Protective Effect of Ginsenoside CK against Autoimmune Hepatitis Induced by Concanavalin A.

Ginsenoside CK, a kind of rare ginsenoside transformed from protopanaxadiol saponins extracted from the genus Panax, has been proven to possess favorable bioactivities such as anti-inflammatory, anti-cancer, anti-diabetes, and hepatoprotective effects. The current study is targeted to determine the effect of ginsenoside CK on hepatitis induced by concanavalin A (Con A). Mice were treated with different dosages of ginsenoside CK for 7 days, and Con A (15 mg/kg) was intravenously injected to induce autoimmune hepatitis (AIH) after the last administration. The results demonstrated that pretreatment with ginsenoside CK (40 mg/kg) could obviously ameliorate the increase in serum indicators related to liver function such as AST, ALT, and ALP, and hepatic lesions induced by Con A. Meanwhile, ginsenoside CK suppressed hepatocyte apoptosis, which was observed in pathological data, and immunoblotting results showed that the expression of Bax, Bcl-2, and other proteins was regulated by CK. Furthermore, the release of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-6 in mice with AIH were lowered by the administration of 40 mg/kg of ginsenoside CK. Furthermore, ginsenoside CK elevated the gene expression of Nrf2 and Sirt1 and augmented downstream target genes such as HO-1. In addition, a significant inhibition effect of the TLR4/NF-κB signal was observed in 40 mg/kg CK-pretreated mice compared with the model group. To sum up, the results indicated that ginsenoside CK has a notable hepatoprotective effect against AIH by activating Sirt1/Nrf2 and suppressing the TLR4/NF-κB signaling pathway.

Identification of Potential Mechanisms of Rk1 Combination with Rg5 in the Treatment of Type II Diabetes Mellitus by Integrating Network Pharmacology and Experimental Validation.

In this study, we aimed to explore the potential targets and functional mechanisms of Rk1 combined with Rg5 (Rk1+Rg5) against type II diabetes mellitus (T2DM). Network pharmacology and molecular docking were used to predict and verify the targets and signaling pathways of Rk1+Rg5 against T2DM. The results were further confirmed by a db/db mouse model and a model using PA-induced L6 cells. According to network pharmacology, a total of 250 core targets of Rk1+Rg5 towards T2DM were identified; the insulin resistance signaling pathways were enriched by KEGG. Results of molecular docking indicated good binding affinity of Rk1 and Rg5 to Akt1. In vivo and in vitro studies further showed that Rk1+Rg5 is an inhibitor of skeletal muscle insulin resistance. The results showed that Rk1+Rg5 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the alleviation of the insulin resistance in skeletal muscles. Finally, the combination activated the Akt signaling pathway and promoted GLUT4 translocation to the cell membrane for glucose uptake. Altogether, our findings, for the first time, demonstrate that the combination of Rk1 and Rg5 could be beneficial for anti-T2DM, possibly involving ameliorated insulin resistance.

Burkholderia ambifaria XN08: A plant growth-promoting endophytic bacterium with biocontrol potential against sharp eyespot in wheat.

Plant growth-promoting bacteria (PGPB) have been considered promising biological agents to increase crop yields for years. However, the successful application of PGPB for biocontrol of sharp eyespot in wheat has been limited, partly by the lack of knowledge of the ecological/environmental factors affecting the colonization, prevalence, and activity of beneficial bacteria on the crop. In this study, an endophytic bacterium XN08 with antagonistic activity against Rhizoctonia cerealis (wheat sharp eyespot pathogenic fungus), isolated from healthy wheat plants, was identified as Burkholderia ambifaria according to the sequence analysis of 16S rRNA. The antibiotic synthesis gene amplification and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analyses were used to characterize the secondary metabolites. The results showed that the known powerful antifungal compound named pyrrolnitrin was produced by the strain XN08. In addition, B. ambifaria XN08 also showed the capacity for phosphate solubilization, indole-3-acetic acid (IAA), protease, and siderophore production in vitro. In the pot experiments, a derivate strain carrying the green fluorescent protein (GFP) gene was used to observe its colonization in wheat plants. The results showed that GFP-tagged B. ambifaria could colonize wheat tissues effectively. This significant colonization was accompanied by an enhancement of wheat plants' growth and an induction of immune resistance for wheat seedlings, which was revealed by the higher activities of polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). As far as we know, this is the first report describing the colonization traits of B. ambifaria in wheat plants. In addition, our results indicated that B. ambifaria XN08 might serve as a new effective biocontrol agent against wheat sharp eyespot disease caused by R. cerealis.

