Objective: To construct a Toll-like receptor 4 (TLR4) gene knockout mouse model using CRISPR / Cas9 technology, and observe the changes in mutant mice’s response to gram-negative bacterial lipopolysaccharide (LPS) stimulation.Methods: One pair of sgRNA fragments were designed and synthesized for exon 2 of TLR4 gene, mixed with mRNA encoding Cas9 and then injected with TLR4 gene knockout mice through fertilized egg microinjection method, and gene knockout homozygous mice were obtained by breeding (TLR4 -/-mice); The response of TLR4-/-mice to inflammatory stress was analyzed by LPS stimulation, and compared with wild-type control (WT) at the molecular and pathological levels.Results: PCR and sequencing showed that exon 2 of TLR4 gene was successfully knocked out in mouse genes. After LPS stimulation, the expressions of inflammatory factors such as IL1β, IL6, MyD88, iNOS and TNFa were detected in the heart of wild-type mice. Significantly up-regulated in liver and lung tissues, but almost no change in TLR4 -/-mice; blood biochemical indicators showed that urea (Crea) and creatinine (Cre) levels in WT mice were significantly increased after LPS stimulation, and TLR4-/-mice had no significant changes before and after stimulation. Pathological analysis also found that TLR4-/-mice were able to resist LPS damage to kidney tissue.Conclusion: TLR4 gene knockout mouse model was successfully constructed using CRISPR / Cas9 technology. The deletion of TLR4 can reduce the response of IL1β, IL6, MyD88, iNOS and TNFa inflammatory factors to LPS stimulation, inhibit LPS-induced inflammatory response and tissue damage.
Objective: This study aims to pinpoint the role of Ozanimod (RPC1063) in the differentiation of oligodendrocyte precursor cell (OPC) and investigate the underlying molecular mechanism.Methods: OPC was directly isolated by immune adsorption and indentified through immunofluorescence and quantitative real time-PCR. Firstly, the mRNA level of S1pr family genes was detected by qRT-PCR during the cerebral cortex development and OPC differentiation. Then, viability of OPC treated with different concentrations of RPC1063 was measured by MTT assay and ATP cell viability assay. Moreover, by means of immunofluorescence, qRT-PCR, and Western blot, we analysed the effects of RPC1063 on the differentiation rate of OPC, and the mRNA or protein level of OPC-differentiation associated genes.Results: High purity of OPC could be obtained by the method of immune adsorption from the cortex of mice. There was no significant effect of RPC1063 on cell viability at concentrations of 0, 0.1, 1, 5, 50, 100nmol/L. Additionally, up-regulation of O4+ or MBP+ cells, and protein level of MBP and mRNA level of Mbp, Sox10, Cnp, or Plp were observed in OPC treated with RPC1063. Moreover, in RPC1063-treated Oli-neu cells, the protein levels of pAKT, p-mTOR and p-4EBP1 were significantly increased at 5min. Besides, the proteins related to p-AKT-mTOR signaling pathway were also significantly increased in OPC. The promotion of RPC1063 on OPC differentiation was weakened by the inhibition of mTOR pathway.Conclusion: RPC1063 promotes the differentiation of oligodendrocyte precursor cell through activating the AKT-mTOR signaling pathway.
The promoters are important components that regulate gene transcription, and key parts in synthetic biology research and cell factory design. Glycolytic pathway and tricarboxylic acid (TCA) cycle are the central metabolisms of carbohydrate catabolism and are strictly regulated by promoter strength. In order to screen some endogenous constitutive promoters with various strength necessary for synthetic biology studies and cell factory design of Escherichia coli, the strength and core structural elements of 27 promoters in glycolytic pathway and TCA cycle of E. coli were systematically studied using the red fluorescent protein (RFP) mCherry as the reporter gene and online analysis software. The results showed that the strength range of these promoters varied greatly, and the strength of the strongest promoter PgapA was 43.6 times that of the weakest promoter PacnA. Moreover, the -10 and -35 sequences of promoters are not exactly same as their consistent sequences, and the spacer between them is 17±3 bp. However, the strength of the promoters was basically consistent with the structural characteristics of the promoters. Using the strongest promoter PgapA, Phosphoenolpyruvate carboxylase and pyruvate kinase were expressed in recombinant E. coli DH5αΔpck, respectively. Their enzyme activity was increased by 0.32 and 1.57 times, respectively, and the production of citric acid was also increased by 124.7% and 75.5%. These promoters with different strength have laid a foundation for the study of synthetic biology and the design of cell factory of E. coli.
