OBJECTIVETo study the effects of fertilizers with the different proportional of nitrogen, phosphorus and potassium on growth and active ingredient of Astragalus membranaceus.

METHODField experiment was conducted based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium. The effects on growth and active ingredient of A. membranaceus were analyzed.

RESULTFertilization promoted the seedling growth and provided abundant supply of nutrition for growth of root, yield and accumulation of active ingredient at the later growth stage, and increased the accumulation of dry matter of stem-leaf and root system. The effect of nitrogen, phosphorus and potassium application on the total dry matter accumulation of A. membranaceus was as following: nitrogen > potassium > phosphorus; the effect on the stem-leaf dry matter accumulation was as following: nitrogen > phosphorus > potassium; the effect on the root dry matter accumulation was as following: nitrogen > potassium > phosphorus. Nitrogen, phosphorus and potassium fertilizer increased the root yield of A. membranaceus. Obviously, the effect on the root yield was as following: nitrogen > potassium > phosphorus. The application of different proportional with nitrogen, phosphorus and potassium increased the content of polysaccharide and astragaloside, but had no distinct effect on the content of total flavonoids. The effect on the content of polysaccharide was as following: potassium > phosphorus > nitrogen, but the effect on the conten,t of astragaloside was as following: nitrogen > potassium > phosphorus.

CONCLUSIONNitrogen and potassium fertilizer application had more important effect on growth, yield and the contents of polysaccharide and astragaloside in A. membranaceus. During medicinal plants cultivation process, it should pay attention to the application of nitrogen fertilizer and potassium fertilizer and make balance application of nitrogen, phosphorus and potassium fertilizer.

Astragalus membranaceus ; drug effects ; growth & development ; metabolism ; Fertilizers ; Nitrogen ; pharmacology ; Phosphorus ; pharmacology ; Potassium ; pharmacology

N-glycanase is a class of deglycosylation enzymes, widely used in the study of N-glycosylation modification of glycoprotein. In this study, an N-glycanase gene (OsPNGase A, XM_015775832) with high GC content (69.48%) was cloned from rice and then the yeast secretory expression vector pPICZ(α)A-OsPNGase A was constructed for the purpose of transformation to Pichia pastoris. After induction in Pichia pastoris SMD1168H, the target protein was purified by DEAE Sepharose and HisTrap HP chromatography, with a yield of 12.3 mg OsPNGase A from 1 L fermentation medium, showing a specific activity of 258 U/mg. SDS-PAGE revealed that the purified OsPNGase A was a single band and showed consistentcy with the expected molecular weight. OsPNGase A could act on transferrin recombinantly expressed in rice, avidin recombinantly expressed in corn and horseradish peroxidase. Furthermore, OsPNGase A showed higher activity than commercial PNGase F towards avidin. OsPNGase A displayed the highest digestion activity at pH 6.0 and 40 °C, and was also active in the neutral and alkaline environment. Despite the fact that OsPNGase A was inhibited by reducing agents and surfactants, it still maintained partial enzymatic activity in 100 mmol/L β-ME or DTT. Therefore, the successful expression of rice OsPNGase A provides a new tool for the study of plant glycoproteins and the establishment of yeast secretion expression system lays the foundation for the preparation of PNGase A.

Skeletal muscle plays a paramount role in physical activity, metabolism, and energy balance, while its homeostasis is being challenged by multiple unfavorable factors such as injury, aging, or obesity. Exosomes, a subset of extracellular vesicles, are now recognized as essential mediators of intercellular communication, holding great clinical potential in the treatment of skeletal muscle diseases. Herein, we outline the recent research progress in exosomal isolation, characterization, and mechanism of action, and emphatically discuss current advances in exosomes derived from multiple organs and tissues, and engineered exosomes regarding the regulation of physiological and pathological development of skeletal muscle. These remarkable advances expand our understanding of myogenesis and muscle diseases. Meanwhile, the engineered exosome, as an endogenous nanocarrier combined with advanced design methodologies of biomolecules, will help to open up innovative therapeutic perspectives for the treatment of muscle diseases.
Exosomes/physiology* ; Muscle, Skeletal/metabolism* ; Cell Communication ; Homeostasis

SG600, SG900 and SG1100 were synthesized by the sol-gel method. Further treatments with increasing temperatures influenced and determined the crystallization degree of the material. Primary cultured osteoclasts were incubated for 4h and 48h on samples. Osteoclast actin labeling was examined by cytochemical staining. The concentrations of Ca and P in culture medium were quantified by colorimetric methods. SEM examined osteoclast morphology and resorption lacuna. Actin staining revealed on all three materials the typical adhesion contact ring. The Ca concentration in the culture medium of SG600 was significantly higher than that in control medium, SG900 and SG1100. Ca and P concentrations were always higher in culture media with the presence of osteoclasts. Morphological studies by scanning electron microsopy(SEM) showed a good adhesion behavior of osteoclasts on all three samples. Well-developed and deep resorption lacunae appearing after the osteoclastic resorption action were detected on all three samples. The synthetic bioglasses with different crystallizations caused different solubility, which seemed to have little effect on the osteoclast resorption behavior. The results of morphological studies on osteoclasts and resorption lacunae clearly demonstrate that the synthetic bioglasses are easily resorbed in vitro by osteoclasts.
Absorbable Implants ; Biocompatible Materials ; Biodegradation, Environmental ; Bone Substitutes ; Cells, Cultured ; Ceramics ; Crystallization ; Osteoclasts ; cytology ; metabolism ; ultrastructure ; Phase Transition