Diversity and Ginsenoside Biotransformation Potential of Cultivable Endophytic Fungi Associated With Panax bipinnatifidus var. bipinnatifidus in Qinling Mountains, China.

To obtain novel fungi with potent ß-glucosidase for minor ginsenoside production, Panax bipinnatifidus var. bipinnatifidus, which is a traditional medicinal plant containing various ginsenosides, was first employed to isolate endophytic fungi in this study. A total of 93 representative morphotype strains were isolated and identified according to ITS rDNA sequence analyses, and they were grouped into three phyla (Ascomycota, Basidiomycota, and Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, and Mucoromycetes), and 24 genera. Plectosphaerella (RA, 19.35%) was the most abundant genus, followed by Paraphoma (RA, 11.83%) and Fusarium (RA, 9.70%). The species richness index (S, 34) and the Shannon-Wiener index (H', 3.004) indicated that P. bipinnatifidus harbored abundant fungal resources. A total of 26 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. In total, 11 strains showed strong ß-glucosidase activities and also presented with the ability of ginsenoside biotransformation with varied glycoside-hydrolyzing pathways. Excitingly, three genera, namely, Ilyonectria, Sarocladium, and Lecanicillium, and all 11 taxa were first found to have the ability to transform ginsenosides in our study. The results indicated that P. bipinnatifidus could be a new fungi resource with potential novel natural compounds with antimicrobial activity and potent ß-glucosidase for varied minor ginsenoside production.

Probiotic Potential of Lactobacillus Strains Isolated From Fermented Vegetables in Shaanxi, China.

The objective of this study was to assess in vitro probiotic potential of Lactobacillus strains derived from artisanal fermented vegetables in Shaanxi, China. In total, 74 acid-producing Gram-positive strains with rod-shaped under the microscope were isolated from 16 samples of spontaneously fermented vegetables. Out of 74 strains, 26 showed high survival rate under low pH and high bile salts conditions and were subjected to molecular identification by 16S rRNA gene sequencing analysis. The results showed that 15 isolates belonged to Lactobacillus plantarum, 9 isolates belonged to Lactobacillus brevis, and the 2 remaining strains belonged to Weissella viridescens. The 24 Lactobacillus strains were investigated for their survival rate to transit simulated gastrointestinal tract, cell surface hydrophobicity, auto-aggregation, co-aggregation with pathogen, adhesion to Caco-2, antimicrobial activity, antibiotics susceptibility, radical scavenging ability, α-glucosidase inhibition, and the cholesterol assimilation. The results showed that the probiotic characteristics were strain-dependent, and several strains exhibited great probiotic potential with specific health benefits, which indicated that they might be excellent candidates for production of functional foods. Interestingly, it was first found that L. plantarum generally had higher antibacterial activities, α-glucosidase inhibition ability, and antibiotics susceptibility compared to L. brevis in this study. The results indicated that Lactobacillus strains isolated from fermented vegetables in Shaanxi, China, could be exploited as a promising novel probiotic source.

Diversity and Antimicrobial Activity of Endophytic Fungi Isolated from Chloranthus japonicus Sieb in Qinling Mountains, China.

