Molecular characterization of adenosine monophosphate deaminase 1 and its regulatory mechanism for inosine monophosphate formation in triploid crucian carp.

Inosine monophosphate (IMP) is the main flavoring substance in aquatic animal, and adenosine monophosphate deaminase1 (AMPD1) gene is a key gene in IMP formation. At present, the research on the mechanism of AMPD1 regulating IMP formation in aquatic animal is still blank. In this study, in order to study the mechanism of AMPD1 regulating IMP formation in fish, the full open reading frame (ORF) of AMPD1 which was 2160bp was obtained for the first time in triploid crucian carp (Carassius auratus). It encoded 719 amino acids with a molecular mass of 82.97 kDa, and the theoretical isoelectric point value was 6.31. The homology analysis showed that the homology of triploid crucian carp and diploid Carassius auratus was the highest, up to 99%. And the phylogenetic tree showed that triploid crucian carp was grouped with diploid Carassius auratus, Culter alburnus, and Danio rerio. And real-time fluorescence quantitative results showed that AMPD1 was expressed specifically in muscle of triploid crucian carp (p < 0.05). The results of detection the localization of AMPD1 in cells indicated that the AMPD1 was mainly localized in cytoplasm and cell membrane. Further, we examined the effects of glutamate which was the promotor of IMP formation on the expression of AMPD1 and the formation of IMP in vivo and in vitro experiments, the results showed that 3% glutamate and 2 mg/ml glutamate could significantly promote AMPD1 expression and IMP formation in triploid crucian carp muscle tissue and muscle cells (p < 0.05). Then we inhibited the expression of AMPD1 in vivo and in vitro experiments, we found the formation of IMP in muscle tissue and muscle cells of triploid crucian carp all were inhibited and they affected the gene expression of AMPK-mTOR signaling pathway. The all results showed that AMPD1 mediated glutamate through AMPK-mTOR signaling pathway to regulate the formation of fish IMP.

Molecular Characterization of LKB1 of Triploid Crucian Carp and Its Regulation on Muscle Growth and Quality.

Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.

Establishment and application of a human osteosarcoma U-2OS cell line that can stably express Cas9 protein.

Osteosarcoma is the most common primary malignant bone tumor, and U-2OS is a common osteosarcoma cell model. The study obtained a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein, and we reported its production method and application. Firstly, we introduced a Cas9 protein expression gene and an antibiotic screening marker gene through CRISPR/Cas9 system to construct a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein. Secondly, as the cell line could stably express Cas9 protein, it was only transfected alone a small sgRNA fragment for related gene editing, we then transfected, respectively, a small ETV4 and MALAT1 sgRNA fragment to U-2OS tool cell line for gene editing. Lastly, the Q-PCR results showed that the transcription levels of ETV4 and MALAT1 were significantly decreased, and western blotting result showed that the translation level of ETV4 was significantly decreased, these results indicated that the constructed U-2OS tool cell line could effectively edit protein-coding gene (ETV4) and long non-coding RNA gene (MALAT1). The results of this study also indicated that the constructed U-2OS tool cell line could greatly improve the efficiency of gene editing. Therefore, the genetic engineering cell line provided by the study is of great significance for studying the pathogenesis and regulatory network of osteosarcoma, and for preventing and treating bone tumor as soon as possible.

A Pragmatic Device Based on a Double-Sided Functional Structure for Efficient Water Harvesting.

Water collection from fog has received much attention to meet the challenges of scarcity of clean drinking water in desert and arid regions. Currently, solar-thermal technology is being used as an efficient, sustainable, and low-cost method for water desalination to produce clean water. To collect the clean water, in recent years, most researchers have designed the structure of water collection surfaces. However, the heat released during the liquefaction process of droplets has an adverse effect on the condensation of droplets, and thus affecting the water collection efficiency. Here, in order to improve water collection efficiency, a radiative cooling layer is introduced on the back of the collection surface to dissipate the heat released during droplet liquefaction. The radiative cooling layer, consisting of poly(vinylidene fluoride-co-hexafluoropropene) embedded with SiO2 and CaMoO4 nanoparticles, can theoretically cool 18.1 °C below the ambient temperature in the daytime. With the addition of cooling coating on the back of the water collection surface, the water harvesting efficiency can be increased by 43-52%. The developed water harvesting device may provide a new pathway to the efficient collection of fresh water.