Abstract. Species-diagnostic anatomical characters of fleshflies are not known for most immature stages or even adults, and an existing key may be incomplete or difûcult for nonspecialists to use. The use of sarcophagids for PMI estimations has been greatly hampered by their highly similar morphological characters. DNA-based method can be used as a supplemental means of morphological method in identification of forensically important sarcophagid flies. However, relying solely on single DNA fragment for delimiting species is considered to be unreliable, especially when the fragment was small. Sequence data of selected regions of the cytochrome oxidase subunit two (COII) and 16S ribosomal RNA (16SrRNA) genes of the most important Chinese fleshfly taxa associated with cadavers are presented, which can be instrumental for implementation of the Chinese Sarcophagidae database. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into five species, which indicated the possibility of separation congeneric species with the short fragments.

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.
Animals ; Cattle ; Neocallimastigales/metabolism* ; Anaerobiosis ; Rumen/microbiology* ; Propionates/metabolism* ; Isobutyrates/metabolism* ; Cellulose/metabolism* ; Fungi ; Starch/metabolism* ; Glucose/metabolism* ; Acetates ; Sucrose/metabolism* ; Cellulases ; Cellulase

We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711. The his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. With the amino acid-p-nitroaniline as the substrate, Lmo1711 hydrolyzed the substrate to free p-nitroaniline monomers, whose absorbance measured at 405 nm reflected the aminopeptidase activity. The specificity of Lmo1711 to substrates was then examined by changing various substrates, and the effect of metal ions on the catalytic efficiency of this enzyme was further determined. Based on the bioinformatics data, Lmo1711 is a member of the M29 family aminopeptidases, containing a highly conserved catalytic motif (Glu-Glu-His-Tyr-His-Asp) with typical structure arrangements of the peptidase family. The recombinant Lmo1711 with a size of about 49.3 kDa exhibited aminopeptidase activity and had a selectivity to the substrates, with the highest degree of affinity for leucine-p-nitroaniline. Interestingly, the enzymatic activity of Lmo1711 can be activated by Cd²⁺, Zn²⁺, and is strongly stimulated by Co²⁺. We here, for the first time demonstrate that L. monocytogenes lmo1711 encodes a cobalt-activated aminopeptidase of M29 family.

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
Aldose-Ketose Isomerases ; biosynthesis ; Arabidopsis ; enzymology ; Arabidopsis Proteins ; biosynthesis ; Cloning, Molecular ; Escherichia coli ; metabolism ; Metals ; Pentosephosphates ; Recombinant Proteins ; biosynthesis

OBJECTIVE： To establish a UPLC fingerprint of Ficus tikoua. METHODS： UPLC method was adopted. The determination was performed on Waters ACQUITY UPLC BEF C18 column with mobile phase consisted of 0.2％ aqueous acetic acid-acetonitrile （gradient elution）； the detection wavelength was 254 nm； the flow rate was 0.1 mL/min； the column temperature was 25 ℃， and sample size was 2 μL. UPLC fingerprints of 10 batches of samples and 2 batches of adulterants were determined by using No. 14 peak as reference. The similarity evaluation was carried out by using the TCM Chromatographic Fingerprint Similarity Evaluation System （2012 edition） so as to determine common peak. The cluster analysis was performed by using SPSS 20.0 software. SIMCA 13.1 software was used to conduct the principal component analysis and orthogonal partial least squares discriminant analysis （OPLS-DA）. RESULTS： There were 28 common peaks in UPLC fingerprint of 10 batches of F. tikoua. The similarity of 10 batches of F. tikoua was between 0.839 and 0.935， and the similarities of the 2 batches of adulterants were 0.503 and 0.173 respectively， which indicated that F. tikoua could be distinguished from adulterants. 10 batches of F. tikoua could be divided into 2 categories by cluster analysis and principle component analysis， and S3-S5， S9 and S10 were grouped into one category， and the remaining batches were grouped into one category. 7 components with a variable importance in projection （VIP） value ＞1 were screened by OPLS-DA analysis. These 7 components may be the main components that caused the quality difference of 10 batches of F. tikoua samples. CONCLUSIONS： Established fingerprint， cluster analysis， principle component analysis and OPLS-DA can be used for the identification and quality control of F. tikoua.