Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3 0 single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.
Osteosarcoma is a common primary bone cancer that there are currently no effective treatment strategies for. Forkhead box M1 (FoxM1) is key in the development of osteosarcoma, and microRNA (miR)-216b serves an antitumor role by targeting FoxM1. Moreover, thiostrepton (TST), a natural thiazole antibiotic, induces antitumor effects and specifically targets FoxM1. Therefore, the present study investigated whether thiostrepton and miR-216b synergistically inhibited osteosarcoma cells by targeting FoxM1. The MTT assay, reverse transcription-quantitative PCR, a dual-luciferase reporter assay and flow cytometry were performed. Compared with the human osteoblast cell line hFOB1.19, miR-216b expression was significantly downregulated in the osteosarcoma cell lines U2OS, MG63 and Saos-2. By contrast, FoxM1 expression was significantly upregulated in osteosarcoma cell lines compared with the hFOB1.19 cell line. The results indicated that miR-216b targeted the 3'-untranslated region of FoxM1. Moreover, the results suggested that miR-216b cooperated with TST to decrease cell cytotoxicity and increase cell apoptosis. In addition, miR-216b cooperated with TST to increase Bax expression and decrease Bcl-2 expression. In conclusion, the combination of TST and miR-216b synergistically promoted osteosarcoma cell cytotoxicity and apoptosis by targeting FoxM1. Therefore, the present study suggested that the combination of TST and miR-216b may serve as a promising therapeutic strategy for osteosarcoma.
In order to solve the problem that the image information is difficult to handle due to the swing of the snake robot’s head during its serpentine locomotion, this paper improves the control function of the general winding gait for the orthogonal joint-connected snake-like robot, and the useful image information is obtained by reducing the swing of the camera-mounted snake’s head based on the improved function. Combining the Sigmoid function with the general function to control the rotation of the yaw joints and applying the Gaussian function to the pitch joints to lift the snake head off the ground, this new control function presented in this paper not only enables the joints to rotate smoothly, but also decreases the swing range of the snake head. Additionally, it can also fasten the robot’s movement. Experiments based on MATLAB and GAZEBO platform demonstrate the superiority and effectiveness of the improved winding gait.
We propose a method of climbing pipe with different diameter, which enables snake robot to divide itself into 3 parts to complete transition on the pipe according to the joints’ state for now. To avoid controlling the robot from building dynamic model, a gait-based open-loop control framework is adopted. An extended kalman filter (EKF) is applied here to estimate the adaptive parameter of gait-based function for different 3 parts and corresponding joints. The adaptive parameter will help snake robot perceive the environment better and climb the pipe more steadily.
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