Objective: The T158M site of MECP2 gene was modified by using single-base editing technique to obtain single site-directed mutant rodent and non-human primate embryos,which lays a foundation for the establishment of an animal disease model simulating Rett syndrome in clinical practice.Methods: Three pairs of T158M-sgRNA plasmids were constructed using single-base editing technique,and co-transfected 293T cells with BE system(BE3 or BE4max)plasmids to screen highly-efficient sgRNAs.In vitro transcribed mRNAs were injected at different concentrations by microinjection to ICR mice and macaque fertilized eggs to measure the embryo development rate and editing efficiency of mouse and macaque and to evaluate the working efficiency of BE3 and BE4max and optimal concentration combination.Results: The mouse embryo development rate and editing efficiency are best when the concentration ratio of BE4max(ng/μl)∶sgRNA(ng/μl)on mouse embryos is 100∶50,and this concentration combination also shows effective editing efficiency on rhesus monkey embryo.Conclusion: The transition of C→T at position T158M of the MECP2 gene were successfully achieved in mouse and macaques embryos,establishing a stable embryo editing system for the later establishment of a T158M mutant RTT animal model.
Objective: To develop a bifunctional tag which can be used for affinity purification of target proteins, and can carry some drug proteins that cannot enter the cell autonomously across cell membrane to exert activity.Methods: Four types of calmodulin binding peptide (CBP) rich in basic amino acids are selected from the literature as candidate tags for fusion expression with green fluorescent protein (EGFP). The CBP with the highest affinity to CaM is screened by CaM affinity binding process. Subsequently, the capacity of the candidate CBP sequences to autonomously transport EGFP recombinant proteins into cell is analyzed and determined by fluorescence microscopy, laser confocal microscopy and flow cytometry. Finally, the novel CBP bifunctional tag is screened then fused and expressed with Apoptin, and its ability to purify recombinant Apoptin is examined after binding with CaM. The ability of this recombinant protein to enter tumor inhibiting cell growth is analyzed by MTT method.Results: Three novel CBP sequences with high affinity to CaM are screened out by CaM-CBP affinity chromatography. The intracellular fluorescence detection results of the recombinant protein showed that the CBP sequences derived from wild-type skeletal muscle myosin light chain kinase (named MLCK) has the best transmembrane efficiency to carry EGFP into cells, and its transportation capacity is much higher than the classic cell-penetrating peptides TAT derived from HIV. The recombinant Apoptin is purified by CaM-CBP affinity chromatography with the affinity tag MLCK and can be transported into cells to exert an anti-tumor activity. Furthermore,MTT assay results show that the 24-hour 50% inhibitory concentrations (IC50) of the recombinant Apoptin on three tumor cells MGC-803, H460, and HeLa are 1.18μmol/L, 1.23μmol/L, and 1.23μmol/L, respectively, which shows that the Apoptin retains partial anti-tumor activity.Conclusion: A novel calmodulin-binding peptide, MLCK, is screened out which can be used in affinity purification due to its high affinity with CaM. Simultaneously, it has high-efficiency transmembrane function which is similar to typical cell-penetrating peptides, and can carry drug proteins into cells autonomously to exert the biological activity of drugs. Therefore, the novel bifunctional tag can be used for both affinity purification of drug proteins and cell membrane transportation, thus it can be widely used in the development of various new drugs.
Oncolytic virus utilizes the characteristics of tumor cells, such as deficiency of antiviral signaling pathway or overexpressing viral receptors, to preferentially infect, replicate in and kill tumor cells, which are emerging as important agents in cancer treatment. Due to the lack of interferon signaling pathways, tumor cells can be specifically targeted by vesicular stomatitis virus (VSV). VSV-based platform shows high prospects, as its efficient replication, broad tissue tropism, low pathogenicity to humans and its small and easy-to-manipulate genome. The virological characteristics of vesicular stomatitis virus and the current research progress of VSV-based oncolytic virus to improve tumor selectivity, extend half-life and increase oncolytic efficacy are discussed in this review.
