance of cofactors can lead to the inhibition of cell growth and production. In the construction of microbial cell factories, it has been considered to be an important strategy to regulate the balance of cofactor metabolism since the cofactor regulation could be employed to improve the efficiency of synthetic pathway of products and thus balance the cell growth and products synthesis, achieving the maximum metabolic flux towards the target products. Common cofactors currently used for metabolic regulation include NAD(P)H/NAD(P)+, coenzyme, and ATP/ADP. The metabolic pathways and functional classifications of these cofactors are reviewed, and the studies on the synthesis regulation of different products in microorganisms using cofactor balancing strategies are summarized. This paper will provide references for the efficient biosynthesis of various compounds.
Sugar substances are widely used in the fields of food, medicine, daily chemical and fermentation, and are of great significance to human health and social development. For a long time, the production of sugar substances has been practically based on the collection and planting-harvesting of plant biomass. Solar energy and carbon dioxide would be fixed in plant photosynthesis process, and stored in the form of sugars (starch, cellulose, and sucrose) in biomass. The bulk sugar feedstock would be extracted from plant biomass, and then be utilized for generation of other sugar products through multiple steps of conversion, refinery, and purification. The dependence on the plant sourced biomass of current sugar production technology leads to the unavoidable risks and drawbacks of long cultivation term, climate & location constraints, and high pre-treatment costs. The development of novel technologies for sugar production is conducive to removing the bottlenecks faced by the traditional modes derived from plant biomass harvesting-refining industry. Cyanobacteria are an important group of photoautotrophic prokaryotic microorganisms and are also supposed to be promising microbial photosynthetic platforms. The development of cyanobacterial synthetic biotechnology has facilitated the direct conversion of carbon dioxide into dozens of natural and non-natural metabolites, of which sugars are a representative group. Cyanobacterial photosynthetic production of sugars is expected to realize the one-step conversion of carbon dioxide into specific sugar products and to refresh the paradigm of current sugar production technology. Sugar metabolites play important roles in the natural photosynthetic metabolic network of cyanobacteria, especially that the operations of several important physiological modules such as the Calvin cycle, glycogen metabolism, and compatible substance metabolism, are mainly promoted by the conversion and metabolism of multiple sugars and sugar-derived metabolites. Although some progress has been made in the synthesis of special sugar substances using natural cyanobacterial resources, such as the successful development and industrial application of the technology of Spirulina large-cultivation based glycerol glucosides production, there are still many challenges in the development and application of this mode. In recent years, the development of synthetic biotechnology has been providing new driving forces for remodeling and expanding the cyanobacterial photosynthetic sugar metabolism networks. Through specific transporter engineering, secretory production of some important sugar products, e.g. sucrose and trehalose, has been achieved, and it significantly relieved the metabolic stress from intracellular storage and increased the product titers. In addition, the sugar secretions would facilitate the development of derived technologies such as in situ separation and extraction of products as well as construction of artificial photosynthesis driven consortium. In combination with metabolic engineering strategies from multiple levels and steps, the production efficiency of cyanobacterial sugars would be significantly enhanced, photosynthetic production of novel sugar products would be achieved, and the updating of the sugar production routes could be expected. This review systematically summarized the progress and challenges in developing and utilizing cyanobacterial photosynthetic sugar production technologies, and discussed the future development prospects and research directions.
DNA assembly and transfer techniques are one of the core enabling technologies for synthetic biology. The increasing degree of complexity in the design and modification of living organisms has led to a growing demand for large DNA assembly and transfer methods. Nowadays,the assembly and transfer techniques of small DNA are well developed, while the manipulation of large DNA in vitro is complicated and inefficient due to DNA high molecular weight and ease to break. This review focuses on advances in large DNA assembly and transfer techniques in Saccharomyces cerevisiae and transfer technology. The methods of one-step assembly and iterative assembly in S. cerevisiae are introduced in detail. The transfer methods from the aspect of transfer in and out are highlighted, and researchers can better understand and choose these methods. In addition, the authors envisage that it is possible to make S. cerevisiae become a universal platform for assembly and transfer of large DNA, which enables large-scale genomic design and modification of more organisms.
After “Brexit”, the UK has accelerated the legislative process in the field of biotechnology, especially for genetic technology. The UK is eager to innovate the original conservative regulatory model of the European Union and give full play to the application value of genetic technology on the premise of ensuring technical safety. To this end, the UK issued the Report on Genetic Technologies, which aims to rebuild a more comprehensive genetic technology regulation system. This report not only analyzes the shortcomings of existing genetic technology governance arrangement, but also points out the direction of genetic technology regulation in the UK in the future. At present, new genetic technologies such as gene editing and synthetic biology are in a period of rapid development. The UK report also provides a reference governance path for the application of these technologies.