The plant Chloranthus japonicus Sieb is known for its anticancer properties and mainly distributed in China, Japan, and Korea. In this study, we firstly investigated the diversity and antimicrobial activity of the culturable endophytic fungi from C. japonicus. A total of 332 fungal colonies were successfully isolated from 555 tissue segments of the medicinal plant C. japonicus collected from Qinling Mountains, China. One hundred and thirty representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, Mucoromycetes), and at least 30 genera. Colletotrichum (RA, 60.54%) was the most abundant genus, followed by Aspergillus (RA, 11.75%) and Diaporthe (RA, 9.34%). The Species Richness Index (S, 56) and the Shannon-Wiener Index (H', 2.7076) indicated that C. japonicus harbored abundant fungal resources. Thirteen out of 130 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Among of these, F8158, which was identified as Trichoderma cf. harzianum, exhibited good antagonistic capacities (the percent inhibition of mycelial growth ranged from 47.72~88.18) for different pathogens and has a potential application in biological control. In addition, it is noteworthy that the strain F8157 (Thanatephorus cucumeris, an opportunistic pathogen) showed antibacterial and antifungal activity, which is reported firstly in this study, and should be investigated further. Taken together, these results indicated that the endophytic fungi from C. japonicus may be of potential interest in screening bio-control agents and discovering of new bioactive compounds.

Isolation, diversity, and antimicrobial activity of fungal endophytes from Rohdea chinensis (Baker) N.Tanaka (synonym Tupistra chinensis Baker) of Qinling Mountains, China.

Endophytic fungi have been emerged as fruitful resources for producing structurally fascinating and biologically active secondary metabolites. However, endophytic fungi from medicinal plants of Qinling Mountains-the most important natural climatic boundary between the subtropical and warm temperate zones of China with an astonishingly high level of biodiversity-have rarely been explored as potential sources of novel fungal species and active secondary metabolites. In this study, a total of 371 fungal colonies were successfully isolated from 510 tissue segments of the medicinal Tupistra chinensis Baker collected from Qinling Mountains, China. Roots of T. chinensis Baker are used as a folk medicine to ameliorate pharyngitis and treat rheumatic diseases. A total of 100 representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), seven classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Microbotryomycetes, Agaricomycetes, Leotiomycetes, Mortierellomycetes), and at least 35 genera. The genera of Collectotrichum (IF, 29.92%), Fusarium (IF, 8.36%), Aspergillus (IF, 8.09%), and Dactylonectria (IF, 5.39%) were most frequently isolated from the tissues of T. chinensis Baker. The Species Richness Index (S, 65) and the Shannon-Wiener Index (H', 3.7914) indicated that T. chinensis Baker harbored abundant fungal resources. Moreover, five isolates were potential new taxa because of low similarity of ITS sequences ranged from 95.09%∼96.61%. Fifteen out of 100 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Two important lead compounds produced by two stains (F8047 and F8075) with high antimicrobial activities were identified using high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-QTOF MS) analyses. In addition, it was noteworthy that the strain F8001, which may be a potential new species, showed antimicrobial activity and should be investigated further. Overall, these results indicated that the endophytic fungi from T. chinensis Baker could be exploited as a novel source of bioactive compounds.

Comparative study of different molecular weight pullulan productions by Aureobasidium pullulans CGMCC No.11602.

Pullulan productions by Aureobasidium pullulans CGMCC No.11602 were conducted using the initial culture (IC) medium and the optimized culture (OC) medium, respectively, in which pullulan with significantly different molecular weights was obtained. Under the IC medium condition, the pullulan molecular weight (M w) reached 288,403 Da with a yield of 64.12 g/L after 96 h fermentation period. However, the pullulan molecular weight was much higher (M w, 3,715,352 Da), while the yield of pullulan was lower (40.12 g/L) using the OC medium. The FTIR spectra indicated that pullulan produced using the IC and OC medium were similar. Transcriptome analysis showed that a total of 871 differentially expressed genes (DEGs) were screened and "N-glycan biosynthesis" and "other types of O-glycan biosynthesis" were the most highly represented differential metabolic pathways (DMPs). Specifically, the genes involved in the two DMPs consistently pointed to glucosyltransferase genes (GTF), all of which were up-regulated in the OC medium when compared with those in the IC medium. Further studies showed that the activity and the relative quantity (RQ) of GTF transcription were remarkable higher, which were coincident with the slower decrease in the molecular weight of pullulan in the OC medium than those in the IC medium during the late stage of fermentation. The results indicated that GTF may be involved in the regulation of pullulan molecular weight.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose.

