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Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts(NSF)cell line using CRISPR/Cas9 genomic editing technology,to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats.Methods We designed four pairs of single guide RNA(sgRNA)sequences targeting exons 1~4 of the NSF HIF-1αgene and successfully constructed an expression plasmid.The plasmid with the optimal sgRNA was identified and transfected into 293T cells,and the supernatant was used for detecting the virus titer.Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein,based on a previous protocol.After transfection,fluorescence signals were observed under a fluorescence microscope,and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1(T7E1)analysis.Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors,demonstrating successful construction of a recombinant plasmid for transfection.T7E1 digestion successfully removed three bands and the target efficiency of sgRNA was 54%.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats(P=0.0019).There were no obvious morphological changes in HIF-1α-knockout cells under the microscope,and gene knockout had no obvious effect on cell proliferation.Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/Cas9 technology,to provide an experimental basis for further studies of the biological function of HIF-1α,as well as the mechanism of hypoxia tolerance in naked mole rats.The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.
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AIM: To investigate the molecular mechanism of lncRNA HOTAIR regulating miR-206 on the proliferation and apoptosis of rheumatoid arthritis synovial cells. METHODS: The synovial tissue from 30 cases of rheumatoid arthritis were collected. Rheumatoid arthritis synovial cells MH7A were cultured. The experiment was divided into si-NC group, si-HOTAIR group, miR-NC group, miR-206 mimic group, si-HOTAIR+NC inhibitor group, si-HOTAIR+miR-206 inhibitor group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of HOTAIR and miR-206 in cells. CCK-8 method to detect cell proliferation; flow cytometry to detect cell apoptosis; Western blot to detect cell protein expression of CyclinD1, p21, Bax and Bcl-2; dual luciferase reporter assay to detect HOTAIR and miR-206 targets To combination relationship. RESULTS: Compared with the healthy control group, the expression level of HOTAIR in patients with rheumatoid arthritis was significantly up-regulated, and the expression level of miR-206 was significantly down-regulated (P<0.05). Compared with the si-NC group, the HOTAIR expression level in the si-HOTAIR group was significantly down-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the miR-NC group, the expression level of miR-206 in the miR-206 mimic group was significantly up-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the si-HOTAIR + NC inhibitor group, the cell survival rate in the si-HOTAIR+ miR-206 inhibitor group were significantly up-regulated, and the apoptosis rate were significantly decrease (P<0.05). CONCLUSION: Inhibiting the expression of HOTAIR and up-regulating the expression of miR-206 can reduce the proliferation of rheumatoid arthritis synovial cells and promote apoptosis.
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p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.
Subject(s)
Ammonia-Lyases/chemistry , Coumaric Acids , Escherichia coli/genetics , TyrosineABSTRACT
Hematoma enlargement is a common harmful event after cerebral hemorrhage, which can lead to deterioration of neurological function and poor outcome. Early detection of high-risk patients can help prevent hematoma enlargement and improve the outcome of patients. Although the exact mechanism of hematoma enlargement is unclear, more and more evidence shows that many imaging signs may be related to hematoma enlargement, such as dot sign, mixed sign, black hole sign, island sign, etc. This article mainly reviews the relevant imaging prediction factors of hematoma enlargement from the perspective of imaging.
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cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.
Subject(s)
Escherichia coli , Metabolism , Fermentation , Industrial Microbiology , Metabolic Engineering , Microorganisms, Genetically-Modified , Promoter Regions, Genetic , Sorbic Acid , MetabolismABSTRACT
BACKGROUND: Icari n as one of the main components of Epimedium has an inhibitory effect on osteoclasts.. OBJECTIVE: To further investigate the influence of icariin on the root absorption of the maxil ary first molar at mesial part during orthodontic treatment in rats. METHODS: Orthodontic root resorption models were established in the left maxil a of rats. Local injection of 200 mg/kg icari n (icari n group) or normal saline (positive control group) was administrated into the left first molar buccal periosteum. The right maxil a of rats served as negative control group that was treated with neither appliance nor drug injection. The mesial distance between bilateral first molars and the contralateral maxil ary incisor was measured before and after the appliance was placed. Mesial surface of the mesial root of bilateral maxil ary first molars was observed using scanning electron microscope. RESULTS AND CONCLUSION: Mesial movement of the maxil ary molars in the icari n group was significantly less than that in the positive control group (P < 0.05). Under the scanning electron microscope, smal absorption lacunae were scattered in the icari n group, while the positive control group showed a large amount of absorption lacunae and they were interconnected into a sheet, showing a stark contrast with the smooth root surface of the negative control group. It is indicated that icari n can inhibit root resorption caused by orthodontic treatment, while reducing the amount of mesial movement of the molar under corrective force.