Autophagy of adipocytes is mainly in the form of Lipophagy and Mitophagy. Lipophagy reduces lipotoxicity of adipocytes and provides substrate for mitochondria by promoting lipolysis. Mitophagy controls the quantity and quality of mitochondria to affect the function of the cells. Excessive accumulation of white adipose tissues and inflammation caused by inappropriate autophagy regulation can lead to obesity and its related metabolic diseases. The conversion of stored white adipose cells into heat-producing beige cells by browning of white adipocytes is one of the current strategies to prevent obesity. The process of browning of white adipose cells is inseparable from the regulation of autophagy. This article reviews the current research progress on the role of two forms of autophagy in browning of white adipose cells, related signaling pathways, and autophagy-regulated inflammation, with a view to providing reference for the study of anti-obesity and related metabolic diseases.
Quorum sensing is a special dynamic metabolic regulation mechanism. It is an environmental signal sensing system used by bacteria to monitor their own population densities. In recent years, with the vigorous development of synthetic biology, breakthroughs have also been made in the artificially synthesized flora and mixed bacteria co-culture technology based on stable flora relations. The quorum sensing system can achieve the purpose of autonomously controlling the relationship between bacteria and bacteria, and its research and application in metabolic engineering has received more and more attention. Based on the overview of quorum sensing, this paper summarizes the dynamic metabolic regulation of single bacteria based on quorum sensing; at the same time, research progress in the dynamic regulation of quorum sensing between Gram-negative and Gram-positive bacteria and co-cultivation is reviewed. In order to provide some suggestions and help for other quorum sensing applications.
Lactic acid bacteria (LAB) is a Gram-positive, obligate anaerobic bacterium, and the main production of lactic acid. It was one of the most important industry strain which was used in food industry, pharmaceutical industry and chemicals industry. In recent years, with the continuous improvement of the industry application of LAB, studies were focused on physiological and biochemical characteristics, acid tolerance characteristic, metabolic pathways and mechanism of lactic acid production of lactic acid bacteria. So, it is necessary to establish a stable and efficient gene-modifying method for analyzing the functions of key genes and geneme network and adjusting the metabolic pathway. The paper reviews the recent progress and future prospects of LAB gene-modifying technologies.
PH is an important factor affecting the growth of microorganisms. In the process of industrial fermentation, the fluctuation of pH regulation could lead to acid / alkali stresses, which affects the growth of microbial cells and the accumulation of target products. In this review, the physiological changes of industrial microorganisms in the pH fluctuation of fermentation system were described, and the main strategies of pH regulation in industrial fermentation and the comparison of them were summarized. Furthermore, the development direction of pH regulation in the future was pointed out, as it can provide new ideas for pH regulation in fermentation industry.
Lignocellulosic resources are abundant renewable resources in nature, and the degradation of lignocellulosic by microorganisms is an important strategy. Based on the study of screening methods and research strategies of lignocellulosic degradation microorganisms all around the world, the advantages, characteristics and application value of lignocellulose degradation by microorganism are summarized and analyzed from single strain, microbial consortium and omics technology: single strain is easy to cultivate but lack of strong degradation ability. Microbial consortium is good at lignocellulose degradation but has poor subculture stability. The omics technology can better explain the mechanism of microbial degradation of lignocellulose, and provide good guidance for screening lignocellulose degradation microorganisms. The strategy of synthetic biology is proposed for screening the Lignocellulose degrading microorganisms, in order to provide some reference for screening the microorganisms which can degrade lignocellulose efficiently.
Objective: Based on the analysis of the current situation and major enterprises of CMO/CDMOS industry in the world, this paper reveals the quality assessment problems existing in the development of CMO/CDMOS industry, and puts forward relevant suggestions for quality assessment.Methods: quantitative analysis and comparative analysis were used to reveal the development status of Immunotherapy and stem cell therapy products and CMO/CDMOS industry.Results: With the development of cell therapy industry, more and more institutions show their interest in cell therapy CMOS / cdmos industry, which is involved by means of creation, acquisition and investment. At present, 48 companies have been identified to provide contract cell or gene therapy manufacturing services.Conclusion: The development of cell therapy industry has led to the prosperity of CMO/CDMOS contract manufacturing agreement. In view of the particularity and complexity of cell therapy, it is also full of challenges to the quality assessment of cell therapy.