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose=80:20) was 0.212h-1, which was significantly higher than those of potato starch hydrolysate alone (0.146h-1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose=80:10:10, 0.166h-1) with 100gL-1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

A novel smart injectable hydrogel prepared by microbial transglutaminase and human-like collagen: Its characterization and biocompatibility.

Various tissue scaffold materials are increasingly used to repair skin defects by cross-linking because of the ability to fill and implant in any form via operation. However, crosslinker residues cannot be easily removed from scaffold materials prepared by chemical crosslinking methods, limiting their use for skin tissue engineering. Here, microbial transglutaminase (MTGase), a nontoxic crosslinker with high specific activity and reaction rate under mild conditions, was employed crosslinks in human-like collagen (HLC) to yield novel smart MTGase crosslinked with human-like collagen (MTGH) hydrogels, which are sensitive to temperature and/or enzymes. Various ratios of MTGase/HLC were performed, and their physicochemical properties were characterized, including the swelling ratio, the elastic modulus, the morphology and the porosity. The degradation behavior and mechanism of MTGase in concentration-dependent manner involved in formation hydrogels were identifying in vitro. The cell attachment in vitro and biocompatibility in vivo were also investigated. The results demonstrated that the use of different concentrations of MTGase to crosslink HLC produced products with different degradation times and biocompatibilities. The 50U/g MTGase-prepared MTGH hydrogels had a higher density of crosslinks, which made them more resistant to degradation by collagenase I and collagenase II. However, 40U/g MTGase-prepared MTGH hydrogels were more suitable for cell attachment. In addition, compared with the Collagen Implant I® (SUM) used in animal experiments, the 40U/g MTGase-prepared MTGH hydrogels had a lower toxicity and better biocompatibility. Therefore, 40U/g MTGase crosslinked with HLC should be used to prepare MTGH hydrogels for potential application as soft materials for skin tissue engineering.

A choline monooxygenase gene promoter from Salicornia europaea increases expression of the beta-glucuronidase gene under abiotic stresses in tobacco (Nicotiana tabacum L.).

A 1312 bp 5' flanking region of Salicornia europaea choline monooxygenase (SeCMO) gene was isolated using the anchored PCR. To investigate the mechanism of regulation for this stress-induced gene, the SeCMO promoter-beta-glucuronidase (GUS) chimeric gene constructs containing five deletions F1, F2, F3, F4 and F5 were introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. The functional properties of each promoter fragment were examined by assaying GUS activity in the leaves of transgenic tobacco treated with abiotic stresses (NaCl, PEG6000 and low temperature). The GUS activity in transgenic tobacco with F2 (-1056 to +8) construct showed highest increase under all the three abiotic stresses. Thus, the study provided a potential promoter induced by the salt, dehydration and cold for the plant genetic manipulation.

Bisintercalator-containing dinuclear iron(III) complex: An efficient artificial nuclease.

Two acridine groups were successfully introduced into di-iron(III) complex. DNA cleavage experiments indicated that complex conjugating bisacridine groups can enhance 300-fold for the cleavage efficiency compared with complex lacking of acridine conjugation. Further ligation assay of DNA segments provided the evidence for hydrolytic mechanism of DNA cleavage.

Efficient enhancement of DNA cleavage activity by introducing guanidinium groups into diiron(III) complex.

Inspired by the structures of natural nucleases, guanidinium groups were introduced into binuclear iron(III) systems. Compared with the corresponding analogue without guanidinium groups, the new diiron(III) system led to considerable rate enhancement on DNA cleavage. The cooperativity between metal ions and guanidine groups was evidenced by the fact that no significant cleavage was observed after incubating pBR322 plasmid DNA with non-metalated ligands or free Fe3+ ion. DNA binding experiments indicated that introduction of positively charged guanidinium groups can obtain more than one order of magnitude enhancement in the affinity of complex with DNA.