Pseudomonas putida KT2440 is a saprophytic, generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene coning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Herein we report an improved scarless gene deletion strategy based on recombineering and I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for 54.8 kb deletion, and the efficiency of RecA mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study and biotechnological and environmental applications of P. putida KT2440.
Zircon has been widely applied in various traditional and emerging fields due to its distinct physiochemical advantages while monodisperse spherical zircon (MSZ) powders hitherto have not been explored for the tremendous challenges in the control of zircon crystallization and growth. Herein, systematic investigations are presented to reveal the paramount role of halide mineralizers upon the crystallization and growth of MSZ powders synthesized under highly acidic hydrothermal environments (pH <0). A formation mechanism is revealed revolving around the complexation of central Zr4+ with the Lewis basic ligand, fluorine, which forms the F‐containing species of [(OH)1–y·Ry] (R = F, Cl) to replace Zr and generates Zr‐deficient zircons as (ZrO4)1‐xSi[(OH)1‐y·Ry]4x. This finding differs much from conventional views on common fluoro‐hydroxylated hydrothermal zircons, which were generally regarded as Si‐deficient silicates from the substitution of fluoro‐hydroxyls for the [SiO4] tetrahedra. A parameter, Kr=4xy, is first proposed as an index to evaluate the stability of hydrothermal zircons. The MSZ powders demonstrate superb thermal stability at high temperature, superior photo‐reflectance, and thermal insulation over normal zircons, promising attractive prospects to extend zircon applications.
Pulmonary veno-occlusive disease (PVOD) is a fatal disease of pulmonary vascular lesions leading to right heart failure. Heritable PVOD (hPVOD) is related to biallelic mutation of EIF2AK4 (encoding GCN2), but its molecular mechanism remains unclear. In this study, we aimed to investigate the pathogenesis of PVOD and to find potential drug targets for PVOD. GCN2 dysfunction led to an enhanced transcription of collagen I gene ( col1a1 and col1a2) through decreasing ATF3-dependent p38 phosphorylation inhibition in PVOD, which promotes the collagen I synthesis in pulmonary arterial smooth muscle cells (PASMCs) and eventually leads to increased collagen deposition in pulmonary artery. Four GCN2 knockout (KO) cell lines (exon 15 or 33 mutation) were successfully constructed by epiCRISPR system. Two induced pluripotent stem cells (iPSCs) were generated by reprogramming peripheral blood mononuclear cells (PBMCs) of PVOD patient. It was also comfirmed that GCN2 dysfunction could lead to increased expression of collagen I in lateral plate mesoderm lineage-smooth muscle cells (LM-SMCs) differentiated from both GCN2 KO cell lines and iPSCs. SB203580 (a specific inhibitor of p38) improved hemodynamics and pulmonary vascular remodeling in mitomycin C (MMC)-induced PVOD rats by right ventricle echocardiography. On the whole, we proposed that GCN2 deficiency decreased ATF3-dependent p38 phosphorylation inhibition in PVOD development and suggested a potential therapeutic reagent of SB203580 for the treatment of the disease.
UAV remote sensing, as a new method of remote sensing, has the characteristics of higher spatial resolution, fine timeliness and high flexibility. It is widely used in the field of natural disaster monitoring, urban planning, resource investigation, and has become one of the indispensable method of remote sensing data acquisition. However, because the UAV remote sensing platform is limited by the flight height and focal length of camera, the acquired image size is smaller, single image can't cover the entire target area. Therefore, image mosaic has become a key technology to solve the problem. Image matching and image fusion are the key techniques of image mosaic. Due to the good robustness of image scaling, translation and rotation, this paper uses the SIFT algorithm to realize image matching of UAV. Since the feature extraction may produce false matches, RANSAC algorithm is applied to the feature point purification points. According to the seam-line in jointing overlap region, weighted fusion algorithm is applied to realize the image seamless splicing.
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