Inhibitory activity of flavonoids fraction from Astragalus membranaceus Fisch. ex Bunge stems and leaves on Bacillus cereus and its separation and purification.

Introduction: Astragalus membranaceus Fisch. ex Bunge is a traditional botanical drug with antibacterial, antioxidant, antiviral, and other biological activities. In the process of industrialization of A. membranaceus, most of the aboveground stems and leaves are discarded without resource utilization except for a small amount of low-value applications such as composting. This study explored the antibacterial activity of A. membranaceus stem and leaf extracts to evaluate its potential as a feed antibiotic substitute. Materials and methods: The antibacterial activity of the flavonoid, saponin, and polysaccharide fractions in A. membranaceus stems and leaves was evaluated by the disk diffusion method. The inhibitory activity of the flavonoid fraction from A. membranaceus stems and leaves on B. cereus was explored from the aspects of the growth curve, cell wall, cell membrane, biofilm, bacterial protein, and virulence factors. On this basis, the flavonoid fraction in A. membranaceus stems and leaves were isolated and purified by column chromatography to determine the main antibacterial components. Results: The flavonoid fraction in A. membranaceus stems and leaves had significant inhibitory activity against B. cereus, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 1.5625 and 6.25 mg/mL, respectively. A. membranaceus stem and leaf flavonoid fraction can induce death of B. cereus in many ways, such as inhibiting growth, destroying cell wall and cell membrane integrity, inhibiting biofilm formation, inhibiting bacterial protein synthesis, and downregulating virulence factor expression. In addition, it was clear that the main flavonoid with antibacterial activity in A. membranaceus stems and leaves was isoliquiritigenin. Molecular docking showed that isoliquiritigenin could form a hydrogen bonding force with FtsZ. Conclusion: A. membranaceus stem and leaf flavonoid fractions had significant inhibitory activity against B. cereus, and the main chemical composition was isoliquiritigenin.

Ultrasound-assisted extraction, optimization, isolation, and antioxidant activity analysis of flavonoids from Astragalus membranaceus stems and leaves.

Astragalus membranaceus is a medicinal and edible species in China, with a variety of biological activities. This study evaluated the reuse potential of A. membranaceus waste as a source of food antioxidants. Antioxidant and antifungal activities of flavonoids, polysaccharides, and saponins from A. membranaceus stems and leaves were evaluated. Results showed that inhibition rate of flavonoids on six tested fungi reaches 100 % at a concentration of 5 mg/mL, and the antioxidant test demonstrated satisfactory antioxidant activity. On this basis, an extremely economical ultrasonic-assisted extraction of flavonoids from A. membranaceus stems and leaves was developed and optimized via response surface methodology (RSM). Optimized conditions included an extraction time of 35 min, ethanol concentration of 75 %, liquid-solid ratio of 40 mL/g, and extraction temperature of 58 °C, in which the extraction yield of flavonoids was 22.0270 ± 2.5739 mg/g. The total flavonoids were separated and purified using activity-guided isolation technology, and frac. ccd with strong antioxidant activity were analyzed via HPLC-MS/MS. Results showed that main components are isoquercitrin and astragalin. This study can provide a potential innovative application for the development of natural food antioxidants from A. membranaceus waste.

Complete Genome Sequencing and Infectious cDNA Clone Construction of Soybean Mosaic Virus Isolated from Shanxi.

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the crossfamily infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

Deltex3 inhibits Epithelial Mesenchymal Transition in Papillary Thyroid Carcinoma via promoting ubiquitination of XRCC5 to regulate the AKT signal pathway.

Background: Papillary thyroid carcinoma (PTC) is one of the most common endocrine malignant tumors. Poor prognoses such as high recurrence rate always appear in PTC patients with cervical lymph node metastasis. The process of ubiquitination plays important roles in PTC. As ubiquitin E3 ligases, Deltex (DTX) family proteins were reported to associate with multiple cancers. However, functions and mechanisms of DTX3 in PTC are currently unknown. Methods: In this study, DTX3 expressions were examined in 114 PTC and paired paracancerous normal tissues through quantitative real-time polymerase chain reaction and western blot. The clinical significances of DTX3 expressions in PTC patients were also investigated. After stable transfection with either short hairpin RNA to knock down DTX3 expression or full-length complementary DNA to upregulate DTX3 expression, changes of malignant phenotypes in two PTC cell lines K1 and TPC-1 were observed using cell viability, flow cytometry, wound healing and transwell assays. Afterwards, altered expressions of epithelial-mesenchymal transition (EMT) and AKT signal pathway related proteins were measured by western blot. Immunoprecipitation and mass spectrometry (IP-MS), immunofluorescence and Co-IP were utilized to identify the possible DTX3 interacting proteins. Results: Both mRNA and protein expressions of DTX3 were lower in PTC tissues and correlated with the presence of cervical lymph node metastasis (P<0.05). DTX3 overexpression inhibited migration and invasion of PTC cells, decreased Vimentin and phosphorylated AKT expressions, but promoted E-cadherin expression (P<0.05). Moreover, knockdown of DTX3 led to opposite changes (P<0.05). Total 46 probable DTX3 interacting proteins were identified by IP-MS. Among them, X-ray repair cross-complementing protein 5 (XRCC5) and NADH: Ubiquinone Oxidoreductase Complex Assembly Factor 5 (NDUFAF5) were verified to be associated with DTX3. Moreover, DTX3 was proved to be co-localized with XRCC5 in nucleus and promote ubiquitination of XRCC5. Conclusions: DTX3 suppresses EMT by partially facilitating ubiquitination of XRCC5 to inhibit AKT signal pathway in PTC.

Characterization of a Strain of Malva Vein Clearing Virus in Alcea rosea via Deep Sequencing.

Malva vein clearing virus (MVCV) is a member of the Potyvirus species, and has a negative impact on the aesthetic development of Alcea rosea. It was first reported in Germany in 1957, but its complete genome sequence data are still scarce. In the present work, A. rosea leaves with vein-clearing and mosaic symptoms were sampled and analyzed with small RNA deep sequencing. By denovo assembly the raw sequences of virus-derived small interfering RNAs (vsiRs) and whole genome amplification of malva vein cleaning virus SX strain (MVCV-SX) by specific primers targeting identified contig gaps, the full-length genome sequences (9,645 nucleotides) of MVCV-SX were characterized, constituting of an open reading frame that is long enough to encode 3,096 amino acids. Phylogenetic analysis showed that MVCV-SX was clustered with euphorbia ringspot virus and yam mosaic virus. Further analyses of the vsiR profiles revealed that the most abundant MVCV-vsiRs were between 21 and 22 nucleotides in length and a strong bias was found for "A" and "U" at the 5'-terminal residue. The results of polarity assessment indicated that the amount of sense strand was almost equal to that of the antisense strand in MVCV-vsiRs, and the main hot-spot region in MVCV-SX genome was found at cylindrical inclusion. In conclusion, our findings could provide new insights into the RNA silencing-mediated host defence mechanism in A. rosea infected with MVCV-SX, and offer a basis for the prevention and treatment of this virus disease.