A α-L-rhamnosidase from Echinacea purpurea endophyte Simplicillium sinense EFF1 and its application in production of Calceorioside B.

Calceorioside B, a multifunctional phenylethanol glycosides (PhGs) derivative, exhibits a variety of notable properties, such as antithrombotic, anti-tumorigenic, anti-neocoronavirus, anti-inflammatory, and neuroprotective effects. However, the large-scale production of calceorioside B is routinely restricted by its existence as an intermediary compound derived from plants, and still unachieved through excellent and activity chemical synthesis. Here, a total of 51 fungal endophytes were isolated from four PhGs-producing plants, and endophyte Simplicillium sinense EFF1 from Echinacea purpurea was identified with the ability to de-rhamnosing isoacteoside to generate calceorioside B. According to the RNA-transcription of EFF1 under the various substrates, a key gene CL1206.Contig2 that undertakes the hydrolysis function was screened out and charactered by heterologous expression. The sequence alignment, phylogenetic tree construction and substrate specificity analysis revealed that CL1206 was a novel α-L-rhamnosidase that belongs to the glycosyl hydrolase family 78 (GH78). The optimum catalytic conditions for CL1206 were at pH 6.5 and 55 °C. Finally, the enzyme-catalyzed approach to produce calceorioside B from 50 % crude isoacteoside extract was explored and optimized, with the maximum conversion rate reaching 69.42 % and the average producing rate reaching 0.37 g-1.L-1.h-1, which offered a great biocatalyst for potential industrial calceorioside B production. This is the first case for microorganism and rhamnosidase to show the hydrolysis ability to caffeic acid-modified PhGs.

Characterization of three novel stem rot pathogens and their antagonistic endophytic bacteria associated with Cistanche deserticola.

Cistanche deserticola is a precious Chinese medicinal material with extremely high health care and medicinal value. In recent years, the frequent occurrence of stem rot has led to reduced or even no harvests of C. deserticola. The unstandardized use of farm chemicals in the prevention and control processes has resulted in excessive chemical residues, threatening the fragile desert ecological environment. Therefore, it is urgent to explore safe and efficient prevention and control technologies. Biocontrol agents, with the advantages of safety and environment-friendliness, would be an important idea. The isolation, screening and identification of pathogens and antagonistic endophytic bacteria are always the primary basis. In this study, three novel pathogens causing C. deserticola stem rot were isolated, identified and pathogenicity tested, namely Fusarium solani CPF1, F. proliferatum CPF2, and F. oxysporum CPF3. For the first time, the endophytic bacteria in C. deserticola were isolated and identified, of which 37 strains were obtained. Through dual culture assay, evaluation experiment and tissue culture verification, a biocontrol candidate strain Bacillus atrophaeus CE6 with outstanding control effect on the stem rot was screened out. In the tissue culture system, CE6 showed excellent control effect against F. solani and F. oxysporum, with the control efficacies reaching 97.2% and 95.8%, respectively, indicating its great potential for application in the production. This study is of great significance for the biocontrol of plant stem rot and improvement of the yield and quality of C. deserticola.

High-Throughput Preparation and Characterization of ZrMoTaW Refractory Multi-Principal Element Alloy Film.

In this work, high-throughput screening technology is applied to four-member refractory multi-principal element alloys (RMPEAs) films with high W content. The exploration of refractory metals such as W is strictly limited by the high melting temperature in this work; a multi-gradient deposition method was introduced to overcome this obstacle. By adjusting the power and distance from the target to the sample, component Zr11Mo11Ta25W53 with the best hardening performance was successfully obtained. The uniformity of the material library was analyzed from the perspectives of phase structure and micromorphology. With the help of Hume-Rothery theory and XRD analysis, it is shown that the film has a stable bcc structure. It is believed that film uniformity, nanoscale size, preferential orientation, surface roughness, and solution mechanism are the pivotal factors to improve hardness performance, especially for high W components. The hardness and modulus of elasticity can reach 20 GPa and 300 GPa, respectively, and the H/Er and H3/Er2 values are 0.067 and 0.065, showing the best wear resistance in many samples.

Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth.

Although aerobic glycolysis (the Warburg effect) is a hallmark of cancer, key questions, including when, how, and why cancer cells become highly glycolytic, remain less clear. For a largely unknown regulatory mechanism, a rate-limiting glycolytic enzyme pyruvate kinase M2 (PKM2) isoform is exclusively expressed in embryonic, proliferating, and tumor cells, and plays an essential role in tumor metabolism and growth. Because the receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) signaling cascade is a frequently altered pathway in cancer, we explored its potential role in cancer metabolism. We identified mTOR as a central activator of the Warburg effect by inducing PKM2 and other glycolytic enzymes under normoxic conditions. PKM2 level was augmented in mouse kidney tumors due to deficiency of tuberous sclerosis complex 2 and consequent mTOR activation, and was reduced in human cancer cells by mTOR suppression. mTOR up-regulation of PKM2 expression was through hypoxia-inducible factor 1α (HIF1α)-mediated transcription activation, and c-Myc-heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent regulation of PKM2 gene splicing. Disruption of PKM2 suppressed oncogenic mTOR-mediated tumorigenesis. Unlike normal cells, mTOR hyperactive cells were more sensitive to inhibition of mTOR or glycolysis. Dual suppression of mTOR and glycolysis synergistically blunted the proliferation and tumor development of mTOR hyperactive cells. Even though aerobic glycolysis is not required for breach of senescence for immortalization and transformation, the frequently deregulated mTOR signaling during multistep oncogenic processes could contribute to the development of the Warburg effect in many cancers. Components of the mTOR/HIF1α/Myc-hnRNPs/PKM2 glycolysis signaling network could be targeted for the treatment of cancer caused by an aberrant RTK/PI3K/AKT/mTOR signaling pathway.

[Molecular mechanism of beta cell apoptosis induced by p58 in high glucose medium].

Type 2 diabetes is a complex disorder with a strong genetic background. CDC2L2 is one of the susceptibility genes of type 2 diabetes in Chinese Han population in northern area. The relationship between CDC2L2 and type 2 diabetes remains unknown. In this paper, the function and its molecular pathway of p58, a protein coded by CDC2L2, in beta cell apoptosis were investigated. INS-1 cells cultured in high glucose (20 mmol/L) medium were divided into control, vector control (transfected with pcDNA3.0) and experimental (transfected with pcDNA3.0-HA-p58) groups. Beta cell apoptosis level was detected by Annexin V-FITC/PI double staining assay. The flow cytometry results showed that in high glucose medium (20 mmol/L), high expression of p58 increased beta cell apoptosis significantly compared with that in blank and vector controls (P<0.01, P<0.05). Western blot revealed that the expressions of Caspase-3, Bax and cytochrome C in cytoplasm increased significantly (P<0.05, P<0.01, P<0.01), whereas the expression of Bcl-2 decreased significantly (P<0.05) in the INS-1 cells with high expression of p58, compared with those in both control groups. However, the Bad and Bik expression levels of INS-1 cells did not show obviously changes compared with those in both controls. The above results suggest that in high glucose condition, p58 may induce INS-1 cell apoptosis through up-regulating the expression of Bax and down-regulating the expression of Bcl-2, since both of them could promote the release of cytochrome C into cytoplasm, and finally activate Caspase-3. These results provide an important basis for the further exploration of the molecular mechanism of beta cell apoptosis induced by CDC2L2.